MASARYK UNIVERSITY
FACULTY OF MEDICINE
DEPARTMENT OF PHARMACOLOGY
DRUG ADDICTION AS A COMORBIDITY OF
PSYCHIATRIC DISORDERS IN ANIMAL MODELS
Habilitation thesis
Annotated publications
PharmDr. Jana Rudá-Kučerová, Ph.D.
Brno 2016
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Originality and conflict of interest statement
Presented original data were acquired as a part of original research performed by the
author together with a team of her colleagues and students.
The research was supported by following projects:
 Czech Ministry of Education - research project: MSM0021622404
 “CEITEC - Central European Institute of Technology” (CZ.1.05/1.1.00/02.0068)
from European Regional Development Fund
 Project provided by the Internal Grant Agency of the Faculty of Medicine at
Masaryk University (InGA): MUNI/11/InGA09/2012
 Project from the SoMoPro II programme, People Programme (Marie Curie action)
of the Seventh Framework Programme of EU according to the REA Grant
Agreement No. 291782, further co-financed by the South-Moravian Region
 Specific University Research Grants, as provided by the Ministry of Education,
Youth and Sports of the Czech Republic in the years 2011 to 2016, numbers:
MUNI/A/0852/2010, MUNI/A/0763/2011, MUNI/A/0701/2012,
MUNI/A/0886/2013, MUNI/A/1116/2014, MUNI/A/1284/2015
The author declares no conflict of interest.
Brno, 28.7.2016
PharmDr. Jana Rudá-Kučerová, Ph.D.
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Acknowledgement
This thesis represents years of work and dedication of a number of people. First I would
like to thank to Prof Alexandra Šulcová who served as my PhD thesis advisor, head of the
department and later the leader of the CEITEC research group Experimental and Applied
Neuropsychopharmacology. At all positions she always helped me to develop new ideas
and collaborations and revised my papers.
I could not perform demanding self-administration experiments without a great support
from my students. I, Petra Amchová and Zuzana Babinská formed a team which was able
to carry out many experiments without confusion, helping each other. With Petra we
started collaborations which broaden our research approaches. Petra introduced the
olfactory bulbectomy model to the Centre of Excellence “Neurobiology of Addiction” at
the University of Cagliari, where Dr. Liana Fattore continued with experiments we
outlined together. Meeting Liana was a great experience as she became also an amazing
mentor who taught me a lot about the art of writing papers. Another person who well
deserves my gratitude is Dr. Magdaléna Šustková-Fišerová. She performed the in vivo
microdialysis experiments with us and helped me a lot with interpretation of our data.
There are many more people who helped me over the years, besides others it is Dr.
Vincenzo Micale who validated the model of schizophrenia at our laboratory, Dr. Amit
Khairnar who was ready to help me anytime and involved me into preclinical research of
Parkinson’s disease.
Animal studies require a lot of daily work in the animal centre which was carried out by
our technicians: Petra Kameníková, Marcela Kučírková, Jana Urbišová and Petra Vrbická.
Special thanks belong to Jaroslav Nádeníček whose interest and remarkable understanding
of animal behaviour solved and prevented many problems and gave me a new motivation.
Last but not least, I would not be able to carry out my research and finish this thesis
without the patience and support of my husband Honza Rudý, who married me despite he
knew how many hours I spend at work.
Besides all the humans I want to express my deep respect and gratefulness to all our rats
that are very patient with us.
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Index
1. INTRODUCTION..........................................................................................................6
2. DEPRESSION AND ADDICTION COMORBIDITY ....................................................8
2.1. Background..............................................................................................................8
2.2. Aims ......................................................................................................................10
2.3. Methods .................................................................................................................11
2.3.1. Animals...........................................................................................................11
2.3.2. Olfactory bulbectomy model of depression (OBX) ..........................................11
2.3.3. IV self-administration (IVSA) surgery and procedures ....................................12
2.3.4. In vivo microdialysis .......................................................................................14
2.3.5. Locomotor activity..........................................................................................15
2.3.6. Forced Swim Test (FST) .................................................................................15
2.3.7. Sucrose preference ..........................................................................................16
2.4. Results ...................................................................................................................18
2.4.1. The effects of methamphetamine self-administration on behavioural
sensitization in the olfactory bulbectomy rat model of depression .................................18
2.4.2. Olfactory bulbectomy increases reinstatement of methamphetamine seeking
after a forced abstinence in rats.....................................................................................29
2.4.3. Reward related neurotransmitter changes in a model of depression: an in vivo
microdialysis study .......................................................................................................38
2.4.4. Enhanced self-administration of the CB1 receptor agonist WIN55,212-2 in
olfactory bulbectomized rats: evaluation of possible serotonergic and dopaminergic
underlying mechanisms.................................................................................................54
2.4.5. Differential characteristics of ketamine self-administration in the olfactory
bulbectomy model of depression in rats ........................................................................74
3. SCHIZOPHRENIA AND ADDICTION COMORBIDITY.........................................108
3.1. Background..........................................................................................................108
3.2. Aims ....................................................................................................................110
3.3. Methods ...............................................................................................................111
3.3.1. Animals.........................................................................................................111
3.3.2. Methylazoxymethanol (MAM) model of schizophrenia.................................111
3.3.1. Alcohol drinking paradigm............................................................................111
3.3.2. IV self-administration (IVSA) surgery and procedures ..................................112
5
3.3.3. Locomotor activity........................................................................................112
3.3.4. Sucrose preference ........................................................................................112
3.4. Results .................................................................................................................113
3.4.1. Reactivity to addictive drugs in the methylazoxymethanol (MAM) model of
schizophrenia in male and female rats.........................................................................113
4. SEX-DEPENDENT SPECIFICITIES IN THE DRUG ABUSE ..................................128
4.1. Aims ....................................................................................................................130
4.2. Methods ...............................................................................................................131
4.2.1. Animals.........................................................................................................131
4.2.1. Ovariectomy and oestrogen supplementation.................................................131
4.2.2. IV self-administration (IVSA) surgery and procedures ..................................131
4.2.3. Locomotor activity........................................................................................131
4.3. Results .................................................................................................................132
4.3.1. Impact of repeated methamphetamine pretreatment on intravenous selfadministration
of the drug in males and estrogenized or non–estrogenized
ovariectomized female rats..........................................................................................132
4.3.2. Sex differences in the reinstatement of methamphetamine seeking after
forced abstinence in Sprague-Dawley rats...................................................................141
5. DISCUSSION.............................................................................................................150
5.1. Depression-addiction comorbidity ........................................................................150
5.2. Schizophrenia-addiction comorbidity ...................................................................155
5.3. Sex-differences in drug abuse...............................................................................157
6. CONCLUSION AND FUTURE PERSPECTIVES.....................................................158
7. LIST OF PAPERS RELATED TO THE HABILITATION THESIS...........................160
7.1. Publications in extenso in journals with IF............................................................160
7.2. Publications in extenso in journals without IF.......................................................162
7.3. Book chapters.......................................................................................................163
8. REFERENCES...........................................................................................................164
9. LIST OF FIGURES ....................................................................................................182
10. APPENDICES............................................................................................................183
6
1. Introduction
Drug addiction is a serious medical and psychosocial problem which leads to organic harm
of the body as well as distortion of the normal functioning of affected persons within the
society and family. Effective treatments are scarce because of variability of motivations
and causes to abuse drugs, comorbidities and different social environments (Winkler et al.,
2013). Drug abuse is clinically a frequent comorbidity of other psychiatric disorders (Testa
et al., 2013, Wedekind et al., 2010). This is particularly well documented in affective
disorders such as anxiety, depression (Volkow, 2004, Pettinati et al., 2013) and
schizophrenia (Koskinen et al., 2009, Mesholam-Gately et al., 2014).
As an explanation of the dual diagnosis of depression and addiction, the self-medication
hypothesis is widely accepted (Hall and Queener, 2007, Khantzian, 1985, Markou et al.,
1998). It explains drug abuse as an attempt of the patient to relieve the monoaminergic
deficits typical for depression. This was clinically confirmed in nicotine (Holma et al.,
2013), methamphetamine (McKetin et al., 2011) and other drugs (Tolliver and Anton,
2015). There is a growing evidence that depression and addiction underlie common
defective neurobiological regulations shared by major depression and withdrawal
syndrome (Markou et al., 1998, Lalanne et al., 2016), namely in dopaminergic (Nestler and
Carlezon, 2006), serotonergic, noradrenergic, cholinergic (Zellner et al., 2011),
glutamatergic (Tzschentke, 2002) and γ-aminobutyric acid (GABA)-ergic (Koek et al.,
2013) systems.
Another psychiatric condition known to be often comorbid with drug addiction is
schizophrenia. Almost 50 % of schizophrenic patients suffer comorbid addiction (Lybrand
and Caroff, 2009) which is linked with substantially higher burden of the disease, shorter
life expectancy (Schmidt et al., 2011, Hartz et al., 2014) and higher suicide attempt rate
(McLean et al., 2011, Melle et al., 2010). The most common drug addiction comorbid with
schizophrenia is nicotine with a prevalence of 70 to 90 % in the patients compared to 26 %
in the general population (Wing et al., 2012, Chambers et al., 2001, Mackowick et al.,
2014, Khantzian, 2016). High prevalence with other substances, such as alcohol (Krystal et
al., 2006, Kalyoncu et al., 2005, Kerner, 2015, Regier et al., 1990), opiates (Kern et al.,
2014), amphetamine psychostimulants (Grant et al., 2012) and cannabis (McLoughlin et
al., 2014) is also alarming. Several lines of evidence support the neurochemical association
7
between schizophrenia and addiction (Volkow, 2009). There is a known risk of triggering
schizophrenia by cannabis especially during adolescence (Kucerova et al., 2014, Caspi et
al., 2005, Hall and Degenhardt, 2015, Semple et al., 2005) and by psychostimulants, e.g.
methamphetamine (Yui et al., 2000, Gururajan et al., 2012) and cocaine (Malave and
Broderick, 2014). This suggests a common distortion of neurobiological mechanisms
underlying both schizophrenia and substance abuse, namely the dopaminergic system
which is known to be aberrant in both schizophrenia and substance abuses. It is possible
that the DAergic dysfunction in patients with schizophrenia disrupts normal reward
pathways predisposing individuals to higher risks for drug abuses (Chambers et al., 2001).
Furthermore, there is a large body of clinical evidence suggesting differential
characteristics of drug abuse in men and women. Despite the absolute number of female
methamphetamine abusers being lower than the male ones, women usually appear more
dependent, show higher escalation rates (Dluzen and Liu, 2008, Becker and Hu, 2008) and
most importantly tend to experience more frequent relapses (Bobzean et al., 2014, Fattore
et al., 2014). The abuse of psychostimulant drugs (cocaine, methamphetamine, etc.) is
currently on the rise among women, and it has been shown that women experience higher
cravings and suffer more relapses than men (Becker and Hu, 2008). These gender specific
differences require specific treatment strategies for men and women (Brecht et al., 2004,
Munro et al., 2006, Terner and de Wit, 2006). This particularly applies to relapseprevention
which represents a key treatment challenge especially for women (Brecht and
Herbeck, 2014).
The experimental work included in this habilitation thesis aims to unravel the relationship
between drug addiction and its psychiatric comorbidities in animal models. These models
may serve in future for testing and development of innovative treatment strategies for dual
disorders. Furthermore, the important factor of sex differences is included in order to
provide information base for development of gender specific treatments. Therefore, this
work is divided into three chapters, first focusing on depression-addiction comorbidity,
second on schizophrenia-addiction comorbidity and last on sex-related differences in drug
taking behaviours.
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2. Depression and addiction comorbidity
2.1. Background
Depression and addiction are frequently comorbid as indicated by a high prevalence of the
secondary addictive disorder in patients with history of major depression and other major
psychiatric disorders (Langas et al., 2010, Testa et al., 2013, Volkow, 2004). The
likelihood of drug addiction and depression to occur together in the same individual is
approximately 5 times greater than what would be expected by the prevalence of each
disorder alone and leads to increased suicide rates among depressive individuals (OrtizGomez
et al., 2014).
One of the theories explaining this comorbidity is the “self-medication hypothesis”, arising
from common risk factors and similarities in the underlying neurobiology of depression
and drug addiction (Hall and Queener, 2007, Khantzian, 1985, Khantzian, 2013, Khantzian
and Albanese, 2009). This theory indicates that a potential monoaminergic deficit in
depression may be relieved by the drug of abuse, thus individuals with depression have
deficits in brain reward systems and may turn to drugs that create euphoric feelings to
compensate for their anhedonia and motivational inadequacy (Baicy et al., 2005, Koob and
Le Moal, 2008, Markou et al., 1998). There is supporting clinical evidence for
methamphetamine (McKetin et al., 2011) and other drugs (McKernan et al., 2015).
Consequently, individuals with affective disorders suffer from higher cravings and
increased relapse rate (Witkiewitz and Bowen, 2010) which is the most demanding
problem faced by clinicians related to the treatment of drug abuse (Schuckit, 2006).
There is growing evidence that common defective neurobiological mechanisms underlie
depression and addiction (Markou et al., 1998). Thus a study of these similarities might
stimulate the future development of innovative antidepressants acting through the reward
circuit of the mesocorticolimbic dopaminergic system (Nestler and Carlezon, 2006).
However, it is essential to differentiate such comorbid disorders into those patients with a
primary diagnosis of depression from those with a primary substance abuse disorder
(Nunes and Rounsaville, 2006).
Despite the high prevalence of drug addiction and depression comorbidity, there are only
few animal models examining drug abuse behaviors in depression and relapse of drug
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addiction. Conformable with the clinical experience, a positive rewarding effect of
amphetamine in rodents subjected to chronic mild stress was shown to be more apparent
than in non-stressed rodents. This evidence suggests that stress influences the vulnerability
for drug-taking behaviour (Lin et al., 2002). Another model used for the study of dual
disorder is the selectively bred rat strain depression-like phenotype with low performance
in the forced swim test (SwLo). However, in this model contradictory data were reported
showing increased consumption of methamphetamine and cocaine in the SwLo line (Weiss
et al., 2008) and opposite effect in a later study (Lin et al., 2012). Unfortunately, this
model was not further tested and the findings remain inconclusive.
The most commonly used model of depression for study of the dual disorder has become
olfactory bulbectomy (OBX) (Kelly et al., 1997, Filip et al., 2013). Bulbectomized rats
were recorded to be hyper-responsive to the locomotor stimulating properties of cocaine
administration. This was explained by the hypersensitivity to the drug induced by OBX
and the model was suggested to be appropriate for investigation of comorbid depression
and addiction disorder (Chambers and Taylor, 2004, Slattery et al., 2007). Validity of the
self-medication hypothesis was further confirmed when bulbectomized rats showed
decreased behavioural depressive-like symptoms when they were treated with nicotine
either intraperitoneally or by self-administration paradigm (Vieyra-Reyes et al., 2008).
Our team has substantially contributed to the research of addiction behaviours in the OBX
model. We developed this rat model of depression and addiction dual disorder where
olfactory bulbectomized animals showed a significantly higher vulnerability in intravenous
drug self-administration of methamphetamine (Kucerova et al., 2012). Later we confirmed
also increased tendency of the OBX rats to reinstate the methamphetamine seeking
behaviour (Babinska et al., 2016). Furthermore, we tested other drugs, specifically
cannabinoid receptor-1 synthetic agonist (Amchova et al., 2014) and ketamine (Babinska
and Ruda-Kucerova, 2016) with similar results. We have also attempted to explain the
underlying neurochemical variables in the nucleus accumbens shell after a drug challenge
(Amchova et al., 2014, Ruda-Kucerova et al., 2015b).
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2.2. Aims
The research on the animal model of the depression and addiction comorbidity aimed to:
1. Establish the rat model of the dual disorder using olfactory bulbectomy as a model
of depression while drug abuse was modelled by intravenous self-administration of
methamphetamine
 Section 2.4.1., Kucerova et al., 2012
2. Further validate the model by assessing the relapse-like behaviour towards
methamphetamine
 Section 2.4.2., Babinska et al., 2016
3. Further validate the model by assessing the basal and methamphetamine influenced
profile of extracellular levels of neurotransmitters in the nucleus accumbens shell,
i.e. the key area of the reward circuit
 Section 2.4.3., Ruda-Kucerova et al., 2015
4. Extend the rat model of the dual disorder to intravenous self-administration of
synthetic CB1 receptor agonist
 Section 2.4.4., Amchova et al., 2014
5. Extend the rat model of the dual disorder to intravenous self-administration of
potential new antidepressant ketamine
 Section 2.4.5., Babinska and Ruda-Kucerova, 2016
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2.3. Methods
2.3.1. Animals
Adult male rats of Wistar, Sprague-Dawley or Lister-Hooded strain were used in the
studies. The rats were housed individually in standard rodent plastic cages. Environmental
conditions during the whole study were constant: relative humidity 50-60 %, room
temperature 23◦C ± 1◦C, inverted 12-hour light-dark cycle. Food and water were available
ad libitum. All experiments were conducted in accordance with all relevant laws and
regulations of animal care and welfare. The experimental protocol was approved by the
Animal Care Committee of the Masaryk University, Faculty of Medicine, Czech Republic,
and carried out under the European Community guidelines for the use of experimental
animals.
2.3.2. Olfactory bulbectomy model of depression (OBX)
Bilateral olfactory bulbectomy is a well-established model of depression with high validity
which closely mimics neurochemical, neuroanatomical, behavioural and endocrine
changes seen in patients with major depression (Song and Leonard, 2005, Harkin et al.,
2003). The bilateral ablation of the olfactory bulbs (Figure 1) was performed in accordance
with the method described by Leonard and Tuite (Leonard and Tuite, 1981) and Kelly et
al. (Kelly et al., 1997).
Figure 1: normal rat brain and OBX rat brain
(Source: author's own figure)
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As validated at our laboratory (Pistovcakova et al., 2008) animals were anaesthetized with
ketamine 50 mg/kg and xylazine 8 mg/kg given intraperitoneally. The top of the skull was
shaved and swabbed with an antiseptic solution, after which a midline frontal incision was
made in the skin on the skull and the skin was retracted bilaterally. After exposure of the
skull, the 2 burr holes 2 mm in diameter were drilled at the points 7 mm anterior to the
bregma and 2 mm lateral to bregma suture (Figure 2). Both olfactory bulbs were removed
by aspiration. The ablation cavity was filled with a haemostatic sponge. Sham operated
rats underwent the identical anaesthetic and drilling procedures as OBX animals, but their
bulbs were left intact. Experiments were carried out 3 weeks after the surgery. At the end
of the experiment, rats were euthanized by a lethal overdose of ether and their brains were
removed for confirmation of the removal of the olfactory bulbs. Rats with an incomplete
bilateral olfactory bulbectomy or with damage to other brain structures were excluded
from data analysis.
Figure 2: location of burr holes drilled for OBX procedure
(Source: Paxinos and Watson, 2007)
2.3.3. IV self-administration (IVSA) surgery and procedures
Under the general anaesthesia with ketamine 50 mg/kg and xylazine 8 mg/kg given
intraperitoneally a permanent intracardiac silastic catheter was implanted through the
external jugular vein to the right atrium. The outer part of the catheter exited the skin in the
midscapular area. The catheters were flushed daily before all the sessions with heparinized
antibiotic to prevent infection and occlusion of the catheter. During this procedure the
13
blood was aspired daily to assess the patency of the catheter, and changes in general
behaviour, weight and other circumstances were recorded. When a catheter was found to
be blocked the animal was excluded from the analysis (Thomsen and Caine, 2005).
Standard experimental cages (Figure 3) with two nose-poke holes allocated on one side of
the cage were programmed by software L2T2 or later Graphic State Notation 3.03
(Coulbourn Instruments, USA) and the IVSA sessions were conducted under the fixed
ratio (FR) schedule of reinforcement1
. Nose-poking in the active hole led to the activation
of the infusion pump and administration of an infusion followed by a timeout, when nosepoking
was recorded but not rewarded. The cage was illuminated by a house light during
the session. The light was flashing when the system was administering infusion (5 sec) and
off during the time-out period to provide environmental cue associated with the drug
infusion. IVSA sessions took place 7 days/week between 8 a.m. and 3 p.m. during the dark
period of the inverted light-dark cycle and after the end rats were returned to their home
cages.
Figure 3: IV self-administration session
(Source: author's own photo)
1
In our collaborating laboratory in Cagliari (Italy) an analogous system (Med Associates,Vermont, USA) was
used. This system is equipped with retractable levers as operandums.
14
After several weeks of stable drug intake the maintenance phase was terminated and rats
were kept in their home cages for 14 days of the forced abstinence period. On day 15, rats
were placed into self-administration chambers for the reinstatement session. The numbers
of responses on the active drug-paired nose-poke and the inactive nose-poke were recorded
but the drug was not delivered. Responses on the active nose-poke are considered to reflect
the reinstatement of drug seeking behaviour, whilst responses on inactive nose-poke reflect
nonspecific locomotor and exploratory activity.
In some studies, self-administration behaviour was initially trained by employing sweet
pellets as a reward. The sessions were conducted in the same experimental boxes as IVSA
studies (Coulbourn Instruments, USA) under the FR1 schedule of reinforcement, where 1
nose-poke lead to activation of a feeder and delivery of a single palatable pellet (BioServ,
sweet dustless rodent pellets, F0021-Purified Casein Based Formula - 45mg). The cage
was illuminated by a house light during the whole session. Self-administration sessions
lasted 30 minutes during the dark period of the inverted light-dark cycle.
2.3.4. In vivo microdialysis
As described in detail earlier (Sustkova-Fiserova et al., 2014, Fiserova et al., 1999), under
ketamine – xylazine anaesthesia (ketamine 100 mg/kg i.p., xylazine 10 mg/kg i.p.), rats
were implanted with a disposable dialysis guide cannula using a stereotaxic instrument.
The guide was randomly alternated on the left and right side. The target site was nucleus
accumbens shell (Paxinos and Watson, 2007, Paxinos and Watson, 1998).
Forty-eight hours after implantation, the probe was inserted into the guide cannula and
artificial cerebrospinal fluid was flushed through the probe at a constant rate of 2.0 μl/min.
After 80 min of habituation to the microdialysis set-up (when dialysate was discarded),
40 μl samples were collected at 20-min intervals. After 3 consecutive baseline samples,
methamphetamine (5 mg/kg in 2 ml) or vehicle (saline 2 ml/kg) was administered
intraperitoneally (at minute 60) and dialysates were collected every 20 minutes (Figure 4).
Total duration of the sampled session was 240 min in animals with administration of
METH and 180 min in animals after saline injection. This is because administration of the
vehicle was not expected to induce any changes so the session was shortened.
The amount of dopamine, serotonin and their metabolites (3-methoxytyramine = 3-MT,
3,4-dihydroxyphenylacetic acid = DOPAC, homovanillic acid = HVA and 5-
15
hydroxyindoleacetic acid (5-HIAA) resp.), as well as glutamate and GABA in the
dialysates were quantified using high-performance liquid chromatography combined with
mass spectrometry (HPLC-MS). The appropriate HPLC-MS determination methods are
described in detail earlier (Syslova et al., 2011).
Figure 4: in vivo microdialysis principle
(Source: https://www.basinc.com/products/iv/MD.html)
2.3.5. Locomotor activity
In brightly lit room, rats were individually tested for locomotor activity using the Actitrack
system (Panlab, Spain)2
. Each Plexiglas arena (45×45×30 cm) was surrounded by 2 frames
equipped with photocells located one above another at 2 and 12 cm over the cage floor.
Animals were placed in the centre of arena and the spontaneous behaviour was tracked for
10 minutes. In the test horizontal locomotor activity (the trajectory calculated by the
system as beam interruptions that occurred in the horizontal sensors) and vertical activity
(number of rearing episodes breaking the photocell beams of the upper frame) were
recorded. At the end of the session, animals were returned to their home cage and arenas
were wiped with 1% acetic acid to avoid olfactory cues (Kucerova and Sulcova, 2008,
Kucerova et al., 2006).
2.3.6. Forced Swim Test (FST)
A modified FST (Detke et al., 1995, Porsolt et al., 1977) was used to measure immobility
of the rats, as described previously (Akinfiresoye and Tizabi, 2013, Tizabi et al., 2012).
Briefly, the rats were individually placed into a plexi-glass cylinder filled with 30 cm of
2
In our collaborating laboratory in Cagliari (Italy) an analogous system Digiscan Animal Activity Analyser
(Omnitech Electronics, USA) was used. The dimensions of arenas were 42×30×60 cm.
16
water (24±1 °C). The sessions were video-taped for later scoring and the water was
changed after every animal. A time-sampling scoring technique was used, whereby the
predominant behaviour, i.e. immobility, swimming or climbing, in each 5-s period of the 5
minutes test was recorded. OBX rats acquire the depressive-like phenotype by surgery,
therefore they should exhibit spontaneous immobility in the forced swim test. Furthermore,
the aim of the test was to assess spontaneous behaviour, i.e. not a drug effect. Hence, there
is no need to have a pre-test exposure to forced swimming the day before to induce
helplessness (Tejani-Butt et al., 2003, Tizabi et al., 2012).
Figure 5: forced swim test procedure
(Source: author's own photo)
2.3.7. Sucrose preference
A two-bottle choice procedure was used to determine the sucrose intake (Chambliss et al.,
2004, Matthews et al., 1995, Romeas et al., 2009). During the 24-h training phase, each rat
was provided in their home cage with two water bottles on the extreme sides of the cage to
adapt rats drinking from two bottles. After training, one bottle was randomly switched to
contain sucrose dissolved in drinking water at concentration of 1% or 2%. The side of
sucrose presentation in the home cage was counterbalanced across rats. At 4h and 24h time
intervals both bottles were removed and the amount of liquid remaining in each bottle was
17
measured. The sucrose preference score was calculated as the percentage of sucrose
solution ingested relative to the total amount of liquid consumed as determined before and
after each test, i.e. sucrose preference = sucrose intake / total liquid (sucrose + water)
intake x 100.
18
2.4. Results
2.4.1. The effects of methamphetamine self-administration on behavioural
sensitization in the olfactory bulbectomy rat model of depression
This study established the rat model of the dual disorder using olfactory bulbectomy as a
model of depression while drug abuse was modelled by intravenous self-administration of
methamphetamine. Furthermore, in order to assess the influence of behavioural
sensitization (Robinson and Berridge, 1993, Robinson, 1984) to methamphetamine, a
chronic exposure to the drug known to induce sensitization (Landa et al., 2005) was
employed.
The results showed that olfactory bulbectomy model of depression increases
methamphetamine intake in the IV self-administration model. This finding correlates well
with the self-medication hypothesis (Hall and Queener, 2007, Khantzian, 1985, Khantzian,
2016) and a previous study with amphetamine (Holmes et al., 2002). Chronic intermittent
pre-treatment with methamphetamine was used to evaluate influence of behavioural
sensitization on the drug intake of olfactory bulbectomized and sham operated rats. Preexposure
to methamphetamine decreased the intake of the drug in the self-administration
in sham operated animals but not in rats subjected to olfactory bulbectomy. This suggests a
differential reactivity to chronic methamphetamine exposure in the OBX model, maybe
due to altered expression of behavioural sensitization in this model of depression.
Kucerova J, Pistovcakova J, Vrskova D, Dusek L, Sulcova A. The effects of
methamphetamine self-administration on behavioural sensitization in the olfactory
bulbectomy rat model of depression. Int J Neuropsychopharmacol. 2012, 15(10): 1503-11.
doi: 10.1017/S1461145711001684.
IF 5.641
Citations (WOS): 8
19
Erratum
Figure 1(b) presented in this paper contains a typographical error in the names of the
groups. The correct version follows.
The eﬀects of methamphetamine
self-administration on behavioural
sensitization in the olfactory bulbectomy
rat model of depression
Jana Kucerova1,2
, Jana Pistovcakova1,2
, Dagmar Vrskova3
, Ladislav Dusek4
and Alexandra Sulcova1
1
CEITEC – Central European Institute of Technology, Masaryk University, Brno, Czech Republic
2
Faculty of Medicine, Department of Pharmacology, Masaryk University, Brno, Czech Republic
3
Faculty of Veterinary Medicine, Department of Pharmacology and Pharmacy, University of Veterinary and Pharmaceutical
Sciences Brno, Brno, Czech Republic
4
Institute of Biostatistics and Analyses of Faculty of Medicine, Masaryk University, Brno, Czech Republic
Abstract
Depression is frequently comorbid with a drug addiction and may seriously complicate its treatment.
Currently, there is no routinely used animal model to investigate this comorbidity. In this study the
eﬀect of repeated administration of methamphetamine on i.v. drug self-administration in an olfactory
bulbectomy model of depression in rats was investigated in order to propose and validate a rat model of
comorbid depression and addiction. Male Wistar rats were either olfactory-bulbectomized (OBX) or
sham-operated. They subsequently underwent a methamphetamine sensitization regime, which consisted
of daily i.p. injections of methamphetamine for a 14-d period; controls received Sal injections at the same
frequency. The i.v. self-administration of methamphetamine (0.08 mg/kg in one infusion) paradigm on a
ﬁxed ratio schedule of reinforcement was performed using operant chambers. A signiﬁcant decrease of the
drug intake was recorded in sham-operated animals pretreated with methamphetamine when compared
to the unpretreated group. This was not apparent in the OBX groups. Both groups of OBX animals
exhibited a higher intake of methamphetamine compared to the corresponding sham-operated groups,
thus conﬁrming the hypothesis of higher drug intake in depressive conditions in this rodent model. The
procedure of behavioural sensitization to methamphetamine decreased the number of self-administered
drug doses per session in the sham-operated rats. It is hypothesized that this phenomenon resulted from
increasing eﬃcacy of the drug after behavioural sensitization caused by repeated methamphetamine
intermittent administration.
Received 16 May 2011; Reviewed 27 June 2011; Revised 14 October 2011; Accepted 17 October 2011
Key words: Depression, methamphetamine, olfactory bulbectomy model, IVSA, Wistar rats.
Introduction
Depression and addiction are frequently comorbid,
as indicated by a high prevalence of the secondary
addictive disorder in patients with a history of major
depression and other major psychiatric disorders
(Langas et al. 2010). This is true for nicotine (Kushnir
et al. 2010), alcohol (Boschloo et al. 2011) and other
drugs of abuse. Drug-dependent subjects suﬀer from
comorbid psychiatric disorder, such as depression,
in approximately 30–50% of cases (Cottencin, 2009;
Davis et al. 2008) and the frequency (data from United
States) tends to increase over time (Compton et al.
2006). The prevalence of depression in drug-addicted
individuals in Europe was recorded as approximately
50% in spite of signiﬁcantly diﬀerent environmental
and social conditions and the type of drugs abused
(Reissner et al. 2011). This comorbidity was deﬁned in
DSM-III (Kessler et al. 1996) and is more accurately
characterized in DSM-IV (Leventhal et al. 2008).
Address for correspondence : Dr J. Kucerova, Masaryk University,
Faculty of Medicine, Department of Pharmacology, Kamenice 5,
625 00 Brno, Czech Republic.
Tel.: +420549494238 Fax: +420549492364
Email: jkucer@med.muni.cz
International Journal of Neuropsychopharmacology, Page 1 of 9. f CINP 2011
doi:10.1017/S1461145711001684
ARTICLE
20
There is growing evidence that common defective
neurobiological mechanisms underlie depression and
addiction (Markou et al. 1998). Thus, a study of these
similarities might stimulate the future development of
innovative antidepressants with a new mechanism of
action through the reward circuit of the mesocorticolimbic
dopaminergic system (Nestler & Carlezon,
2006). However, it is essential to diﬀerentiate such
comorbid disorders into those patients with a primary
diagnosis of depression from those with a primary
substance abuse disorder (Nunes & Rounsaville, 2006).
The self-medication hypothesis was developed as
a possible explanation of the frequent comorbidity
of depression or anxiety with psychostimulant abuse
linked to enhanced monoaminergic neurotransmission.
Thus, it is hypothesized that symptoms
related to a potential monoaminergic deﬁcit in depression
may be relieved by the drug of abuse (Hall &
Queener, 2007; Khantzian, 1985). Evidence supporting
this hypothesis comes from the ﬁnding that antidepressant
treatment of substance abuse is more
eﬀective in depressed than non-depressed individuals
(Markou et al. 1998; Wohl & Ades, 2009).
Conformable with clinical experience, animal
models were employed, ﬁnding positive rewarding
eﬀects of amphetamine in rodents subjected to chronic
mild stress more apparent than in non-stressed
rodents. This evidence suggests that stress inﬂuences
the vulnerability for drug-taking behaviour (Lin et al.
2002). There is evidence of an increase of dopamine
receptor density in the ventral striatum (Cairncross
et al. 1975), which correlates with enhanced reinforcing
properties of drugs of abuse in the chronic lesion
model of depression evoked by bilateral olfactory
bulbectomy (OBX) (Kelly et al. 1997). Bulbectomized
rats were recorded to be hyper-responsive to the
locomotor-stimulating properties of cocaine administration.
This was explained by the hypersensitivity to
the drug induced by OBX and the model was suggested
to be appropriate for investigation of comorbid
depression and addiction disorder (Chambers et al.
2004; Slattery et al. 2007). The self-medication hypothesis
was further conﬁrmed when bulbectomized
rats showed decreased behavioural depressive-like
symptoms when they were treated with nicotine either
i.p. or by self-administration paradigm (Vieyra-Reyes
et al. 2008).
Behavioural sensitization to drugs of abuse
(Fukushiro & Frussa-Filho, 2010) and the related
adaptations in striatal neurotransmission (particularly
dopaminergic) are thought to play an important role
in certain aspects of addiction, such as a tendency to
relapse following abrupt drug withdrawal (Ohmori
et al. 2000; Shuto et al. 2008). Pharmacotherapeutic
support in drug addiction is also challenged by
heterogeneity of the disorder in humans (Alguacil et al.
2011) and, by being dependent on possible distress in
early life, is complicated to assess and measure in
rodent models (Zellner et al. 2011). Moreover, there is a
lack of rodent models, taking into account comorbid
psychiatric disorders that are common in clinical
practice (Wong et al. 2010). Therefore, the aim of this
study was to utilize the i.v. drug self-administration
(IVSA) model, which is known to be reliable for testing
dependence potential and abuse liability of drugs
(Collins et al. 1984), together with the OBX model of
depression (Kelly et al. 1997) in order to compare
methamphetamine (Meth) intake in male rats subjected
to repeated drug pretreatment shown to induce
behavioural sensitization (Landa et al. 2005). From the
self-medication hypothesis, the anticipated outcome of
this study is: (a) a decrease of the total number of Meth
doses self-administered by rats per experimental
session as indicated by our previous studies (Kucerova
et al. 2009); (b) an enhancement of Meth intake as
a reﬂection of the depressive-like state induced by
bilateral OBX.
Method
Animals
Adult male albino Wistar rats weighing 180–220 g at
the beginning of the experiment were purchased from
Biotest Ltd (Konarovice, Czech Republic). The rats
were housed in sections of four (two bulbectomized
and two sham-operated) in standardized rat plastic
cages. After catheter implantation surgery was performed,
the rats were housed individually in standard
plastic cages (dimensions: 20.5 cmr36 cm, height
16.5 cm). Environmental conditions during the whole
study were constant: relative humidity 50–60%;
temperature 23¡1 xC; reversed 12-h light/dark cycle
(lights on 17:00 hours). Food and water were available
ad libitum. All experiments were conducted in accordance
with relevant laws and regulations of animal care
and welfare. The experimental protocol was approved
by the Animal Care Committee of the Masaryk
University Faculty of Medicine, Czech Republic and
carried out under the European Community guidelines
for the use of experimental animals.
Drugs and treatments
Meth from Sigma Chemical, Co., USA was used
for both initial drug pretreatment and in the IVSA
model. The administration of Meth prior to IVSA was
2 J. Kucerova et al.
21
according to the following dosing regimen, which
was successfully used in previous studies carried out
at our laboratory (Landa et al. 2005, 2008) to induce
behavioural sensitization: 0.5 mg/kg.d i.p. for 14 d,
administered in home cages. The identical volume and
route of administration of saline (Sal) solution was
used for all control treatments. The Meth dose available
in the operant cage for IVSA was 0.08 mg per infusion.
The maximum number of infusions obtainable
in one session was set to 50, which was a procedure
producing reinforcing eﬀects in the same model of
IVSA in our laboratory (Vinklerova et al. 2002).
OBX surgery
At the beginning of the study the rats were randomly
divided into two groups. The bilateral ablation of the
olfactory bulbs was performed in one half of the rats in
accordance with the method described by Kelly et al.
(1997), Leonard & Tuite (1981) and Song & Leonard
(2005). The other group received a sham operation
consisting of all surgical procedures except olfactory
bulb ablation. Animals were anaesthetized with
50 mg/kg ketamine and 8 mg/kg xylazine given i.p.
(Narkamon 5%; SPOFA a.s., Czech Republic and
Rometar 2%; SPOFA a.s.). The top of the skull was
shaved and swabbed with an antiseptic solution, after
which a midline frontal incision was made in the skin
on the skull and the skin was retracted bilaterally.
After exposure of the skull, two burr holes, 2 mm in
diameter, were drilled at the points 7 mm anterior to
the bregma and 2 mm lateral to the bregma suture.
Both olfactory bulbs were removed by aspiration. Care
was taken to avoid damage to the frontal cortex. The
ablation cavity was ﬁlled with a haemostatic sponge.
The skin above the lesion was closed with suture and
the antibacterial neomycin and bacitracin powder
(Framykoin pulv.; Infusia a.s., Czech Republic) was
applied. Sham-operated rats underwent the identical
anaesthetic and drilling procedures as OBX animals,
but their bulbs were left intact. Experiments were
carried out 3 wk after the surgery when hyperlocomotion
induced by the OBX method was assessed
following standard methodology (Pistovcakova et al.
2008). During this period, the animals were handled
daily to eliminate aggressiveness, which could otherwise
arise (Leonard & Tuite, 1981; Song & Leonard,
2005). At the end of the experiment, rats were killed by
a lethal overdose of ether and their brains were removed
for conﬁrmation of the removal of the olfactory
bulbs. Rats with an incomplete bilateral OBX or with
damage to other brain structures were excluded from
data analysis.
IVSA surgery and procedures
IVSA procedures including surgery were started 3 wk
after OBX surgery. The animals were allowed 1 wk
recovery and then repeated administration of Meth
was performed for 2 wk. Under general anaesthesia
with 50 mg/kg ketamine and 8 mg/kg xylazine given
i.p. (Narkamon 5%; SPOFA a.s. and Rometar 2%;
SPOFA a.s.) in combination with isoﬂurane inhalation
for induction to anaesthesia, a permanent intracardiac
silastic catheter was implanted through the external
jugular vein to the right atrium. The outer part of
the catheter exited the skin in the midscapular area.
A small nylon bolt was ﬁxed on the skull with dental
acrylic to stainless steel screws embedded in the
skull; this served as a tether to prevent the catheter
from being pulled out while the rat was in the
self-administration chamber. The catheters were
ﬂushed daily before all the sessions with heparinized
cephalosporine (Vulmizolin 1.0 inj sicc; Biotika a.s.,
Slovak Republic) solution (0.05 mg/kg cephalosporine
dissolved in Sal with 2.5 IU/kg heparin) and ﬁnally
0.05 ml heparin (Heparin Leciva inj. sol. 1r10 ml/
50 IU) solution (5 IU) to prevent infection and occlusion
of the catheter. During this procedure, blood
was aspired daily to assess the patency of the catheter
and changes in general behaviour, weight and other
circumstances were recorded. When a catheter was
found to be blocked, the animal was excluded from the
analysis.
IVSA protocol
Standard experimental cages with two nose-poke
holes located on one side of the cage were programmed
by L2T2software (Coulbourn Instruments,
USA) and the IVSA sessions were initially conducted
under the ﬁxed ratio (FR) schedule of reinforcement,
starting at FR1 (each correct response reinforced).
FR requirements were raised (e.g. FR2 – two correct
responses required, FR3 – three correct responses
required, etc.) when the animal fulﬁlled the following
conditions for three consecutive sessions: (a) at least
70% preference of the drug-active nose-poke; (b)
minimum intake of 10 infusions per session; (c) stable
intake of the drug (maximum 10% deviation). Active
nose-pokes led to the activation of the infusion pump
and administration of a single infusion followed by
30 s time-out, while the other nose-pokes were recorded
but not rewarded. The cage was illuminated
by a house light during the session. The light was
twinkling when administering infusion and oﬀ in the
time-out. The IVSA sessions lasted 90 min and took
place daily (including weekends) regularly between
Methamphetamine i.v. self-administration in OBX rats 3
22
07:00 and 16:00 hours during the dark period of the
reversed light cycle. After the session the animals were
returned to the home cage.
Experimental groups
There were 9–10 rats per experimental group at the
beginning of the experiment. However, due to complicated
surgical procedures and the nature of
the OBX operation, a signiﬁcant number of the
subjects was lost or excluded from analysis for diﬀerent
reasons.
The ﬁnal groups as statistically analysed were as
follows:
(a) SH Sal group (n=7): sham-operated (not OBX)
rats with 14 d of Sal (placebo) pretreatment.
(b) SH Meth group (n=5): sham-operated rats with
14 d of Meth pretreatment.
(c) OBX Sal group (n=6): OBX rats with 14 d of Sal
(placebo) pretreatment.
(d) OBX Meth group (n=7): OBX rats with 14 d of
Meth pretreatment.
Statistical data analysis
Standard robust descriptive statistics were used for
the analysis; categorical variables were described as
number of cases and percentage of categories; continuous
variables as median and 5th–95th percentile
range. Baseline time series in dosage of Meth was
summarized as daily number of infusions and as area
under the dosage time curve, calculated per day.
The area under the curve concept was adopted as a
quantitative measure integrating information on how
much and how long the intake of the drug remained
signiﬁcant (Dahlquist & Bjo¨rck, 2008; Mason &
Graham, 2002). Non-parametric tests (Kruskal–Wallis
test, Mann–Whitney U test) were used to compare
diﬀerent experimental groups. A non-parametric approach
was used due to proven non-normal sample
distribution of analysed datasets.
Special attention was focused on variability in
the Meth dosage time series. Day-to-day consecutive
diﬀerences in injection intake were calculated and
summarized as mean absolute diﬀerence (MAD).
Autocorrelation coeﬃcient of ﬁrst order (R1) was estimated
as a measure of potential mutual dependence
of consecutive dosage points. For each experimental
animal, Ljung– Box test statistics (Ljung & Box, 1978)
was computed to verify the null hypothesis of
independence (overall randomness) in dosage time
series; statistical signiﬁcance was assessed using x2
distribution. Individually estimated autocorrelation
coeﬃcients and Ljung–Box p values were then summarized
for the whole experimental groups as
median, minimum and maximum values.
Statistical analyses were computed using SPSS
19.0.1 (IBM Corporation, USA). A value of p<0.05 was
recognized as the boundary of statistical signiﬁcance
in all applied tests.
Results
Table 1 summarizes all accessible quantitative and
qualitative measures reached from the baseline time
series of Meth injections. It appears that both the
absolute number of infusions per day and 1 d-related
quantiﬁed area under time dosage curve (Fig. 1) reveal
the same trend and reﬂect similar diﬀerences among
the experimental groups. The greatest diﬀerence
occurred between bulbectomized rats and shamoperated
rats, in that OBX rats signiﬁcantly increased
Meth intake (p=0.014). This was also consistently
conﬁrmed within groups pretreated repeatedly by
Meth (SH Meth vs. OBX Meth, p=0.040) as well
as within the control group (SH Sal vs. OBX Sal,
p=0.046).
Comparison of animals repeatedly pretreated by
Meth to control group (Sal) revealed signiﬁcantly decreased
Meth intake in the Meth group (p=0.044).
This trend was signiﬁcantly conﬁrmed in shamoperated
animals (p=0.047), while no exact statistically
signiﬁcant diﬀerence was observed in OBX
animals (p=0.102).
Preference in taking Meth was recorded as a qualitative
measure on each day of baseline experiment;
however, with no signiﬁcant diﬀerence among experimental
groups (Table 1, Fig. 2).
Table 2 displays the main experimental results from
the viewpoint of variability and randomness of Meth
intake time series. It appears that sham-operated
animals (regardless of type of pre-treatment) showed
less variable day-to-day intake than animals in the
OBX group (measured as day-to-day MAD, also expressed
in % of overall mean intake). The individual
intake time series also appeared to be more random in
the OBX group as compared to the SH group; none
of the ﬁrst order autocorrelation coeﬃcients and
individual tests for randomness were signiﬁcant in
OBX animals. On the other hand, sham-operated rats
showed a less random Meth intake with a relatively
high ﬁrst order autocorrelation (SH Sal: 0.298 with
maximum 0.594, SH Meth: 0.386 with maximum
0.548). The Ljung–Box test did not demonstrate overall
randomness of time of intake in 45% of sham-operated
animals, but not in any of the OBX-treated animals.
4 J. Kucerova et al.
23
Discussion
In the present study, the rewarding eﬀect of Meth was
apparent in all experimental groups by achieving a
high preference (%) of the active nose-poke. However,
no signiﬁcant variability was recorded in nose-poke
responding among the groups (Table 1, column Preference).
This indicates that there was no diﬀerence in rewarding
eﬀects of the drug in particular animal groups.
The present study also conﬁrms our previous ﬁndings
that male Wistar rats repeatedly pre-exposed to
Meth (14 daily doses of 0.5 mg/kg) self-administer a
signiﬁcantly lower number of Meth infusions under
a FR schedule of reinforcement (0.08 mg/infusion)
compared to animals pretreated with Sal (Kucerova
et al. 2009, 2010). The decreased drug-seeking behaviour
in this model can be considered as a sign of behavioural
sensitization, suggesting higher rewarding
properties of Meth in previously sensitized animals
similarly as recorded elsewhere when using amphetamine
and cocaine (de Vries et al. 1998; Lorrain et al.
2000). However, repeated intermittent pretreatment
with Meth decreased subsequent drug intake only in
the sham-operated animals. In the OBX group the same
trend was apparent, but it did not reach statistical
signiﬁcance due to increased behavioural variability.
The bulbectomized animals showed a higher day-today
variability in Meth intake as compared to shamoperated
animals. The intake time series were also
more frequently random in the OBX group in comparison
to the control group. This larger variation
could be partially attributed to the cognitive impairment
reported in the OBX animals (Kelly et al. 1997)
and to a reduced number of animals that were lost due
to failure of the catheter maintenance combined with
surgical complications.
There is a lack of data describing the inﬂuence of
OBX on self-administration of dependence-producing
drugs. In support of the self-medication theory, it has
been shown that nicotine (Vieyra-Reyes et al. 2008),
alcohol (Chiang et al. 2008) and cocaine (Slattery et al.
2007) are able to decrease the symptoms of depression
induced by OBX. However, to date, comorbid drug
addiction and OBX-induced depressive conditions
was, according to available literature, studied only by
Holmes et al. (2002). In that study, male Sprague–
Dawley rats self-administered signiﬁcantly more infusions
of a low dose of amphetamine (12 mg/infusion
of D-amphetamine sulfate) after OBX than shamoperated
controls. This diﬀerence was only present at
Table 1. Comparison of experimental groups in baseline characteristics
Group
Time-related proﬁle of infusionsa
Preferencea
(%)
No. of infusions
per day
Infusions in
time: AUC/d
Pretreatment by Sal
SH Sal (n=7) 13 (10–26) 14.0 (2.2) 79.6 (39.2–94.3)
OBX Sal (n=6) 18 (13–34) 20.3 (3.1) 84.8 (46.3–97.0)
Pretreatment by Meth
SH Meth (n=5) 11 (10–12) 10.1 (0.8) 79.4 (61.3–95.0)
OBX Meth (n=7) 15 (13–23) 15.9 (2.3) 75.2 (53.4–94.1)
Statistical comparisonsb
SHrOBX p=0.014 p=0.724
SH SalrOBX Sal p=0.046 p=0.946
SH MethrOBX Meth p=0.040 p=0.911
SalrMeth p=0.044 p=0.481
SH SalrSH Meth p=0.047 p=0.933
OBX SalrOBX Meth p=0.102 p=0.705
Sal, Saline; SH Sal, sham-operated [not olfactory-bulbectomized (OBX)] rats with 14 d of Sal (placebo) pretreatment; OBX Sal,
OBX rats with 14 d of Sal (placebo) pretreatment; Meth, methamphetamine; SH Meth, sham-operated rats with 14 d of Meth
pretreatment; OBX Meth, OBX rats with 14 d of Meth pretreatment.
a
No. of infusions and preference: median (5th–95th percentile range); area under curve (curve: cumulative proﬁle of infusions
in time) per 1 d (AUC/d). Values are shown as mean (S.E.).
b
Statistical signiﬁcance (p value) of diﬀerences among given groups in AUC/d and in recorded preference; tested by
Mann–Whitney U test.
Methamphetamine i.v. self-administration in OBX rats 5
24
25
(a) (b)
(c) (d)
20
15
10
5
0
25
20
15
10
5
0
SH-Sal OBX-Sal
20.3014.00
SH-Sal OBX-Meth
10.10
*
*
*
14.00
1-drelatedquantifiedarea
undertimedosagecurve
25
20
15
10
5
0
25
20
15
10
5
0
SH-Meth OBX-Meth
15.9010.10
OBX-Sal OBX-Meth
15.9020.30
1-drelatedquantifiedarea
undertimedosagecurve
Fig. 1. The diﬀerences in methamphetamine (Meth) intake among experimental groups. (a) A signiﬁcant increase (p=0.046) of
Meth intake in olfactory bulbectomized (OBX) rats compared to sham-operated (SH) animals, both without Meth pretreatment;
(b) a signiﬁcant decrease (p=0.047) of Meth intake in SH animals with a history of Meth administration (sensitized) compared to
non-sensitized rats; (c) a signiﬁcant increase (p=0.040) of Meth intake in the OBX rats compared to SH animals, both at
conditions of Meth pretreatment; (d) statistical comparison of diﬀerences in Meth intake of OBX animals with and without
history of Meth administration (n.s.). Mean values and corresponding S.E. are shown in the graph. Statistical signiﬁcance of
diﬀerences among given groups were evaluated as area under the dosage time curve, calculated per day; tested by
Mann–Whitney U test. Sal, saline.
100
90
79.60 79.40
84.80
75.20
80
70
60
50
40
%ofactivenose-pokepreference
30
20
10
0
SH-Sal SH-Meth OBX-Sal OBX-Meth
Fig. 2. Minimal, maximal and average (values presented on the graph) percentage of active nose-poke preference among all
experimental groups. SH-Sal, sham-operated [not olfactory-bulbectomized (OBX)] rats with 14 d of saline (Sal) (placebo)
pretreatment; SH-Meth, sham-operated rats with 14 d of Meth pretreatment; OB-Sal, OBX rats with 14 d of Sal (placebo)
pretreatment; OB-Meth, OBX rats with 14 d of Meth pretreatment. Comparison of preferences did not show statistically
signiﬁcant diﬀerences. Statistical signiﬁcance of recorded preference was tested by Mann–Whitney U test.
6 J. Kucerova et al.
25
low doses of amphetamine (Holmes et al. 2002) and the
experiment did not take into account the inﬂuence of
behavioural sensitization.
In our study, low-dose Meth had a similar eﬀect to
amphetamine (Holmes et al. 2002). This also corresponds
with self-medication reported in patients with
depression (Hall & Queener, 2007; Khantzian, 1985).
Based on these ﬁndings, it is suggested that the
beneﬁcial eﬀect of various antidepressants, particularly
selective serotonin reuptake inhibitors (SSRIs),
reduce drug self-administration in animal studies, as
shown in the case of alcohol (O’Brien et al. 2011),
morphine (Raz & Berger, 2010), amphetamine (Yu et al.
1986) and Meth (Reichel et al. 2009). However, so far
there is no strong clinical evidence to suggest the
routine use of antidepressants in the treatment of drug
dependence (Silva de Lima et al. 2010).
According to our knowledge, to date there is no
study describing the eﬀects of SSRIs or other antidepressants
on spontaneous drug intake in a selfadministration
model in which OBX animals were
used. Behavioural, immunological and neurochemical
eﬀects induced by the OBX model can be eliminated
by antidepressant treatment (Kelly et al. 1997; Song &
Leonard, 2005). The results of the present study indicate
that the OBX model may be a valid model for the
investigation of drugs of abuse.
There is a need to distinguish appetitive and
consummatory behaviour in drug administration.
Consummatory behaviour is innate and leads to the
satisfaction of basic needs, such as eating, drinking,
sexual behaviour or ‘drug-taking’. Appetitive behaviour
is characteristic of exploration of environment,
motivation and learning processes – ‘drug-seeking’
(Craig, 1917). These two types of behaviour can be
distinguished pharmacologically. Thus, amphetamine
self-administration was shown to suppress consummatory
behaviour (eating) at a dose that did not aﬀect
appetitive behaviour (Foltin, 2005). These aspects of
behaviour are reﬂected in changes in mesolimbic
brain areas that are centres for appetitive and consummatory
behaviour (Gan et al. 2010).
In summary, this study demonstrates that the OBX
model of depression increases Meth intake in the IVSA
model of consummatory drug intake. This ﬁnding
correlates well with the self-medication hypothesis
to explain the relationship between depressive and
addictive disorders as an attempt to relieve symptoms
of monoaminergic deﬁcit in depression by selfadministering
a psychostimulant drug (Khantzian,
1985). Moreover, in this experiment, chronic intermittent
pretreatment with Meth was used to evaluate
the inﬂuence of behavioural sensitization on the drug
intake of OBX and sham-operated rats. Pre-exposure
to Meth subsequently decreased the intake of the
drug in self-administration in sham-operated animals
but not in rats subjected to OBX. Further studies are
necessary to describe the speciﬁcities of OBX animals
in the drug self-administration paradigm and consequently
to validate the model of comorbid depression
and drug addiction.
Acknowledgements
We thank Professor Brian Leonard (Galway, Ireland)
for his kind help with preparation of this manuscript.
Table 2. Time-related proﬁle of methamphetamine (Meth) intake (number of infusions): analysis of variability in time series
Group
Day-to-day ﬂuctuations First-order autocorrelation coeﬃcient (R1)b
MAD (S.E.)a
MAD in % of
mean (range) R1 median (range)
p value median
(range)
Pretreatment by Sal
SH Sal (n=7) 4.29 (1.02) 27.4 (15.0–53.7) 0.298 (0.145 to 0.594) 0.451 (0.004–0.845)
OBX Sal (n=6) 5.96 (0.64) 33.8 (18.4–64.5) 0.108 (x0.085 to 0.281) 0.450 (0.245–0.450)
Pretreatment by Meth
SH Meth (n=5) 2.71 (0.91) 24.5 (16.2–48.2) 0.392 (0.189 to 0.548) 0.045 (0.006–0.624)
OBX Meth (n=7) 7.15 (2.42) 47.6 (26.6–83.8) 0.144 (0.021 to 0.305) 0.422 (0.101–0.921)
Sal, Saline; SH Sal, sham-operated [not olfactory-bulbectomized (OBX)] rats with 14 d of Sal (placebo) pretreatment; OBX Sal,
OBX rats with 14 d of Sal (placebo) pretreatment; SH Meth, sham-operated rats with 14 d of Meth pretreatment; OBX Meth, OBX
rats with 14 d of Meth pretreatment.
a
Mean absolute diﬀerence (MAD) of day-to-day Meth intake (measured as number of infusions; supplied with S.E.).
b
Autocorrelation coeﬃcients and p values in Ljung–Box test for randomness in intake time series: individually based estimates
were summarized as median and range within experimental groups.
Methamphetamine i.v. self-administration in OBX rats 7
26
This work was supported by the Czech Ministry of
Education – research project: MSM0021622404 and by
the project ‘CEITEC – Central European Institute of
Technology’ (CZ.1.05/1.1.00/02.0068) from European
Regional Development Fund.
Statement of Interest
None.
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Methamphetamine i.v. self-administration in OBX rats 9
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2.4.2. Olfactory bulbectomy increases reinstatement of methamphetamine
seeking after a forced abstinence in rats
This experiment was designed to further validate the model by assessing the relapse-like
behaviour towards methamphetamine after a short maintenance phase of
methamphetamine self-administration. The forced abstinence paradigm with single
reinstatement session was chosen to assess methamphetamine seeking behaviour (Fuchs et
al., 2006, Reichel and Bevins, 2009, Yahyavi-Firouz-Abadi and See, 2009). This paradigm
mimics the human treatment very well, because the patient usually discontinues the drug
abuse in the drug rehabilitation centre and for some time does not have access to the drug
related environments. Therefore, in the model animals do not have access to the operant
box for some time and then they are re-introduced to the box again for one session with no
drug availability (Reichel and Bevins, 2009, Fuchs et al., 2006, Yahyavi-Firouz-Abadi and
See, 2009). Thus the motivation of drug response behaviour is not influenced by any
training procedures and the recorded variable is stimulation of both active and inactive
operandums. This provides information of the animals’ motivation to seek the drug.
Results of this study indicate that the methamphetamine self-administration and forced
abstinence in the olfactory bulbectomized rats is a valid model of increased relapse-like
behaviour in depressive-like rats. Therefore, we proposed this approach to test drugs
intended to suppress the reinstatement to the drug seeking behaviour, as it seems to be
relevant for downwards translation of human drug relapse and ultimately for developing
innovative treatment strategies.
Babinska Z, Ruda-Kucerova J, Amchova P, Merhautova J, Dusek L, Sulcova A.
Olfactory bulbectomy increases reinstatement of methamphetamine seeking after a forced
abstinence in rats. Behav Brain Res. 2016, 297: 20-7, doi: 10.1016/j.bbr.2015.09.035.
IF (2015) 3.002
Citations (WOS): 0
Behavioural Brain Research 297 (2016) 20–27
Contents lists available at ScienceDirect
Behavioural Brain Research
journal homepage: www.elsevier.com/locate/bbr
Research report
Olfactory bulbectomy increases reinstatement of methamphetamine
seeking after a forced abstinence in rats
Zuzana Babinskaa,b
, Jana Ruda-Kucerovaa,b,∗
, Petra Amchovaa,b
, Jana Merhautovab
,
Ladislav Dusekc
, Alexandra Sulcovaa
a
Experimental and Applied Neuropsychopharmacology Group, CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic
b
Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
c
Masaryk University, Institute of Biostatistics and Analyses of Faculty of Medicine, Kamenice 3, 625 00 Brno, Czech Republic
h i g h l i g h t s
• Further validation of an animal model of depression-addiction dual disorder.
• Olfactory bulbectomy increases reinstatement of METH seeking behavior.
• Forced abstinence is a valid translational approach to model drug relapse.
a r t i c l e i n f o
Article history:
Received 6 August 2015
Received in revised form
21 September 2015
Accepted 25 September 2015
Keywords:
methamphetamine
self-administration
reinstatement
olfactory bulbectomy
depression
Sprague-Dawley rats
a b s t r a c t
Drug addiction is commonly associated with depression and comorbid patients also suffer from higher
cravings and increased relapse rate. To address this issue preclinically we combined the olfactory bulbectomy
(OBX) model of depression and intravenous methamphetamine self-administration procedure
in rats to assess differences in relapse-like behavior.
Male Sprague-Dawley rats were divided randomly into two groups; in one group the bilateral olfactory
bulbectomy (OBX) was performed while the other group was sham operated. After recovery, intracardiac
catheter was implanted. Intravenous self-administration procedure was conducted in operant boxes
using nose-poke operandi (Coulbourn Instruments, Inc., USA) under ﬁxed ratio 1 schedule of reinforcement.
Methamphetamine was available at dose 0.08 mg/kg/infusion. After stable methamphetamine
intake was maintained, a period of forced abstinence was initiated and rats were kept in their home-cages
for 14 days. Finally, one reinstatement session was conducted in operant boxes with no drug delivery.
In the reinstatement session the mean of 138.4 active nose-pokes was performed by the OBX group,
while the sham group displayed 41 responses, i.e. 140 % and 48 % of basal nose-poking during maintenance
phase in OBX and sham operated group respectively. OBX group also showed signiﬁcantly more
passive nose-pokes indicating hyperactive behavioral traits in bulbectomized rats. However, the % of
active operandum preference was equal in both groups.
Olfactory bulbectomy model signiﬁcantly increased reinstatement of methamphetamine seeking
behavior. This paradigm can be used to evaluate potential drugs that are able to suppress the drug-seeking
behavior.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
The likelihood of drug addiction and depression to occur
together in the same individual is approximately 5 times greater
than what would be expected by the prevalence of each disorder
∗ Corresponding author at: Masaryk University, CEITEC, Kamenice 5, 625 00 Brno,
Czech Republic.fax: N/A
E-mail address: jkucer@med.muni.cz (A. Sulcova).
alone [1,2] and leads to increased suicide rates among depressive
individuals [3]. A widely accepted theory to explain depression
and drug addiction comorbidity is the “self-medication hypothesis”,
arising from common risk factors and similarities in the
underlying neurobiology of depression and drug addiction [4,5].
This theory indicates that individuals with depression have deﬁcits
in brain reward systems and may turn to drugs that create
euphoric feelings to compensate for their anhedonia and motivational
inadequacy [6–8] and there is supporting clinical evidence
for methamphetamine [9] and other drugs [10]. Consequently,
http://dx.doi.org/10.1016/j.bbr.2015.09.035
0166-4328/© 2015 Elsevier B.V. All rights reserved.
30
Z. Babinska et al. / Behavioural Brain Research 297 (2016) 20–27 21
individuals with affective disorders suffer from higher cravings and
increased relapse rate [11] which is the most demanding problem
faced by clinicians [12]. Despite the high prevalence of drug addiction
and depression comorbidity, there are only few animal models
examining drug abuse behaviors in depression and relapse of drug
addiction [13]. Thus, the composite animal model used here should
provide a basis for investigation of the mechanisms underlying the
interaction between depression and addiction [14].
Bilateral olfactory bulbectomy is a well-established model of
depression with high face, construct, and predictive validity which
closely mimics neurochemical, neuroanatomical, behavioral and
endocrine changes seen in patients with major depression [15].
Our team has developed a rat model of depression and addiction
dual disorder where olfactory bulbectomized animals showed
a signiﬁcantly higher vulnerability in methamphetamine intravenous
self-administration (IVSA) paradigm [14] and differential
dopamine and serotonin release in nucleus accumbens shell after
methamphetamine challenge [16]. Similar ﬁndings were reported
earlier also for self-administration of amphetamine [17] and for
self-administration and dopamine release induced by CB1 receptor
agonist WIN55,212-2 [18]. Interestingly, this behavioral effect was
not replicated in a similar study with cocaine [19].
To mimic relapse in the IVSA paradigm, extinction training is
usually employed when the animal still has a regular access to the
operant box but the drug delivered by infusion pump is replaced by
vehicle. After reaching a speciﬁc extinction criteria, one last session
is conducted and the reinstatement of the drug seeking behavior
is primed by an environmental factor (stress, cue) or a drug
dose [20]. In the OBX model of depression combined with drug
IVSA Frankowska et al. (2014) and Amchova et al. (2014) proved
signiﬁcantly later extinction of cocaine- and CB1 agonist-seeking
behavior in the OBX rats. However, this approach does not mimic
the human situation as the patient usually discontinues the drug
taking in a different, not drug-related environment. Therefore, a
forced abstinence paradigm was suggested as more translational.
In this model the animal does not have access to the operant selfadministration
and is kept in the home cage for certain time period
[21,22].
The aim of this study was to assess relapse-like behavior in
the OBX model of depression after short maintenance phase of
methamphetamine self-administration. We have chosen the highly
translational forced abstinence paradigm and we expected higher
methamphetamine seeking behavior of the OBX rats in the reinstatement
session.
2. Methods
2.1. Animals
Twenty male albino Sprague-Dawley rats (8 weeks old, with
weight range of 200-225 g at the beginning of the experiment) were
purchased from Charles River (Germany). The rats were housed
individually in standard rodent plastic cages. Environmental conditions
during the whole study were constant: relative humidity
50-60 %, room temperature 23 ◦C ± 1 ◦C, inverted 12-hour lightdark
cycle (6 a.m. to 6 p.m. darkness). Food and water were available
ad libitum. There were two experimental groups: SHAM = sham
operated rats (n=8 at the beginning of the study) and OBX = olfactory
bulbectomized rats (n=12 at the beginning of the study). All
experiments were conducted in accordance with all relevant laws
and regulations of animal care and welfare. The experimental protocol
was approved by the Animal Care Committee of the Masaryk
University, Faculty of Medicine, Czech Republic, and carried out
under the European Community guidelines for the use of experimental
animals.
2.2. Drugs and treatments
Methamphetamine (METH) from Sigma Chemical, Co., St Louis,
MO, USA available in the operant cage for IV self-administration
was 0.08 mg/kg per infusion with the maximum number of infusions
obtainable in one session set to 50 as was routinely used in
our laboratory [14,23].
2.3. Olfactory bulbectomy surgery
At the beginning of the study the rats were randomly divided
into two groups and the bilateral ablation of the olfactory bulbs
was performed in accordance with the standard method [24] as
described earlier [14,18]. In brief, animals were anaesthetized with
ketamine 50 mg/kg and xylazine 8 mg/kg given intraperitoneally.
The top of the skull was shaved, swabbed with an antiseptic solution,
after which a midline frontal incision was made in the skin
on the skull. After exposure of the skull, 2 burr holes were drilled
at the points 7 mm anterior to the bregma and 2 mm lateral to
bregma suture. Both olfactory bulbs were aspirated while paying
particular attention not to damage the frontal cortex. Prevention of
blood loss was achieved by ﬁlling the dead space with a haemostatic
sponge. The skin above the lesion was closed with suture and the
antibacterial neomycin and bacitracin powder was applied. Sham
operated rats underwent the identical anaesthetic and drilling
procedures as OBX animals, but their bulbs were left intact. Afterwards
animals were treated with non-steroidal anti-inﬂammatory
meloxicam (0.2 ml/kg SC). A period of 14 days was allowed for the
recovery from the surgical procedure. During this period, animals
were handled daily for few minutes to eliminate aggression, which
could otherwise arise [15,25]. At the end of the experiment, rats
were euthanized by an anaesthetic overdose and the brains were
dissected for conﬁrmation of the successful removal of the olfactory
bulbs. Animals with incomplete removal of the olfactory bulbs
were eliminated from the analysis.
2.4. Intravenous drug self-administration surgery
The IV self-administration catheter was implanted after recovery
from the OBX surgery following standard procedure described
earlier [14,18,23]. In brief, animals were deeply anesthetized with
IP injections of 50 mg/kg ketamine plus 8 mg/kg xylazine. Catheter
was inserted 3.7 cm [26] into the right external jugular vein to
the right atrium and securely sutured. A subcutaneous tunnel was
made and the catheter exited the skin in the midscapular area. Since
the implantation, the catheters were ﬂushed daily by heparinized
0.05 g/kg cefazolin dissolved in saline with 2.5 IU/kg heparin and
ﬁnally 0.05 ml heparin solution (5 IU/kg) to prevent infection and
occlusion of the catheter. When a catheter was found to be blocked
or damaged, the animal was excluded from the analysis.
2.4.1. Intravenous self-administration protocol
Methamphetamine self-administration was conducted as previously
described [14,23] in 10 standard experimental boxes
(30 × 25 × 30 cm, Coulbourn Instruments, USA) using nose-poking
as operandum. Each cage was provided with two nose-poke holes
allocated on one side and programmed by software Graphic State
Notation 3.03 (Coulbourn Instruments, USA). Nose-pokes in the
active hole led to the activation of the infusion pump and administration
of a single infusion followed by a 10 sec timeout, while
nose-poke stimulation was recorded but not rewarded, i.e. ﬁxed
ratio (FR) schedule of reinforcement. Speciﬁcally, training sessions
were initially conducted under a FR-1 schedule of reinforcement.
When the animal fulﬁlled the following acquisition criteria for three
consecutive sessions: a) at least 70 % preference of the drug-paired
active nose-poke, b) minimum intake of 10 infusions per session, or
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c) stable intake of the drug (maximum 10 % deviation) ﬁxed-ratio
1 was then raised to FR-2. Infusions were delivered by a syringe
within an automatic infusion pump located outside the chamber.
The infusion pumps were connected to liquid swivels which were
ﬁxed to the catheters via polyethylene tubing withinside a metal
spring tether. The cage was illuminated by a house light during the
session. The light was ﬂashing when infusion was being administered
(5 sec) and off during the time-out period. Self-administration
sessions lasted 90 minutes and took place 7 days/week between 8
a.m. and 3 p.m. during the dark period of the cycle.
After 14 days of stable methamphetamine intake at FR-2 the
maintenance phase was terminated and rats were kept in their
home cages for the 14 days of the forced abstinence period. As
described earlier, on the day of reinstatement, rats were placed into
self-administration chambers for the last session taking 90 minutes
[27]. The numbers of responses on the active drug paired
nose-poke and the inactive nose-poke were recorded but the drug
was not delivered. Responses on the active nose-poke are considered
to reﬂect reinstatement of drug seeking behavior, while
responses on inactive nose-poke are interpreted to reﬂect general
locomotor and exploratory activity.
2.5. Statistical Data analysis
Primary data were summarized using arithmetic mean and
standard error of the mean estimate. IV self-administration data
during the 14 days of maintenance were analysed at individual
days by t-test and at 5 day intervals by mixed ANOVA model
with Greenhouse-Geisser correction. Data from the reinstatement
session were analysed by t-test or Mann-Whitney U test for nonparametric
data and mixed ANOVA model. Active operandum
preferences were compared by unpaired t-test, Welch corrected.
Fig. 1. Maintenance of METH self-administration Fig. 1A shows a comparison of SHAM (n=7) and OBX (n=7) groups in mean ±SEM active nose-poking during 14 days of
maintenance phase. The groups differ signiﬁcantly only in the day 7 (*p=0.037). Fig. 1B reﬂects mean ±SEM number of passive nose-pokes during maintenance phase (n.s.).
Fig. 1C depicts mean ±SEM number of infusions over the whole maintenance phase. The groups differ signiﬁcantly from the day 5 to day 9 (*p=0.024). Fig. 1D shows mean
±SEM dose of METH self-administered in mg/kg. The groups differ signiﬁcantly again only in the day 7 (**p=0.006). Fig. 1E reﬂects percent of active nose-poke preference
(n.s.).
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Z. Babinska et al. / Behavioural Brain Research 297 (2016) 20–27 23
Fig. 2. Reinstatement of methamphetamine self-administration Fig. 2A shows mean ±SEM number of active nose-pokes in the reinstatement session in SHAM (n=6) and
OBX (n=5) animals, maintenance data are included for comparison. OBX rats performed signiﬁcantly more nose-pokes in the reinstatement (t-test, ***p=0.0001), speciﬁcally
Fig. 2B depicts mean ±SEM number of active nose-pokes in the reinstatement session. Fig. 2C shows mean ±SEM number of passive nose-pokes during reinstatement session
in SHAM and OBX rats (Mann-Whitney U test, *p=0.0175). Fig. 2D indicates mean ±SEM percent of active nose-poke preference during the reinstatement session (n.s.).
Statistical analyses were computed using SPSS 19.0.1 (IBM Corporation,
2010). A value p<0.05 was recognized as boundary of statistical
signiﬁcance in all applied tests.
3. Results
3.1. Maintenance of methamphetamine self-administration in
SHAM and OBX rats
The maintenance of METH taking behavior was assessed in
terms of mean number of nose-pokes, infusions self-administered
per session and by the mean METH dose per session in mg/kg.
Fig. 1A shows mean number of active nose-pokes obtained per
daily session during the maintenance phase in SHAM and OBX
rats. ANOVA revealed no signiﬁcant effects over the whole period
of maintenance with an exception of day 7 (p=0.037), where OBX
group exhibited more active operant responses. Non drug-paired
(passive) nose-poking is a measure of locomotor-exploratory
activity of the animal and is generally quite high in animals selfadministering
METH due to psychostimulant properties of this drug
[18]. As expected, mean numbers of passive nose-pokes were variable
but there was no difference between the groups recorded,
ANOVA, n.s. (Fig. 1B). Fig. 1C depicts mean number of infusions
and ANOVA revealed signiﬁcantly more infusions in OBX rats in
the middle of the maintenance period (days 5 to 9, p=0.024). It
should be noted that the number of nose-poke responses does not
match the number of infusions delivered in our paradigm. This is
always the case when the system uses nose-poke operandi (and
in some cases levers which do not retract after infusion delivery).
Besides number of infusions we proposed to evaluate also METH
dose per kilogram of body weight as more exact measure of actual
drug intake. Therefore, Fig. 1D indicates mean METH dose in mg/kg
showing no difference over the maintenance phase except higher
intake in the OBX animals on the day 7 (ANOVA, p=0.006). The fact
that the general pattern of METH taking behavior was similar in
both groups is further supported by active nose-poke preference
data, ANOVA, n.s. (Fig. 1E).
3.2. Reinstatement of methamphetamine self-administration in
SHAM and OBX rats
After the 2 week-long period of forced abstinence one last reinstatement
session was performed with no drug availability. The
only measure of the drug-seeking behavior was the number of
active operandum responses. Fig. 2A and 2B report the mean number
of active nose-pokes obtained during the reinstatement session
in SHAM and OBX rats. For easy comparison the maintenance data
shown on Fig. 1A are also included in the Figure 2A. SHAM and OBX
rats show signiﬁcant difference in the responding during reinstatement
session (138.4 active nose-pokes in OBX group vs 41 in SHAM
group, t-test, p=0.0001). Mean ±SEM number of active nose-pokes
in the reinstatement session are summarized on the Figure 2B. OBX
group reached mean 138.4 while SHAM rats 41 active nose-pokes
(t-test, p≤0.0001).
To assess the locomotor-exploratory activity we evaluated the
differences in the passive nose-pokes between SHAM and OBX
group (Figure 2C). The comparison revealed signiﬁcant differences:
41.2 passive nose-pokes in OBX group and 11.7 passive
33
24 Z. Babinska et al. / Behavioural Brain Research 297 (2016) 20–27
Fig. 3. Responding patterns in the reinstatement session The ﬁgure shows temporal responding patterns of all animals in the study. The line indicates the increasing
cumulative record of nose-poking while the vertical bars indicate the ﬂashing light as a cue for infusion delivery. Infusions were not delivered in this session but the protocol
was kept equal as during maintenance. The caption of each ﬁgure refers to the speciﬁc number of animal in the SHAM/OBX group. The protocol used in the reinstatement
session was the same as for maintenance where the maximum number of infusions is set for safety reasons (prevention of overdose). This limit is not necessary in the
reinstatement session but the session was conducted under the same conditions the animals were used to. We have detected signiﬁcant difference between sham and OBX
animals despite this restriction. If we would have not limit the maximum number of infusions in this session the difference would probably be even higher.
nose-pokes in SHAM group (Mann-Whitney U test, p=0.0175).
However, Figure 2D shows percent of active nose-poke preference
in the reinstatement session, revealing no signiﬁcant differences
between the groups (t-test), proving equal active operandum preference
among the groups.
In order to evaluate possible qualitative differences in the
operant responding between the groups we assessed temporal
nose-poking patterns. Whole OBX group ﬁnished the reinstatement
session prematurely, due to reaching the maximum of
operant responding set previously for the maintenance sessions.
Responding in the OBX animals was higher during the whole ses-
34
Z. Babinska et al. / Behavioural Brain Research 297 (2016) 20–27 25
Fig. 3. (Continued).
sion. SHAM group showed high responding in the beginning of the
session only, followed by long sequences of non-responding in one
half and scarce responding in the other half of the group (Fig. 3).
4. Discussion
In the present study we report that bulbectomized rats displayed
signiﬁcantly increased reinstatement of METH seeking
behavior indicating higher vulnerability to relapse and trend
towards higher drug intake during maintenance phase. We have
shown earlier that OBX animals self-administer more METH infusions
than SHAM controls [14]. However, as observed in this study,
the mean number of active nose-pokes, METH infusions and drug
dose in mg/kg were stable with only a trend towards increase in
OBX group in the middle of the maintenance phase. Therefore, this
study only partially replicates our previous ﬁndings. The reason
is probably due to the length of the acquisition and maintenance
phases together, i.e. total duration of drug exposure. Kucerova et al.
35
26 Z. Babinska et al. / Behavioural Brain Research 297 (2016) 20–27
(2012) reported increased METH intake at FR-3 where the selfadministration
lasted approximately 2 to 3 months due to training
and longer maintenance phase. However, the primary goal of this
experiment was to evaluate relapse-like behavior, therefore, the
allowed period of maintenance was shorter (2 weeks, considered
as chronic drug intake in most of preclinical studies [28,29]) and
the total length of the self-administration including training was
approximately 1 month. Another confounding factor might be the
effect of rat strain since Kucerova et al. (2012) employed Wistar
rats and this study works with Sprague-Dawley strain. Strain difference
is known to be an inﬂuential factor in assessing drug taking
behavior [30]. Regarding strain differences it has also been proved
that Sprague-Dawley rats tend to self-administer more than Wistar
rats [31] which might disguise the difference induced by the OBX
surgery. Moreover, Frankowska et al. (2014) did not prove higher
cocaine intake in OBX Wistar rats in her study using stable dose of
cocaine (0.5 mg/kg/infusion) in up to FR-5 and following extinction
phase, when drug is replaced by vehicle as opposed to our study
using forced abstinence paradigm. The difference in drug intake
between the groups vanishes when using higher doses of cocaine or
amphetamine [17,19], therefore the speciﬁc choice of dose could be
an important factor. Low dose amphetamine 0.10 mg/kg/infusion)
or methamphetamine (0.08 mg/kg/infusion) resulted in higher
drug self-administration in OBX rats [14,17], while a rather high
dose of amphetamine (0.25 mg/kg/infusion) led to the same active
lever pressing activity in both OBX and SHAM Sprague-Dawley rats
[17]. It should be noted that olfactory bulbectomy also increases
voluntary 10% [32] and 20% ethanol consumption (manuscript in
preparation) and also IV self-administration of CB1 receptor agonist
WIN55,212-2 [18] suggesting that olfactory bulbectomy leads
to increased response patterns to a variety of abused substances.
The main ﬁnding of this study is the increased vulnerability to
reinstatement of METH seeking behavior in the OBX rats in terms of
absolute number of active nose-pokes and percent of mean basal
nose-poking during the maintenance phase. Interestingly, during
the reinstatement session the number of passive nose-pokes was
signiﬁcantly increased in the OBX group. This may be explained
mainly by increased motivation to obtain the drug and also by
known hyperactive behavioral traits in OBX rats [15,33,34]. In line
with this hypothesis, the data on the active nose-poke preference
are showing no difference between OBX and SHAM rats. However,
the temporal responding patterns show marked differences
between the groups. OBX rats made high number of nose-pokes
during the whole session while SHAM animals responded either
less during the whole session or just at the beginning. This probably
reﬂects the increased motivation to obtain the drug in the
OBX group. In line with the present data, other animal models
of depression also show higher vulnerability to relapse. The
social defeat-induced persistent stress paradigm with depressivelike
symptomatology led to increased motivation to obtain alcohol
[35]. Moreover, the model of unconditioned foot-shock stress has
been proved to reinstate previously extinguished alcohol-seeking
behavior to a higher extent than alcohol or stress alone [36].
Additionally chronic restraint stress model of depression during
abstinence phase promotes nicotine seeking after extinction of
nicotine self-administration [37]. Thus, all these models seem to
be at least in partial accordance with the clinical situation.
In summary, reinstatement phenomena induced by drug
dose, stress or drug-related cues occurring in rats resembles
human relapse [28,38]. On the other hand, there are also
important differences between animal and human situation, as
the drug discontinuation motive in humans is mostly punishment/incarceration
or lack of drug availability in rehabilitation
clinic while animal models mostly employ extinction training.
Therefore, it is crucial to interpret the data from preclinical
studies using the extinction-reinstatement design considering
this limitation. This study indicates that the methamphetamine
self-administration and forced abstinence is a valid model of
increased relapse-like behavior in OBX rats which closely mimics
the human situation. Therefore, we propose this approach to test
drugs intended to suppress the reinstatement to the drug seeking
behavior, as this approach relevant seems to be more relevant for
downwards translation of human drug relapse and ultimately for
developing innovative treatment strategies.
Acknowledgements
This work was supported by the project “CEITEC - Central
European Institute of Technology” (CZ.1.05/1.1.00/02.0068) from
European Regional Development Fund, project of speciﬁc research
at the Masaryk University (MUNI/A/1116/2014) and the Internal
project of the Faculty of Medicine at Masaryk University
(MUNI/11/InGA09/2012).
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38
2.4.3. Reward related neurotransmitter changes in a model of depression: an
in vivo microdialysis study
This study was systematically assessed the extracellular levels and turnover of dopamine
and serotonin and levels of glutamate and GABA in the OBX rat model of depression in
the nucleus accumbens shell, i.e. the main reward-related area (Di Chiara et al., 2004). To
this goal, the in vivo microdialysis technique was used (Sustkova-Fiserova et al., 2014) and
the neurotransmitter levels were assayed both at baseline conditions and after a challenge
dose of methamphetamine.
The findings indicated a different baseline condition: significantly decreased basal levels
of dopamine, serotonin and their metabolites and increased levels of glutamate and GABA
in the OBX rats. Furthermore, dopamine and serotonin turnover was elevated when
calculated from the amounts of the neurotransmitters and their respective second-step
metabolites. After acute methamphetamine challenge (5 mg/kg, i.p.) a significantly higher
release of dopamine, serotonin and their metabolites was detected in OBX rats; however,
glutamate levels were lower and GABA was not found to be different from sham control
animals. These findings were in accordance with a differential behavioural profile in the
OBX rats.
Importantly, a dose-response assessment of dopamine release and changes induced by
chronic methamphetamine administration are of high interest. Chronic drug administration
data are needed for further elucidation of the underlying pathophysiological processes
shared by the depressive-like phenotype and reward.
Ruda-Kucerova J, Amchova P, Havlickova T, Jerabek P, Babinska Z, Kacer P, Syslova
K, Sulcova A, Sustkova-Fiserova M. Reward related neurotransmitter changes in a model
of depression: An in vivo microdialysis study. World J Biol Psychiatry. 2015, 16(7): 521-
35. doi: 10.3109/15622975.2015.1077991.
IF 4.159
Citations (WOS): 1
THE WORLD JOURNAL OF BIOLOGICAL PSYCHIATRY, 2015
http://dx.doi.org/10.3109/15622975.2015.1077991
ORIGINAL INVESTIGATION
Reward related neurotransmitter changes in a model of depression: An in vivo
microdialysis study
Jana Ruda-Kucerova1,2
, Petra Amchova1,2
, Tereza Havlickova3
, Pavel Jerabek3
, Zuzana Babinska1,2
, Petr Kacer4
,
Kamila Syslova4
, Alexandra Sulcova1
& Magdalena Sustkova-Fiserova3
1
Experimental and Applied Neuropsychopharmacology Research Group, CEITEC - Central European Institute of Technology,
Masaryk University, Brno, Czech Republic, 2
Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czech Republic,
3
Department of Pharmacology, Third Faculty of Medicine, Charles University, Prague, Czech Republic, and 4
Laboratory of Medicinal
Diagnostics, Department of Organic Technology ICT, Prague, Czech Republic
ABSTRACT
Objectives: The self-medication hypothesis assumes that symptoms related to potential
monoaminergic deficits in depression may be relieved by drug abuse. The aim of this study was
to elucidate the neurotransmitter changes in a rat model of depression by measuring their levels in
the nucleus accumbens shell, which is typically involved in the drug of abuse acquisition
mechanism. Methods: Depression was modelled by the olfactory bulbectomy (OBX) in Wistar male
rats. In vivo microdialysis was performed, starting from the baseline and following after a single
methamphetamine injection and behaviour was monitored. The determination of neurotransmitters
and their metabolites was performed by high-performance liquid chromatography combined
with mass spectrometry. Results: OBX animals had lower basal levels of dopamine and serotonin
and their metabolites. However, g-aminobutyric acid (GABA) and glutamate levels were increased.
The methamphetamine injection induced stronger dopamine and serotonin release in the OBX rats
and lower release of glutamate in comparison with sham-operated rats; GABA levels did not
differ significantly. Conclusions: This study provides an evidence of mesolimbic neurotransmitter
changes in the rat model of depression which may elucidate mechanisms underlying intravenous
self-administration studies in which OBX rats were demonstrated to have higher drug intake
in comparison to intact controls.
ARTICLE HISTORY
Received 16 January 2015
Revised 21 May 2015
Accepted 23 July 2015
KEY WORDS
Depression, in vivo
microdialysis,
methamphetamine,
olfactory bulbectomy, rats
Introduction
Drug abuse is clinically a frequent comorbidity of other
psychiatric disorders including mood disorders (Testa
et al. 2013). As an explanation of the dual diagnosis of
depression and addiction, the self-medication hypothesis
is widely accepted (Hall and Queener 2007). It
explains drug abuse as an attempt of the patient to
relieve the monoaminergic deficits typical for depression.
This was clinically confirmed in nicotine (Holma
et al. 2013), methamphetamine (McKetin et al. 2011) and
other drugs. There is growing evidence that depression
and addiction underlie common defective neurobiological
regulations shared by major depression and
withdrawal syndrome (Markou et al. 1998), namely in
dopaminergic (DA; Nestler and Carlezon 2006), serotonergic
(5-HT), noradrenergic (NA), cholinergic (Zellner
et al. 2011), glutamatergic (Tzschentke 2002) and
g-aminobutyric acid (GABA)-ergic (Koek et al. 2013)
systems.
Bilateral olfactory bulbectomy (OBX) is a wellestablished
model of depression (Harkin et al. 2003;
Song and Leonard 2005). Our team has developed a rat
model of depression and addiction dual disorder where
OBX animals showed a significantly higher vulnerability
in methamphetamine self-administration paradigm
(Kucerova et al. 2012). This finding was reported earlier
also for amphetamine (Holmes et al. 2002) and later for
CB1 receptor agonist (Amchova et al. 2014) and in a
slightly different operant paradigm also partially for
cocaine (Frankowska et al. 2014). However, there is
only fragmentary knowledge about the neurochemical
changes in the reward-related brain areas in the OBX rats
which could contribute to the explanation of these
behavioural effects.
OBX rats were recorded to have increased basal DA
levels in ventral and dorsal striatum but at the same time
decreased NA levels (Masini et al. 2004). Regarding
serotonin, in vivo microdialysis data demonstrate that
CONTACT: Jana Ruda-Kucerova, Masaryk University, CEITEC, Kamenice 5, 625 00 Brno, Czech Republic. Tel: +420 549 494 238. jkucer@med.muni.cz
! 2015 Taylor & Francis
39
the OBX procedure decreases serotonin levels but not its
turnover in the basolateral amygdala and dorsal hippocampus.
DA and NA (van der Stelt et al. 2005), and
impaired serotonin functioning was described also in
frontal cortex (PFC) and mid-brain (Song and Leonard
2005). The glutamatergic system is known to be strongly
influenced by the OBX surgery as density of the NMDA
receptors was shown to be elevated in the PFC. This is
supposed to contribute to the typical hyperactive
response of the OBX animals to novel environment
(Song and Leonard 2005) together with enhanced
striatal glutamate neurotransmission (Ho et al. 2000).
However, the GABA-ergic system was shown to be
hyperactive in the amygdaloid cortex and there is an
increased turnover and density of GABA-A receptors
(Song and Leonard 2005).
Drugs of abuse are consistently reported to strongly
influence the reward system of the brain, specifically the
nucleus accumbens shell (NACSh) and not the core
(Di Chiara et al. 2004). This was shown for cannabinoids,
opioids (Tanda et al. 1997), psychostimulants (Pontieri
et al. 1995) and nicotine (Pontieri et al. 1996). Regarding
glutamate, amphetamine was shown to increase its
levels in NAC and PFC, but methamphetamine in the
same study failed to do so (Shoblock et al. 2003). Striatal
glutamate transmission was increased after a high dose
of methamphetamine which could be the cause
of neurotoxicity (Nash and Yamamoto 1992). The role
of GABA is also substantial in the methamphetamine
effect, as GABA-agonistic drugs injected to NAC were
reported to suppress reinstatement of the methamphetamine-seeking
behaviour (Rocha and Kalivas 2010).
Functioning of neurotransmitter systems was assayed
repeatedly in the OBX model, but the reward-related
regions have not yet been described. The aim of this
study was, therefore, to assess systematically the neurotransmitter
levels and turnover in the OBX rat model of
depression in the nucleus accumbens shell. To this goal,
the in vivo microdialysis technique was used and the
neurotransmitter levels were assayed both at baseline
conditions and after a dose of methamphetamine.
Furthermore, the behavioural profile was established
during the in vivo microdialysis session.
Methods and materials
Animals
Thirty adult male albino Wistar rats weighting 180–220 g
at the beginning of the experiment were purchased
from Velaz (Konarovice, Czech Republic). After the
bulbectomy surgery, the rats were housed individually.
Environmental conditions were constant: relative humidity
50–60%, temperature 22–24 
C, and a 12-h light–dark
cycle, the experiments were carried out during the first
half of the light phase. Food and water were available ad
libitum. Due to incomplete olfactory bulbectomy, three
animals were excluded from analysis, thus the study was
completed by 13 sham operated and 14 olfactory
bulbectomized rats (OBX). The experimental groups
were: SHAM-SAL (sham-operated rats treated with
saline as a vehicle (n¼5)), SHAM-METH (sham-operated
rats treated with 5 mg/kg METH (n¼8)), OBX-SAL (olfactory
bulbectomized rats treated with saline (n¼6)) and
OBX-METH (olfactory bulbectomized rats treated with
5 mg/kg METH (n¼8)). All experiments were conducted
in accordance with relevant laws and regulations of
animal care and welfare. The experimental protocol was
approved by the Expert Committee for Protection of
Experimental Animals of the Third Faculty of Medicine,
Charles University, Prague, Czech Republic, and experiments
were performed in accordance with the Animal
Protection Act of the Czech Republic (No. 246/1992 Sb.)
and carried out under the European Community guidelines
for the use of experimental animals.
Drugs and treatments
Methamphetamine (METH) from Sigma Chemical Co.
(St Louis, MO, USA) was used for acute intraperitoneal
(i.p.) administration during in vivo microdialysis session
in a dose of 5 mg/kg in 2 ml of sterile saline, and 2 ml/kg
i.p. of saline was used as a vehicle.
Olfactory bulbectomy surgery
At the beginning of the study the rats were randomly
divided into two groups and the bilateral ablation of the
olfactory bulbs was performed in accordance with the
standard method (Kucerova et al. 2009,2012; Amchova
et al. 2014). In brief, animals were anaesthetized with
ketamine 50 mg/kg and xylazine 8 mg/kg i.p., the skull
was shaved, swabbed with an antiseptic solution. A
midline frontal incision was made in the skin and after
exposure of the skull, two burr holes were drilled 7 mm
anterior to the bregma and 2 mm lateral. Olfactory bulbs
were removed by aspiration and the cavity filled with a
haemostatic sponge. The skin was sutured and the
antibacterial neomycin/bacitracin powder was applied.
Sham operated rats underwent the identical procedures
as OBX animals, but their bulbs were left intact.
Microdialysis experiments were carried out 3 weeks
after the surgery.
In vivo microdialysis
Surgery. As described in detail earlier (Sustkova-Fiserova
et al. 2014), under ketamine–xylazine anaesthesia
2 J. RUDA-KUCEROVA ET AL.
40
(ketamine 100 mg/kg i.p., xylazine 10 mg/kg i.p.), rats
were implanted with a disposable dialysis guide cannula
(MAB4 probes, Agnthos, Sweden) using a stereotaxic
instrument (StoeltingCo). After taking the co-ordinates
with a guide mounted on the stereotaxic holder (NACSh:
A: +2.0 mm and L: ±1.2 mm from bregma and V: 6.2 mm
from occipital bone) (Paxinos and Watson 2007), the
guide was slowly lowered into the brain and secured to
the skull with dental cement and an anchoring screw.
The guide was randomly alternated on the left and right
side. After completion of the microdialysis experiments,
the successful bulbectomy was confirmed and placement
of the dialysis probe was verified histologically
(Figure 1).
Microdialysis and chemical analysis assay. Forty-eight
hours after implantation, the probe (MAB4, 2 mm active
cuprophane membrane, Agnthos, Sweden) was inserted
into the guide cannula and artificial cerebrospinal fluid
(Ringer’s solution; 147 mM NaCl, 2.2 mM CaCl2 and
4.0 mM KCl; adjusted to pH 7.0) was flushed through the
probe at a constant rate of 2.0 ml/min (Univentor 864
Syringe Pump, Agnthos, Sweden). After 80 min of
habituation to the microdialysis set-up (when dialysate
was discarded), 40-ml samples were collected at 20-min
intervals in small ice-cooled polyethylene test tubes
containing 12 ml HCl 0.1 mM, to prevent catecholamine
hydrolysis. After three consecutive baseline samples,
methamphetamine (5 mg/kg in 2 ml) or vehicle (saline
2 ml/kg) was administered intraperitoneally (at 60 min)
and dialysates were collected every 20 min. Total
duration of the sampled session was 240 min in animals
with administration of METH and 180 min in animals
after saline injection. This is because administration of
the vehicle was not expected to induce any changes so
the session was shortened. Immediately following
collection, the samples were frozen at –70 
C. The
amount of dopamine, serotonin and their metabolites 3methoxytyramine
(3-MT), 3,4-dihydroxyphenylacetic acid
(DOPAC), homovanillic acid (HVA) and 5hydroxyindoleacetic
acid (5-HIAA), respectively, as well
as glutamate and GABA in the dialysates were quantified
using high-performance liquid chromatography combined
with mass spectrometry (HPLC-MS). The appropriate
HPLC-MS determination methods were described in
detail earlier (Syslova et al. 2011). In brief, the
microdialysates were first lyophilised (to concentrate
the inherent substances), the lyophylisation residue was
dissolved in methanol and thus induced suspension of
precipitated salts was briefly centrifuged and the supernatant
was immediately analysed using LC-ESI–MS/MS
(following specific procedure). The LC–MS system consisted
of a chromatograph Accela 1250 autosampler and
a TSQ Vantage mass spectrometer (all Thermo Scientific,
USA). The analytes were separated on a GeminiÕ
NX-C18
(5 mm at 110 A˚) LC Column (150 Â 2 mm) using a mobile
phase (solvent A: aqueous solution of acetic acid (pH 2);
solvent B: methanol) with a gradient elution flow rate of
150 ml/min. The conditions on the mass spectrometer
were optimised and were as follows: spray voltage 3000
V, temperature of ion transfer tube 350 
C, temperature
of H-ESI vaporiser 350 
C, sheath gas pressure (nitrogen)
35 psi, flow of auxiliary gas (nitrogen) 10 arbitrary units.
The data were acquired and processed using Xcalibur
2.1.0 software (Thermo Scientific, USA).
Behavioural assay
Behaviour was studied simultaneously, while microdialysis
measurements were performed as described earlier
(Fiserova et al. 1999; Sustkova-Fiserova et al. 2014). Three
behavioural categories were distinguished: immobility
(sedation, eyes closed, akinesia, and reduced responsiveness
to environmental cues), locomotion (nonstereotyped
activity, sniffing, grooming, rearing, and
walking) and stereotyped activity (stereotypical head
movements, confined gnawing, licking and stereotypical
sniffing). Behavioural categories were scored every 20
min (at each microdialysis interval) by an observer who
was unaware of the manipulation and treatment that
each rat had received. The percentage of time spent by
the animal in each behavioural category was calculated
for each 20-min interval. Behavioural changes were
monitored during the entire dialysis period (60 min
baseline and 3 h following methamphetamine or 2 h
following saline injection).
Statistical data analysis
For comparison of basal neurotransmitter levels
between OBX versus SHAM rats (in the in vivo
microdialysis study), one-way analysis of variance
(ANOVA) followed by Bonferroni t-test was used. The
metabolic turnover was evaluated by t-test in parametric
data and Mann–Whitney U-test in non-parametric data.
Raw values for neurotransmitters and their metabolites
were further transformed into percentage of baseline
levels (mean of the three values prior to treatment). For
statistical analysis of differences between OBX versus
SHAM rats in time-related changes in the course of the in
vivo microdialysis study, the two-way analysis of variance
for repeated measures (ANOVA) followed by
Bonferroni t-test was used. In this ANOVA analysis,
the group of rats (SHAM vs. OBX) was entered as
the between-group factor and the time-points as
repeated within-subject measure (to compare all treatments
to baseline).
THE WORLD JOURNAL OF BIOLOGICAL PSYCHIATRY 3
41
For evaluation of the behavioural data, a t-test was
used for each time point and a mixed ANOVA model for
paired data was also used in order to assess the within
subject effects with rat group regarding interaction
differences and SHAM/OBX between subject effects.
The data were analysed using SigmaStat 3.5 (Systat
Software, Inc., USA) (neurochemical data) and SPSS,
version 2.0 (behavioural data). Results are presented as
the arithmetic mean and standard error of the mean
estimate (±SEM). A value P50.05 was recognised as
boundary of statistical significance in all applied tests.
Results
Basal NACSh neurotransmitter/metabolite levels
There were recorded highly significant differences in the
basal levels of all assayed neurotransmitters and their
metabolites. As shown on the Figure 2, extracellular
levels of dopamine, serotonin and their metabolites in
NACSh were decreased in the olfactory bulbectomy
model of depression. On the other hand, glutamate and
GABA levels were significantly increased. The exact
concentration values are summarised in Table I.
Figure 1. Location of dialysis probes within the nucleus accumbens shell (NACSh). Schematic locations of probe tips in rats, which
were included in analyses of accumbens neurotransmitter concentrations (the solid lines indicate the dialysing portions) as described
in the atlas of Paxinos and Watson (2007). On the left, for each section, the distance from bregma (in mm) is indicated.
4 J. RUDA-KUCEROVA ET AL.
42
Extracellular turnover of dopamine and serotonin
in the NACSh
For analysis of dopamine and serotonin turnover metabolic
ratios were calculated as follows: for each animal
baseline value of the metabolite concentration (mean of
20-, 40- and 60-min interval values) was divided by mean
baseline concentration value (analogously mean of 20-,
40- and 60-min values) of the neurotransmitter. Before
the calculation the concentration data were all passed to
pg/ml units. In this way relative data were obtained
(expressed as a mean±SEM). As shown on the Figure 3 in
DA metabolism there was found a statistical difference
between SHAM and OBX animals only in the increased
HVA/DA ratio – which could suggest an increased
turnover of DA (P50.01). Production of 3-MT and
DOPAC was not found to be significantly different. As
for the 5-HT metabolism, 5-HIAA (similarly to HVA in DA
pathway, a second step metabolite of 5-HT) production
was found to be significantly increased (P50.05).
NACSh neurotransmitter/metabolite changes
induced by methamphetamine administration
The changes of the neurotransmitter/metabolite levels
are expressed as percentage of the mean baseline values
Figure 2. Baseline levels of the specific neurotransmitters/metabolites in the NACSh. The bars depict the mean±SEM values of
extracellular concentrations (pg/ml or ng/ml) of the specific neurotransmitters and their metabolites in the NACSh in the SHAM
(n¼12) and OBX (n¼14) rats during all three baseline measurements (at 20-, 40- and 60-min intervals). One-way ANOVA followed by
Bonferroni t-test: ***P50.001 in all measures, F values are presented in Table 1. Note that in order to show the SEM error bars clearly
the y-axis in the graphs does not always begin at zero value.
Table I. Baseline neurotransmitter/metabolite levels in baseline measurements at sampling intervals 20, 40
and 60 min.
Mean baseline values OBX effect
20 min 40 min 60 min P value F(1,79) value
DA Sham 56.92±1.71 55.54±2.32 57.69±2.11 50.001 71.251 #
(pg/ml) OBX 45.79±0.98 44.64±1.39 45.07±1.07
3-MT Sham 134.00±1.29 135.46±0.68 132.08±0.67 50.001 985.739 #
(pg/ml) OBX 100.79±1.46 103.07±1.34 98.07±1.30
DOPAC Sham 3.87±0.02 3.87±0.01 3.84±0.02 50.001 2231.631 #
(ng/ml) OBX 3.24±0.01 3.25±0.01 3.23±0.01
HVA Sham 4.52±0.02 4.53±0.01 4.53±0.02 50.001 260.768 #
(ng/ml) OBX 4.29±0.01 4.34±0.01 4.33±0.02
5-HT Sham 98.54±5.81 97.31±6.17 98.23±6.55 50.001 76.177 #
(pg/ml) OBX 66.64±0.99 66.64±1.37 66.64±1.89
5-HIAA Sham 430.31±4.82 432.54±4.38 430.77±3.48 50.001 961.212 #
(pg/ml) OBX 344.79±2.06 345.86±2.18 345.36±2.51
GLU Sham 130.85±2.31 127.31±2.28 129.77±2.04 50.001 241.426 "
(ng/ml) OBX 153.50±1.67 153.93±1.32 152.57±1.32
GABA Sham 6.81±0.01 6.83±0.02 6.82±0.02 50.001 146.549 "
(ng/ml) OBX 7.06±0.02 7.03±0.03 7.05±0.03
THE WORLD JOURNAL OF BIOLOGICAL PSYCHIATRY 5
43
(at 20-, 40- and 60-min intervals) at the specific timepoint
(from 80-min interval on). Figure 4 pools graphs
showing the accumbens release of dopamine, 3-MT,
DOPAC and HVA after an acute dose of both vehicle
and 5 mg/kg METH in SHAM and OBX rats. As expected,
acute METH induced an immediate strong release of
dopamine in both sham-operated and OBX animals as
compared to their respective baselines. However, a
significantly higher percent of release was recorded in
OBX rats when the relative values were compared
between SHAM-METH and OBX-METH groups (at peaks
approximately 400 vs. 500%, respectively). Following the
dopamine release, levels of its metabolites were also
changed. HVA values show a delayed moderate increase,
as this metabolite follows the formation of 3-MT and
DOPAC. There were no significant changes after saline
dose.
Figure 5 pools graphs showing accumbens release of
serotonin, its metabolite 5-HIAA, glutamate and GABA
after treatments. Acute dose of METH induced a release
of serotonin and 5-HIAA in both sham-operated and OBX
animals (P50.001). Similarly, as in the case of dopamine,
Figure 3. Dopamine and serotonin turnover in the NACSh. The graphs show mean baseline concentration value of the metabolite
divided by mean baseline concentration value of the neurotransmitter. In the DA metabolism a statistically significant difference
between SHAM and OBX animals was found in the increased HVA/DA ratio what indicates that OBX rats produce more HVA than
SHAM controls. Similarly, in the 5-HT metabolism, 5-HIAA production was found to be significantly higher which indicates that OBX
rats produce more 5-HIAA than SHAM controls. The statistical significance was determined by t-test in dopamine (parametric data)
and Mann–Whitney U-test in 5-HT (non-parametric data): **P50.01, *P50.05.
6 J. RUDA-KUCEROVA ET AL.
44
Figure 4. Methamphetamine-induced release of dopamine and its metabolites in the NACSh. The graphs show the mean±SEM of
relative values of concentration (% of mean baseline value) of dopamine and its metabolites in the NACSh in all groups and
treatments. The horizontal line shows significant change in the neurotransmitter level induced by the METH treatment versus baseline
in both SHAM-METH and OBX-METH groups in the time points below it with indication of significance level. (In case of HVA the short
line indicates lower P value in the SHAM-METH group in the 220-min time point.) Asterisks indicate significant differences between
groups, respectively in SHAM-METH versus OBX-METH groups, ***P50.001, **P50.01, *P50.05, two-way ANOVA for repeated
measures followed by Bonferroni t-test. Note that the y-axis scales differ among the graphs.
THE WORLD JOURNAL OF BIOLOGICAL PSYCHIATRY 7
45
Figure 5. Methamphetamine-induced release of serotonin, its metabolite, glutamate and GABA in the NACSh. The graphs show the
mean±SEM of relative values of concentrations (% of mean baseline value) of serotonin, its metabolite, glutamate and GABA in the
NACSh in all groups and treatments. The horizontal line shows significant change in the neurotransmitter level induced by the METH
treatment versus baseline in both SHAM-METH and OBX-METH groups in the time points below it with indication of significance level.
(In case of 5-HIAA the short line indicates a non-significant effect of METH in the SHAM-METH group starting from the 200-min time
point.) Asterisks indicate significant differences between groups, respectively in SHAM-METH versus OBX-METH groups, ***P50.001,
**P50.01, *P50.05, two-way ANOVA for repeated measures followed by Bonferroni t-test. Note that the y-axis scales differ among the
graphs.
8 J. RUDA-KUCEROVA ET AL.
46
a significantly higher percent of release was recorded in
OBX rats (at peaks approximately 240 vs. 280%, respectively).
Formation of 5-HIAA was enhanced in the OBXMETH
as compared to the SHAM-METH group 1 h after
methamphetamine injection and remained significantly
higher till the end of the microdialysis session. 5-HIAA
levels in the SHAM-METH group fell down to the
baseline levels in the last three measurements. Acute
dose of METH led also to a strong accumbens release of
glutamate in both groups (P50.001) and normalised at
the same time before the end of the microdialysis
session. A significant difference in percentage of glutamate
release was recorded in OBX rats when
compared to the control group. In OBX-METH group
the glutamate peak was lower than in the SHAM-METH
animals (at peaks approximately 140 vs. 160%,
respectively).
Accumbens GABA levels were significantly decreased
by the METH treatment in both groups with no
difference between OBX and SHAM rats. There were
no significant changes after saline administration in any
neurotransmitter.
Behavioural assessment
We have recorded significantly altered behavioural
profile in the OBX rats over the course of the
microdialysis session, including both basal differences
(increased locomotion) and response to methamphetamine
dose (in comparison to SHAM rats), depicted in
Figure 6. As expected, methamphetamine induced
significant stereotyped behaviour and proportionally
decreased immobility in both groups. However, OBX rats
were recorded to show more stereotypies and lower
immobility than SHAM controls (expressed in percentage
of total behaviour). Furthermore, locomotion in OBX
was higher at the beginning and lower at the end of the
session as compared to SHAM which corresponds to
higher METH effects in this group in terms of increased
locomotion and longer stereotyped behaviour. Saline, as
vehicle, did not induce any significant behavioural
changes in either SHAM or OBX animals and did not
provoke any stereotyped behaviour.
Discussion
As one of the core symptoms of depression is anhedonia,
the mesolimbic dopaminergic reward circuit of the
brain is believed to be dysregulated and contributes to
the high incidence of comorbid drug abuse (Nestler and
Carlezon 2006). Furthermore, clinically it is known that
moderate enhancement of DA function is at least
partially responsible for antidepressant activity of some
drugs, e.g., bupropione (Quesseveur et al. 2013). These
findings are supported by some preclinical studies which
showed that similarly, as in our present study, DISC1Q31L
(disrupted-in-schizophrenia-1 protein) mutant
mice which have depression-like behaviours were
found to have reduced levels of dopamine, serotonin
and norepinephrine in the NAC (Lipina et al. 2013). This
could indicate that lower monoamine levels in the NAC
are linked to the depressive-like phenotype. However,
the baseline DA level varies greatly in different brain
regions, because in another in vivo microdialysis study,
OBX rats unexpectedly exhibited significantly higher
basal DA levels and lower NE levels in both ventral and
dorsal striatum (Masini et al. 2004). In our earlier study
with Lister-hooded rats we did not record any basal
difference in NACSh DA levels in OBX and sham rats
which was probably caused by low sample size – four
animals per group (Amchova et al. 2014), while the
present study provides robust baseline data on 14 OBX
and 13 sham-operated rats.
D1 and D2 receptor densities in the ventral striatum of
the OBX animals, as another measure of DA-ergic system
functioning, were found to be increased (Holmes 1999),
which is in accordance with lower basal levels of DA.
However, the matter remains inconclusive: one study
found a decreased expression of the D1 receptor in the
striatum (nucleus accumbens, caudate/putamen and
olfactory tubercle) in the OBX, with an increase after
chronic antidepressant but not cocaine treatment (Taoka
et al. 2006). However, an autoradiographic study showed
no difference (Sato et al. 2010). There could be some
strain- or gender-specific issues that could shed light
into the discrepancy, but these have not yet been
evaluated.
Furthermore, DA metabolic turnover evaluated in this
study was found to be significantly increased in the
second step metabolite – HVA and a trend to decrease
the production of 3-MT and increase DOPAC was found.
These latter ones are the first-step DA metabolites,
forming two alternative pathways using two different
enzymes: catechol-O-methyl transferase (COMT) and
monoaminooxidase (MAO). This could indicate certain
adjustments in the DA metabolism induced by OBX
surgery in terms of higher MAO activity. However, there
is high interindividual variability in both SHAM and OBX
groups and we have not included in this study any more
measures to assess the situation more specifically.
Resultantly, this hypothesis does not go beyond a
speculation. Previously, a mouse OBX model exhibited
a decrease of the dopamine turnover in the hypothalamus
which was not apparent in the striatum, PFC and
hippocampus. However, the validity of this finding may
be compromised by the fact that an ex vivo assay was
THE WORLD JOURNAL OF BIOLOGICAL PSYCHIATRY 9
47
Figure 6. Behavioural scores during microdialysis session. The graphs indicate the percentage of time spent in each behavioural
category (lomomotion, immobility or stereotyped behaviour) during the appropriate 20-min sampling intervals in SHAM and OBX
animals after saline and methamphetamine treatment. The baseline point is a mean of all three measurements before the injection of
SAL/METH, injection indicated by arrows). The SAL-treated animals finished the microdialysis session earlier which is reflected in the
behavioural record as well. Asterisks indicate significant differences between the SHAM and OBX groups (a mixed ANOVA model),
***P50.001, **P50.01, * P50.05.
10 J. RUDA-KUCEROVA ET AL.
48
used to evaluate this finding (Hellweg et al. 2007).
Hence, further elucidation by more sensitive methodological
means (e.g., evaluation of MAO and COMT
activity, number of DA transporters, etc.) need to be
used in order to explain the mechanisms underlying
lower DA levels and higher turnover.
The self-medication hypothesis was repeatedly confirmed
in drug self-administration experiments where
OBX animals self-administer higher doses of amphetamine
(Holmes et al. 2002), methamphetamine
(Kucerova et al. 2012) and CB1 agonist (Amchova et al.
2014). This study shows higher methamphetamineinduced
DA release and metabolism in OBX animals.
However, earlier we recorded an abolished DA release in
OBX animals after a challenge dose of the CB1 agonist
WIN55,212-2 (WIN) (Amchova et al. 2014). The explanation
could lie in the challenge dose of the respective
drug. Amchova et al. (2014) reported a DA release
after 0.3 mg/kg of WIN, which is a dose typically selfadministered
by sham-operated rats. The WIN dose
recorded to be self-administered by OBX rats was
approximately 0.5 mg/kg. Therefore, it could be concluded
that the dose used in the microdialysis study
simply is not high enough to produce DA release in OBX
rats, which compensates the lower DA tonus in the
reward pathway by self-administering a higher dose.
This study employed 5 mg/kg of methamphetamine in
order to produce a robust neurotransmitter release.
However, the usual METH dose self-administered by
sham/OBX animals is approximately 1.8/3 mg/kg,
respectively (Amchova et al. 2014), so the possible
explanation is a ceiling effect of this dose. The differences
presented in this paper are shown as relative
values (% of baseline in each animal). When absolute
(concentration) values are compared, strong baseline
dependence is apparent and the peak concentration
values are similar in sham and OBX groups. Notably, selfadministration
studies have a chronic nature, in contrast
to cited microdialysis experiments. METH is known to
induce DA release in the NACSh after acute administration,
but the repeated treatment leads to a decrease of
DA release (Broom and Yamamoto 2005). The neuroplastic
adjustments induced by OBX surgery were never
investigated in this correlation, thus, the lowered
reactivity induced by chronic administration can be
more pronounced in the OBX model and lead to the
increase of the self-administered dose.
The reduced amount of serotonin throughout the
brain is a well-known symptom of major depression
and impaired serotonergic signalling after OBX surgery is
one of the key aspects of validity of this model (Song
and Leonard 2005). In both OBX mice and rats, a
decrease of serotonin content has been consistently
reported in many depression-related brain regions:
frontal cortex, nucleus accumbens, hippocampus,
corpus striatum and basolateral amygdala (van der
Stelt et al. 2005; Hellweg et al. 2007; Pudell et al.
2014). These changes can be reversed by chronic
antidepressant treatment (Harkin et al. 2003; Song and
Leonard 2005). The evidence of hypo-serotonergic
depressive-like symptoms following OBX surgery is
further supported by the compensatory 5-HT hyperinnervation
of the PFC and increased numbers of 5-HT
transporters (Harkin et al. 2003; Song and Leonard 2005).
Brain serotonin turnover was found to be significantly
elevated in depressed patients before treatment (Barton
et al. 2008). However, these data might be misleading
as the 5-HT turnover is not easy to measure in human
subjects and possible seasonal effects may have
contributed to these results (Luykx et al. 2013).
Furthermore, the interpretation is complicated due to
different turnover definitions used by the authors either
as a metabolic ratio (Barton et al. 2008) or directly as a
5-HIAA level (Luykx et al. 2013). Apart from that,
numerous other factors may influence levels of 5HT
and 5-HIIA, such as availability of tryptophan, activity of
tryptophan hydroxylase, formation of melatonin, activity
of other retrograde signalling systems, etc. In the same
ex vivo study mentioned above in DA, a mouse OBX
model showed a decrease of the serotonin turnover
(calculated as a ratio) in the hippocampus, frontal cortex
and hypothalamus (Hellweg et al. 2007). Similar
findings were found in hippocampus of OBX Wistar
rats (Pudell et al. 2014). However, in our study we
recorded an increase of serotonin turnover in the NACSh
and currently there are no other NAC-specific data to
compare available. Furthermore, OBX rats showed a
lower rate of 5-HT synthesis under basal conditions
without impairment of the synthetic capacity in the
hippocampus and basolateral amygdala (van der Stelt
et al. 2005) which is in accordance with our data on the
basal serotonin level. However, in some limbic areas the
synthesis was reported to be enhanced in this model:
the cingulate, the medial forebrain bundle, the hippocampus
and the thalamus (Watanabe et al. 2003).
Serotonin levels are known to be enhanced especially
by MDMA (ecstasy) to a higher extent than dopamine
levels, but other amphetamines (as well as other drugs)
induce strong serotonin release in multiple brain regions
as shown in numerous studies (Schenk 2009; Matsumoto
et al. 2014). As expected, we have recorded this effect in
both SHAM and OBX rats. However, similar to case of
dopamine, a higher 5-HT and 5-HIAA relative release was
found after a challenge dose of methamphetamine in
the OBX group. When absolute (concentration) values
are compared, strong baseline dependence is apparent
THE WORLD JOURNAL OF BIOLOGICAL PSYCHIATRY 11
49
and the peak concentration values are actually higher
in the SHAM than the OBX group.
It has been sufficiently confirmed that the concentrations
of monoamine neurotransmitters (including dopamine
and serotonin) in the microdialysates reflecting
their presence in the extracellular compartment represent
an overflow of neuronally released transmitters
from the synapses. For example, infusion of tetrodotoxin
or calcium antagonist (Cd2+
) results in substantial
reduction of these monoamine levels in the dialysates
(Westerink and de Vries 1989; Sharp et al. 1990; Morari
et al. 1993). However, in case of GABA and glutamate the
situation is more complicated. Therefore, in our previous
experiments (Sustkova et al., in preparation) we tested
the sensitivity of basal GABA and glutamate levels in the
NACSh to the Ca2+
presence in the perfusion solution.
Temporary replacement (1 h 20 min) of Ca2+
in Ringer’s
solution with Cd2+
resulted in an immediate and steep
reduction of GABA in the NACSh dialysates (drop to
maximum 8 % of basal levels). This indicates that GABA
release in the NACSh in our experiment is Ca2+
dependent.
In the case of glutamate, we did not observe any
decrease; on the contrary, glutamate concentrations
showed a dramatic increase during Cd2+
perfusion
(increase to maximum 284 % of basal levels) which
began to decrease as soon as perfusion with normal
Ringer’s solution was resumed, although glutamate
levels did not recover to basal values. These results are
in accordance with similar data from the striatum (Miele
et al. 1996) and indicate that in our experiment
glutamate is not derived from exocytotic release.
Therefore, similar to Miele et al. (1996) we assume that
the glial glutamate release might be considered.
Excitatory and inhibitory systems including glutamate
and GABA are imbalanced in OBX animals (Song and
Leonard 2005). The condition of the glutamatergic
system in the model is usually reported as hypoactive
in different brain regions (Harkin et al. 2003; Song and
Leonard 2005), but the typical hyperactive response to a
novel environment seen in OBX rats has been hypothesised
to result from increased glutamate levels in the
striatum recorded in OBX but not in sham-operated rats
while performing the microdialysis study during open
field test (Ho et al. 2000). In other microdialysis studies,
glutamate basal levels were found to be no different
from SHAM control in the PFC, but acute riluzole (a drug
with multiple indirect glutamate antagonistic mechanisms)
decreased glutamate levels in OBX rats only
(Takahashi et al. 2011) suggesting higher sensitivity of
the glutamate system in the OBX model. Our data on the
higher extracellular levels of glutamate in the NACSh in
the OBX rats contrast with these findings; however, direct
comparison is not possible for the lack of available data.
The GABA-ergic system is reported mostly as hyperactive
in the OBX model, specifically in the amygdaloid
cortex an increased GABA turnover and density of GABAA
receptors were found together with decreased
density of GABA-B receptors (Song and Leonard 2005).
This evidence is supported by our present data at basal
conditions. Moreover, GABA-B-positive allosteric modulators
have been proposed as a promising treatment for
drug addiction (Filip et al. 2014), probably via inhibition
of the mesolimbic DA pathway.
As repeatedly demonstrated, glutamatergic
(Tzschentke 2002) and GABA-ergic systems are involved
in the reward processes and their role is especially
important in repeated drug intake contributing to the
biological basis of sensitisation. We have shown here a
lower glutamate release after METH challenge in OBX
rats in comparison to their SHAM counterparts. There is
an apparent mutual modulation mechanism between
dopamine, glutamate and GABA in the NAC
(Schoffelmeer et al. 2000). Therefore, the neurotransmitter
changes seen after METH challenge dose in this study
are probably interdependent. This is supported by a
microdialysis study during cocaine self-administration
(Wydra et al. 2013). However, our acute dose-related
data show similar level of decrease in GABA levels in
both SHAM and OBX group.
As expected, in this study we have recorded a
significantly altered behavioural profile in the OBX rats
over the course of the microdialysis session. Hyperactive
response of the OBX rats to novel environment is a
widely established measure of success of OBX surgery
validated in numerous laboratories (Harkin et al. 2003;
Song and Leonard 2005). Also, the locomotor response
to acute dose was repeatedly found to be increased in
the OBX model after cocaine (Slattery et al. 2007;
Eisenstein et al. 2009) or amphetamine (Romeas et al.
2009). Increased locomotion correlates with dopaminergic
signalling in the striatum and NAC (Do et al. 2012),
so the presented behavioural and neurochemical data in
this study are in full accordance with previous reports.
Furthermore, we have observed an increase of stereotypical
behaviour after METH dose in the OBX group
compared to the SHAM control. Methamphetamineinduced
stereotypies have been shown to correlate also
with the DA firing in the NAC – interestingly only NAC
core and not shell (Morra et al. 2010).
In summary, this study provides comprehensive data
on extracellular levels of four neurotransmitter systems
in the nucleus accumbens shell, the main reward-related
area (Di Chiara et al. 2004) in the OBX rat model of
depression. The most important finding is the different
baseline condition: significantly decreased basal levels
of dopamine, serotonin and their metabolites and
12 J. RUDA-KUCEROVA ET AL.
50
increased levels of glutamate and GABA in OBX rats.
After acute methamphetamine administration we
detected a significantly higher release of dopamine,
serotonin and their metabolites in OBX rats; however,
glutamate levels were lower and GABA was not found
to be different from SHAM control. These findings are
further supported by a differential behavioural profile in
the OBX rats. This study provides comprehensive data on
extracellular levels of four neurotransmitter systems in
the nucleus accumbens shell, the main reward-related
area. However, a dose–response assessment of DA
release and changes induced by chronic methamphetamine
administration are of high interest. Furthermore,
chronic drug administration data are needed to further
elucidate the underlying pathophysiological processes
shared by the depressive-like phenotype and reward.
Acknowledgements
This work was supported by the project ‘‘CEITEC – Central
European Institute of Technology’’ (CZ.1.05/1.1.00/02.0068)
from European Regional Development Fund, projects of
specific research at the Masaryk University (MUNI/A/0886/
2013 and MUNI/A/1116/2014), project PRVOUK P34,
‘‘Operational Programme Prague – Competitiveness’’
(CZ.2.16/3.1.00/22197, CZ.2.16/3.1.00/21537) and ‘‘National
Programme of Sustainability’’ (NPU I (LO) MSMT – 34870/
2013). The funding has not influenced the process of the paper
production including among others the data interpretation and
submission. The authors are grateful to Ladislav Dusek for his
help with statistical analysis of behavioural data, Jan Jurica and
Gabriela Dovrtelova for help with turnover data interpretation
and Vanessa Raileanu (Toronto, Canada) for the help with
manuscript preparation and proof reading.
Statement of interest
None to declare.
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54
2.4.4. Enhanced self-administration of the CB1 receptor agonist WIN55,212-2
in olfactory bulbectomized rats: evaluation of possible serotonergic and
dopaminergic underlying mechanisms
The aim of this study was to extend the rat model of the dual disorder to intravenous selfadministration
of synthetic CB1 receptor agonist WIN55-212,2, (WIN) in the olfactory
bulbectomy model of depression. Since cannabinoid self-administration was shown to be
associated to an increased dopaminergic transmission in the shell of the nucleus
accumbens (Fadda et al., 2006), the in vivo microdialysis technique was used in order to
test whether OBX and SHAM rats displayed similar increase in dopamine levels within the
nucleus accumbens shell in response to a challenge dose of WIN (0.3 mg/kg). This dose is
equivalent to the mean daily amount of the drug typically self-administered by trained rats
(Fattore et al., 2001, Fattore et al., 2007). Furthermore, the 5-HT1B receptor is greatly
involved in the modulation of both depression and drug intake (Miszkiel et al., 2012,
Murrough et al., 2011), therefore we tested the effect of the 5-HT1B agonist CGS-12066B
(CGS) on WIN self-administration in OBX and SHAM Lister Hooded rats and Sprague
Dawley rats self-administering methamphetamine.
Findings of this study showed that olfactory bulbectomy markedly increases selfadministration
of WIN, possibly through a reduction of its rewarding effects to which
animals compensate by increasing WIN intake. A decreased dopamine neurotransmission
in the nucleus accumbens shell might contribute to this compensatory behaviour. CGS did
not show any influence on drug taking behaviour in any strain and drug.
Amchova P, Kucerova J, Giugliano V, Babinska Z, Zanda MT, Scherma M, Dusek L,
Fadda P, Micale V, Sulcova A, Fratta W, Fattore L. Enhanced self-administration of the
CB1 receptor agonist WIN55,212-2 in olfactory bulbectomized rats: evaluation of possible
serotonergic and dopaminergic underlying mechanisms. Front Pharmacol. 2014, 5: 44. doi:
10.3389/fphar.2014.00044.
IF 3.802
Citations (WOS): 5
ORIGINAL RESEARCH ARTICLE
published: 20 March 2014
doi: 10.3389/fphar.2014.00044
Enhanced self-administration of the CB1 receptor agonist
WIN55,212-2 in olfactory bulbectomized rats: evaluation of
possible serotonergic and dopaminergic underlying
mechanisms
Petra Amchova1,2
, Jana Kucerova1,2
*, Valentina Giugliano3
, Zuzana Babinska1,2
, Mary T. Zanda3
,
Maria Scherma3
, Ladislav Dusek4
, Paola Fadda3,5,6
, Vincenzo Micale1
, Alexandra Sulcova1
,
Walter Fratta3,5,6
and Liana Fattore5,7
1
Central European Institute of Technology, Masaryk University, Brno, Czech Republic
2
Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
3
Section of Neuroscience and Clinical Pharmacology, Department of Biomedical Sciences, University of Cagliari, Monserrato, Italy
4
Institute of Biostatistics and Analyses of Faculty of Medicine, Masaryk University, Brno, Czech Republic
5
Center of Excellence “Neurobiology of Addiction,” University of Cagliari, Monserrato, Italy
6
National Institute of Neuroscience (INN), University of Cagliari, Monserrato, Italy
7
CNR Institute of Neuroscience-Cagliari, National Research Council-Italy, Monserrato, Italy
Edited by:
Maria Grazia Cascio, University of
Aberdeen, UK
Reviewed by:
Jean-Philippe Guilloux, Université
Paris-Sud, France
Viviana Trezza, Roma Tre University,
Italy
Marcello Solinas, Institut National de
la Santé et la Recherche Medicale,
France
*Correspondence:
Jana Kucerova, Central European
Institute of Technology, Masaryk
University, Kamenice 5, 62500 Brno,
Czech Republic
e-mail: jkucer@med.muni.cz
Depression has been associated with drug consumption, including heavy or problematic
cannabis use. According to an animal model of depression and substance use disorder
comorbidity, we combined the olfactory bulbectomy (OBX) model of depression with
intravenous drug self-administration procedure to verify whether depressive-like rats
displayed altered voluntary intake of the CB1 receptor agonist WIN55,212-2 (WIN,
12.5 μg/kg/infusion). To this aim, olfactory-bulbectomized (OBX) and sham-operated
(SHAM) Lister Hooded rats were allowed to self-administer WIN by lever-pressing under
a continuous [ﬁxed ratio 1 (FR-1)] schedule of reinforcement in 2 h daily sessions. Data
showed that both OBX and SHAM rats developed stable WIN intake; yet, responses in
OBX were constantly higher than in SHAM rats soon after the ﬁrst week of training.
In addition, OBX rats took signiﬁcantly longer to extinguish the drug-seeking behavior
after vehicle substitution. Acute pre-treatment with serotonin 5HT1B receptor agonist,
CGS-12066B (2.5–10 mg/kg), did not signiﬁcantly modify WIN intake in OBX and SHAM
Lister Hooded rats. Furthermore, acute pre-treatment with CGS-12066B (10 and 15 mg/kg)
did not alter responses in parallel groups of OBX and SHAM Sprague Dawley rats
self-administering methamphetamine under higher (FR-2) reinforcement schedule with
nose-poking as operandum. Finally, dopamine levels in the nucleus accumbens (NAc) of
OBX rats did not increase in response to a WIN challenge, as in SHAM rats, indicating
a dopaminergic dysfunction in bulbectomized rats. Altogether, our ﬁndings suggest that
a depressive-like state may alter cannabinoid CB1 receptor agonist-induced brain reward
function and that a dopaminergic rather than a 5-HT1B mechanism is likely to underlie
enhanced WIN self-administration in OBX rats.
Keywords: WIN55212-2, cannabinoid, methamphetamine, olfactory bulbectomy, depression, drug dependence,
serotonin, dopamine
INTRODUCTION
Many psychiatric disorders including depression, schizophrenia,
and anxiety are frequently associated to drug addiction
(Langas et al., 2010; Testa et al., 2013). Recently, clinical associations
between depression and marijuana smoking have been
reported (Horwood et al., 2012; Lev-Ran et al., 2013). Yet,
whether cannabis abuse in depressed patients antedates the disorder
onset or is a consequence of its course is still to be
determined. Several hypotheses have been offered to explain
the high rates of marihuana smoking in people with depression
including genetic factors, environmental inﬂuences, and
self-medication. Recently, a genetically conditioned hypersensitivity
to elicit cannabis dependence was evaluated in a depressive
population, and although the outcome was not fully conclusive
authors suggested that the links between cannabis use and depressive
symptoms are conditional on the individual’s genetic makeup
(Otten and Engels, 2013). Social difﬁculties such as limited economical
resources, impaired interpersonal skills, social isolation,
or stressful events may also trigger both depression and cannabis
abuse (Baker et al., 2010). On the other hand, cannabis use has
been proposed to serve as a self-medication in depressed patients
(Degenhardt et al., 2003), although some studies excluded that
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55
Amchova et al. Enhanced WIN55,212-2 self-administration in depressive-like rats
subjects with prior depression experience symptom relief after
smoking cannabis (Arendt et al., 2007).
The self-medication theory was developed on the basis
of the monoaminergic hypothesis of depression, according to
which depression is associated with a reduced monoaminergic
transmission, in particular noradrenaline and serotonin
(5-HT) (Rotenberg, 1994; Prins et al., 2011). In fact, symptoms
related to monoaminergic deﬁcits (as in depression) may
be relieved by a variety of abused drugs (Khantzian, 1985;
Hall and Queener, 2007; Becker et al., 2012; Holma et al.,
2013). Accordingly, stimulant-dependent patients with depressive
disorders reduce their abuse of stimulants when treated
with antidepressants to a greater extent than non-depressed
(stimulant-dependent) individuals (Markou et al., 1998; Wohl
and Ades, 2009). Similar to other abused drugs, cannabis induces
release of dopamine (DA) in the mesolimbic reward pathway
(Oleson and Cheer, 2012), thus elevating mood and improving
wellbeing. However, it also signiﬁcantly affects bioavailability
of serotonin. Notably, genetic deletion of the cannabinoid
CB1 receptor reduces the functionality of the brain serotonin
system (Mato et al., 2007), while chronic CB1 receptor
antagonism induces a depression-like phenotype (Beyer et al.,
2010).
Majority of available studies aimed at investigating the role
of endocannabinoid system in animal models of depression
reported a decreased activity of the endocannabinoid system
(Micale et al., 2013a,b). Pharmacological and genetic blockade of
cannabinoid CB1 receptors result in symptoms that mimic those
seen in depression (Ashton and Moore, 2011). In keeping with
this, stimulation of CB1 receptors exerts an antidepressant-like
effect similar to that induced by the antidepressants desipramine
and ﬂuoxetine in the rat forced-swim test (Hill and Gorzalka,
2005) and the olfactory bulbectomy (OBX) rat model of depression
(Rodriguez-Gaztelumendi et al., 2009), respectively. These
antidepressant effects are antagonized by administration of CB1
receptor antagonists leading back to depression-like phenotypes
(Hill and Gorzalka, 2005). All these and other preclinical evidence
strengthen the involvement of the endocannabinoid system
in depressive-like states. However, no data are available on the voluntary
consumption of cannabinoid receptor agonists in animal
models of depression.
The multi-faceted effects of 5-HT are mediated by at least
14 receptor subtypes (Hoyer et al., 1994). Among them, the
5-HT1B receptor subtype has recently attracted scientiﬁc attention
for its potential role in modulating addictive behaviors
(Pentkowski et al., 2012; Neisewander et al., 2013). Serotonin 5HT1B
receptors are widely distributed in the brain where function
as both autoreceptors and heteroreceptors, that mediate release
of serotonin and other neurotransmitters (Barnes and Sharp,
1999; Moret and Briley, 2000; Pytliak et al., 2011; Cai et al.,
2013). A number of human and animal studies demonstrated a
causal link between altered 5-HT1B receptor activity and development
of neuropsychiatric conditions, including depression and
drug addiction. For example, lower functioning of 5-HT1B receptors
was found in patients suffering major depressive disorders
(Murrough et al., 2011), while a polymorphism at the 5-HT1B
receptor gene (HTR1B) was reported to be associated signiﬁcantly
with alcoholism (Lappalainen et al., 1998). In animal models of
drug addiction, stimulation of the 5-HT1B receptor was shown
to induce antidepressant effects (Tatarczynska et al., 2004) and to
decrease the number of behavioral responses for alcohol (Grant
et al., 1997; Maurel et al., 1999; Tomkins and O’Neill, 2000),
d-amphetamine (Fletcher and Korth, 1999), intracranial selfstimulation
(Hayes et al., 2009) and the positive reinforcing effects
of cocaine in rats (Harrison et al., 1999). More importantly,
administration of the 5-HT1B receptor agonist CGS-12066B
into the nucleus accumbens (NAc) core was shown to decrease
operant responses for ethanol but not sucrose solution in rats
(Czachowski, 2005) indicating a selective effect of this compound
on drug-induced responses. However, systemic administration
of CGS-12066B did not reduce cocaine self-administration
in rats (Parsons et al., 1996), which implies a certain drug
selectivity of this compound in attenuating self-administration
behavior.
The main aim of this study was to investigate the intravenous
self-administration of the CB1 receptor agonist WIN55-212,2,
(WIN), using lever-pressing as operandum under a continuous
[ﬁxed ratio 1 (FR-1)] schedule of reinforcement, in a wellestablished
rat model of depression, the bilateral OBX. Given
the signiﬁcant differences observed in WIN self-administration
between OBX and SHAM rats, we decided to perform pilot
experiments in an attempt to shed some light on possible underlying
mechanisms. Therefore, since the 5-HT1B receptor has been
recently involved in the modulation of both depression and drug
intake, we tested the effect of the 5-HT1B agonist CGS-12066B
(CGS) on WIN self-administration in OBX and sham-operated
(SHAM) Lister Hooded rats displaying depressive-like phenotypes.
To further investigate the role of 5-HT1B receptor in drugtaking
behavior and verify its effect on the self-administration
of a different drug, in different strains of rats and under dissimilar
experimental conditions, we tested the CGS compound
in OBX and SHAM Sprague Dawley rats self-administering
methamphetamine (METH) as previously reported (Kucerova
et al., 2012). CGS was chosen because of its high selectivity
to 5-HT1B receptors (Neale et al., 1987) and its reducing
effects on amphetamine (Fletcher and Korth, 1999) and alcohol
self-administration (Tomkins and O’Neill, 2000; Czachowski,
2005). Finally, since cannabinoid self-administration was shown
to be associated to an increased DA transmission in the shell
of the NAc (Fadda et al., 2006), we used the in vivo microdialysis
technique to test whether OBX and SHAM rats displayed
similar increase in DA levels within the NAc shell in
response to a challenge of WIN at a dose (0.3 mg/kg) mimicking
daily mean amount of the drug typically self-administer by
trained rats.
MATERIALS AND METHODS
ANIMALS
Adult male Lister Hooded rats weighting 250–270 g at the
beginning of the experiment (9 weeks old) were purchased
from Harlan-Nossan (Italy) and housed four per cage at the
Animal Facility of the Department of Biomedical Sciences,
University of Cagliari, Italy. Rats were provided with free access
to water and food and maintained on a reversed 12/12 h
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Amchova et al. Enhanced WIN55,212-2 self-administration in depressive-like rats
light/dark cycle (lights on 7 p.m.) with constant room temperature
(22 ± 2◦C) and humidity (60%). The experimental
protocols were approved by the local Animal Care Committee
at the Department of Biomedical Sciences, University of Cagliari,
Italy.
Adult male albino Sprague Dawley rats weighting 220–240 g at
the beginning of the experiment (8 weeks old) were purchased
from Charles River (Germany) and housed individually at the
Animal Facility of the Department of Pharmacology, Masaryk
University in Brno, Czech Republic. Animals were maintained on
a reversed 12/12 h light/dark cycle (lights on 5 p.m.) with constant
relative humidity of 50–60% and temperature of 23 ± 1◦C, and
food and water available ad-libitum. The experimental protocols
were approved by the Animal Care Committee of the Faculty of
Medicine, Masaryk University, Czech Republic.
All experiments were carried out in strict accordance with the
E.C. Regulations for Animal Use in Research (CEE No. 86/609)
and local acts.
DRUGS AND TREATMENTS
For self-administration training, WIN55-212,2 (R-[2,3-dihydro-
5-methyl-3-[(morpholinyl) methyl]-pyrrolo[1,2,3-de]-1,4benzoxazinyl]-(1-naphthalenyl)-methanone
mesylate), (WIN,
RBI, USA) was freshly dissolved in one drop of Tween 80, diluted
in heparinized (1%) saline solution and made available at the
dose of 12.5 μg/kg/infusion (volume of infusion: 100 μl), as
previously described (Fattore et al., 2001). To ensure sterility,
fresh drug solutions were ﬁltered by 0.22 μm syringe ﬁlters prior
to use. For microdialysis testing, WIN solution was prepared
as described above and administered intravenously at the dose
of 0.3 mg/kg (volume of injection: 1 ml/kg). This drug dose
was selected on the basis of the daily amount of WIN typically
self-administered by male Lister Hooded rats under the same
experimental conditions (Deiana et al., 2007; Fattore et al., 2007;
Spano et al., 2010). Importantly, this dose of WIN was also shown
to signiﬁcantly increase DA release in the shell part of the NAc of
rats (Tanda et al., 1997).
Methamphetamine (METH, Sigma Chemical Co., St Louis,
MO, USA) was dissolved in saline sterile solution and made
available at dose of 0.08 mg/infusion as previously described
(Vinklerova et al., 2002).
CGS-12066B, 7-triﬂuoromethyl-4(4-methyl-1-piperazinyl)pyrrolo[1,2-a]-quinoxaline
dimaleate (CGS, R&D systems,
Abingdon, Oxon, UK) was dissolved in saline and administered
intraperitoneally (i.p.) at doses ranging from 2.5 to 15 mg/kg
(volume of injection: 2 ml/kg), and administered 20 min before
starting the session. These drug doses were selected on the basis
of their ability to acutely reduce self-administration behavior
in rats in a dose-dependent manner (Parsons et al., 1996).
Treatments were assigned on the basis of a Latin square design
whereby at least three training sessions separated two consecutive
testing sessions to allow for assessment of carryover effects. Each
animal was tested once with each drug dose and once with saline
in a counterbalanced manner, i.e., the order of presentation of
different treatments was varied between animals.
All antibiotics and anesthetics were purchased as sterile solutions
from local distributors.
OLFACTORY BULBECTOMY (OBX) SURGERY
At the beginning of the behavioral and neurochemical
experiments, rats were randomly divided into two groups:
OBX and SHAM rats. The bilateral ablation of the olfactory
bulbs was performed as previously described (Kucerova et al.,
2012). Animals were anaesthetized with isoﬂuran 2% (Italy) or
i.p. injections of 50 mg/kg ketamine plus 8 mg/kg xylazine (Czech
Republic). The top of the skull was shaved and swabbed with an
antiseptic solution. Then, midline frontal incision was made on
the skull and the skin was retracted bilaterally. Two burr holes,
2 mm in diameter, were drilled in the frontal bone 7 and 7.5 mm
anterior from the bregma, 1.5 and 2 mm lateral to bregma suture
for rats weighing 230 ± 10 g and 260 ± 10 g, respectively. Both
olfactory bulbs were removed by aspiration paying particular
attention to not damage the frontal cortex. Prevention of blood
loss from the ablation cavity was achieved by ﬁlling the dead
space with a hemostatic sponge. The skin above the lesion was
closed with suture. Finally, bacitracin plus neomycin powder was
applied to prevent bacterial infection. SHAM rats underwent
identical anesthetic and drilling procedures but their bulbs were
left intact.
A period of at least 20 days was allowed for the recovery from
the surgical procedure and the development of the depressivelike
syndrome. During this period, animals were handled daily
for few minutes to eliminate aggressiveness, which could otherwise
arise (Leonard and Tuite, 1981; Song and Leonard, 2005).
Before starting either drug self-administration training or microdialysis
experiments, animals were tested in the sucrose preference
and motor activity test for anhedonia and hyperactive locomotor
response to a novel environment, respectively, (Song and
Leonard, 2005).
SUCROSE PREFERENCE TEST
After 20 days of recovery from the OBX surgery, Lister-Hooded
animals were transferred into single housing with free access
to food. A two-bottle choice procedure was used to determine
baseline sucrose intake. During the 24-h training phase, all rats
were provided in their home cage with two water bottles on
the extreme sides of the cage to adapt for drinking from two
bottles. After training, one bottle was randomly switched to contain
2% sucrose solution, a concentration known to provide a
robust sucrose preference (Muscat and Willner, 1989). The side
of sucrose presentation in the home cage was counterbalanced
across rats. At 4 and 24 h time intervals both bottles were removed
and the amount of liquid remaining in each bottle was measured.
After 4 h, the relative position of the bottles was inverted,
i.e., they were switched from one side of the cage to the other
to avoid perseveration effects. The sucrose preference score was
calculated as the percentage of sucrose solution ingested relative
to the total amount of liquid consumed as determined before
and after each test, i.e., sucrose preference = sucrose intake/total
liquid (sucrose + water) intake × 100.
LOCOMOTOR ACTIVITY TEST
A day after conclusion of the sucrose preference test, the validity
of OBX lesions was further conﬁrmed by assessing increased
activity in a brightly lit novel environment. Rats were individually
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Amchova et al. Enhanced WIN55,212-2 self-administration in depressive-like rats
tested for locomotor activity using the Digiscan Animal Activity
Analyser (Omnitech Electronics, USA) as previously described
(Castelli et al., 2013). Each operant cage (42 × 30× 60 cm) was
equipped with two sets of 16 photocells located at right angles
to each other projecting horizontal infrared beams 2.5 cm apart
and 2 cm above the cage ﬂoor. The outside of the four walls
was covered with aluminium foil and two 90-W light bulbs were
located at diagonally opposed corners to provide bright illumination.
Rats were brought into the testing room individually, placed
in the center of the box, and allowed to move freely for 10 min.
Locomotor activity was deﬁned from measurement of sequential
infrared beam breaks recorded at every 5 min after placing
the animals individually in the cage. During the 10-min test the
following behavioral parameters were measured:
- Horizontal activity: The total number of beam interruptions
that occurred in the horizontal sensors;
- Vertical activity: The total number of beam interruptions that
occurred in the vertical sensors; that is the number of times the
animal rose onto its hind legs with the front limbs either against
the wall or freely in the air (number of rearing episodes);
- Total distance (cm): The horizontal distance travelled by the
animal (dependent on animal’s trajectory).
At the end of the session, animals were gently removed from
the Plexiglas boxes and returned to their home cage. Boxes were
wiped with H2O2 between sessions to prevent olfactory cues.
INTRAVENOUS DRUG SELF-ADMINISTRATION SURGERY
At the end of the motility test, OBX and SHAM animals were
deeply anesthetized with isoﬂuran 2% (in Italy) or i.p. injections
of 50 mg/kg ketamine plus 8 mg/kg xylazine (in the Czech
Republic). Under aseptic conditions, a permanent intracardiac
silastic catheter was implanted through the external jugular vein
to the right atrium. The outer part of the catheter exited the skin
in the midscapular area. After surgery, each animal was allowed
for recovery, individually, in its home cage with food and water
freely available. On the following 6–7 days, each rat received an
intravenous infusion of gentamicin (0.16 mg/kg, Italy) or heparinized
cephazoline (Vulmizolin 1.0 g, Czech Republic) solution
followed by 0.1 ml of a heparinized (1%) sterile saline solution
to prevent infection and occlusion of the catheter. During recovery,
changes in general behavior and body weight were monitored.
When a catheter was found to be blocked or damaged, the animal
was excluded from the analysis. Once completely recovered from
surgery, food-restriction condition was applied, where rats were
fed with 20 g/day of standard rat chow given in the home cage
immediately after each session.
INTRAVENOUS SELF-ADMINISTRATION
WIN55-212,2 self-administration was conducted in 12 operant
chambers (29.5 × 32.5 × 23.5 cm, Med Associates, Vermont,
USA) using lever-pressing as operandum under a continuous
(FR-1) schedule of reinforcement, i.e., each active response was
reinforced. Each chamber was encased in a sound and light attenuating
cube. In addition, chambers had a ventilation fan, and
a front panel equipped with two retractable levers (each 4 cm
wide) positioned 12 cm apart, 8 cm from the grid and extending
1.5 cm into the box. A white stimulus light was placed
above each lever and a red house light was located on the
opposite wall. Intravenous infusions of WIN were delivered by
a software-operated infusion pump (Med Associates, Vermont,
USA) through a counterbalanced single-channel swivel and an
extra length of plastic tubing enclosed in a metal spring connecting
the swivel to the catheter ﬁtting on the animal’s back. Pressure
on the lever, deﬁned as active, resulted in: (i) extinction of the
house light and illumination of the stimulus light above the active
lever for 15 s; (ii) retraction of both levers; and (iii) activation of
the infusion pump for 5.8 s delivering 0.1 ml intravenous infusion
of drug solution. On completion of the 15 s timeout period,
levers were re-extended into the chamber, stimulus light went out
and the house light was switched on. Pressure on the other lever,
deﬁned as inactive, was not coupled to any successive event, but
was always recorded to provide an index of basal activity levels.
Assessment of schedules and data collections was programmed
by means of a computer using the MED Associates MED-PC
software package. Throughout each phase of the study, locomotor
activity was monitored within the operant chambers, which
were equipped with a series of photocells located 3.5 cm above the
cage ﬂoor. The number of photocell beam breaks was recorded
and served as a measure of general horizontal locomotor activity.
Self-administration sessions lasted 120 min and took place 7
days/week between 9 a.m. and 1 p.m. during the dark period of
the cycle.
Acquisition training was carried out until steady baseline of
drug intake was reached. Response was considered stable when
animals displayed accurate discrimination between the active and
inactive lever. Acquisition was deﬁned as the number of active
lever-presses >15 and not differing by more than 20% for three
consecutive days. Rats not meeting the acquisition criterion were
excluded from the subsequent phases of the study. Only rats
developing a stable pattern of WIN intake were allowed to continue
daily self-administration sessions until day 30. Then, extinction
condition was introduced by replacing WIN with sterile
vehicle solution (1% Tween 80 in saline solution) which allowed
responses to be recorded without drug consequences. All other
experimental parameters were left unchanged; therefore pressure
on the active lever resulted in an infusion of 0.1 ml of vehicle
accompanied by the presentation of the stimulus light previously
paired with WIN delivery. Drug-reinforced behavior was considered
extinguished when the maximum number of responses on
the active lever was ≤10 and the total number of lever presses
(i.e., active + inactive) in a single test session was ≤20.
Methamphetamine self-administration was conducted as previously
described (Kucerova et al., 2009, 2012) in 10 standard
experimental (30 × 25× 30 cm, Coulbourn Instruments, USA)
boxes using nose-poking as operandum under a ﬁnal FR-2 schedule
of reinforcement, i.e., animal had to make two consecutive
nose-pokes on the active hole to obtain a single drug infusion.
Each cage was provided with two nose-poke holes allocated on
one side and programmed by software Graphic State Notation
3.03 (Coulbourn Instruments, USA). Speciﬁcally, training sessions
were initially conducted under a FR-1 schedule of reinforcement.
Fixed-ratio requirement was then raised to FR-2 when the
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Amchova et al. Enhanced WIN55,212-2 self-administration in depressive-like rats
animal fulﬁlled the following conditions for three consecutive sessions:
(a) at least 70% preference of the drug-active nose-poke,
(b) minimum intake of 10 infusions per session, or (c) stable
intake of the drug (maximum 10% deviation). Nose-pokes in the
active hole led to the activation of the infusion pump and administration
of a single infusion followed by a 10 s timeout, while
nose-pokes in the inactive hole were recorded but not rewarded
and reset the count of active nose pokes back to zero. The cage
was illuminated by a house light during the session. The light was
ﬂashing when administering infusion and off during the timeout
period. Self-administration sessions lasted 90 min and took
place 7 days/week between 7 a.m. and 2 p.m. during the dark
period of the cycle. Acquisition criteria were the same as for WIN
self-administration behavior.
In both apparatuses, assignment of the active (drug-paired)
and the inactive (not drug-paired) levers/holes was counterbalanced
between rats and remained constant for each subject
throughout all the experiments. CGS testing was performed on
Lister Hooded and Sprague Dawley rats self-administering WIN
and METH, respectively, during the maintenance phase of the
self-administration training, i.e., once animals stabilized their
drug intake.
MICRODIALYSIS SURGERY AND PROCEDURE
A separate batch of drug-naïve Lister Hooded male rats was
used for the in vivo microdialysis study. Rats were anesthetized
with 2% isoﬂurane and placed in a stereotaxic frame (David
Kopf Instruments, Tujunga, CA, USA). The skull was exposed
and a small hole was drilled on the right side. A concentric
self-made microdialysis probe with a 2 mm dialyzing surface
length (AN 69AF; Hospal-Dasco, Bologna, Italy; cut-off 40,000
Da, in vitro recovery about 30%) was inserted vertically into the
shell of the NAc (coordinates from bregma, AP: +1.7, L: ±0.7,
V: −8.2) according to the Paxinos anatomical atlas (Paxinos
and Watson, 1998) and then ﬁxed to the skull using dental
acrylic cement. During the same surgery session, rats were
implanted with intravenous catheters as previously described,
which allowed intravenous administration of WIN. Starting 24 h
from implantation of the dialysis probe, artiﬁcial cerebrospinal
ﬂuid (147 mM NaCl, 4 mM KCl, 1.5 mM CaCl2, pH 6–6.5) was
pumped through the dialysis membrane at a constant rate of
2.5 μL/min with a CMA/100 microinjection pump (Carnegie
Medicine, Sweden). Dialysate samples (50 μL) were collected
every 20 min and directly injected into a high performance
liquid chromatography system in order to quantify DA. The
system consisted of an isocratic pump (ESA model 580; ESA,
Chelmsford, Massachusetts), a 7125 Rheodyne injector connected
to a Hewlett Packard (Waldbronn, Germany) series 1100 column
thermostat with a reverse phase column (LC18 DBSupelco,
5 μm, 4.6 × 150 mm), and an ESA Coulochem II detector. The
ﬁrst electrode of the detector analytical cell was set at 400 mV
and the second at −180 mV; column temperature was set at
30◦C. The mobile phase, delivered at 1.0 mL/min, consisted of
50 mM/L sodium acetate, 0.073 mM/L Na2 ethylenediaminetetraacetic
acid, 0.35 mM/L 1-octanesulfonic acid, 12% methanol,
pH 4.21, with acetic acid. In this condition the sensitivity of
the assay for DA was 2 fM/sample. Only results deriving from
rats with correctly positioned dialysis probes were included in
statistical analysis of data. The location of the probe was determined
histologically at the end of each experiment by examining
coronal brain sections (50 μm) stained with cresyl violet.
STATISTICAL DATA ANALYSIS
At the end of the study, rats were anesthetized by isoﬂuran inhalation
and decapitated. Their brains were removed for conﬁrmation
of the ablation of the olfactory bulbs. Only rats with a complete
removal of both olfactory bulbs with no damages to the frontal
cortex were included for data analysis.
Primary data were summarized using arithmetic mean and
standard error of the mean estimate. Statistical analysis of differences
between OBX and SHAM rats in time-related differences
used One-Way and Two-Way analysis of variance (ANOVA)
with repeated measures model. In Two-Way repeated measures
ANOVA, the group of rats was entered as the between-group
factor and the time-points as repeated within-subject measure.
One-Way ANOVA was used when comparing the time-points
within given experimental group of rats.
Time-related changes and differences between OBX and
SHAM groups during drug self-administration after acute pretreatment
with the 5-HT1B receptor agonist CGS-12066B were
analyzed using Two-Way repeated measures ANOVA.
Microdialysis data were analyzed using One-Way or Two-Way
ANOVA (treatment × time), followed by Tukey’s or Bonferroni’s
post-hoc test comparison procedures, respectively. The level of
statistical signiﬁcance was set at p < 0.05.
Statistics were calculated using the statistical package SPSS
(version 2.0).
RESULTS
BEHAVIORAL PROFILE AFTER OBX SURGERY (LISTER HOODED RATS)
To verify the development of a depressive-like phenotype in OBX
lesioned animals, basal behavioral differences between SHAM
and OBX rats were established by measuring sucrose preference
and locomotor activity. Anhedonia and hyperactive response to a
novel brightly lit environment are two major features of OBX rats
consistently described by previous studies (Kelly et al., 1997; Song
and Leonard, 2005; Romeas et al., 2009).
As shown in Figure 1, OBX rats consumed signiﬁcantly lower
proportion of sucrose than SHAM rats (p < 0.001) after both 4
and 24 h from sucrose solution presentation, which conﬁrmed
a reduced hedonic response as a consequence of ablation of the
olfactory bulbs. Differences between the two dependent variables
were tested using independent Student t-test.
Figure 2 illustrates results from the motor activity test conducted
in brightly lit conditions. In all locomotor measures (i.e.,
horizontal activity, vertical activity, and total distance travelled),
statistically signiﬁcant differences were detected at 5 min of measurement,
a time-point representing response to novel environment
(horizontal activity, p < 0.001; vertical activity, p < 0.05;
total distance travelled, p < 0.01). At the 10 min time-point (little
effect of novelty), a signiﬁcant difference was only present
in vertical activity measure (p = 0.032). Differences between the
two independent variables were tested using independent Student
t-test.
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Amchova et al. Enhanced WIN55,212-2 self-administration in depressive-like rats
FIGURE 1 | OBX rats display anhedonia. The sucrose preference test in
SHAM (n = 6) and OBX (n = 7) Lister Hooded rats. Data are percentages
(±s.e.m.) of sucrose solution consumed at two time-points, i.e., after 4 and
24 h from the beginning of the test. Student t-test, ∗∗∗p < 0.001.
WIN 55-212,2 SELF-ADMINISTRATION IN LISTER HOODED RATS
Figure 3A shows responses of SHAM and OBX rats on the active
lever during the acquisition phase of WIN self-administration
training. Repeated measures ANOVA revealed no signiﬁcant
effects over the ﬁrst 8 days of training, whereas from day 9 onward
a signiﬁcantly higher active lever-pressing rate was observed in
OBX compared with SHAM rats (repeated measures ANOVA:
α < 0.001). In contrast, inactive lever-pressing rates of OBX and
SHAM rats were statistically indistinguishable throughout the
30 days of training and remained constantly below 6 responses
per session starting from the ﬁrst week of training, with the
sole exception of the initial 4 days of training (data available as
Supplementary Figure 1A). This indicates that the increase in
the rate of responding observed in OBX rats was not due to an
unspeciﬁc effect, as further conﬁrmed by the ﬁnding of no significant
differences between OBX and SHAM animals in the basal
motor activity during the self-administration daily sessions, as
measured by the mean number of interruptions of the photocell
beams located inside the boxes (mean activity over the maintenance
phase: 989 ± 41 and 1005 ± 27 for OBX and SHAM rats,
respectively).
In accordance with this, the total amount of WIN consumed
by OBX rats during the maintenance phase, i.e., once animals
stabilized drug intake, was signiﬁcantly higher than that consumed
by SHAM rats. More speciﬁcally, mean WIN intake during
the last 7 days of training before extinction was signiﬁcantly
higher (+105%) in OBX than in SHAM rats (repeated measures
ANOVA: α < 0.001) (Figure 3B). However, the percentages
of rats meeting acquisition criteria for WIN self-administration
in OBX and SHAM groups were similar, being 85.5 and 86.8%,
respectively.
Furthermore, OBX and SHAM rats displayed clear cut differences
in the time course of operant behavior even when saline was
substituted for WIN, i.e., during extinction training (Figure 3C).
Analysis of response on the active lever by repeated measures
ANOVA showed signiﬁcant differences between OBX and SHAM
animals (α = 0.012 from day 1 to 7; α = 0.004 from day 8 to
FIGURE 2 | OBX rats display hyperactivity in a novel aversive
environment. Spontaneous horizontal and vertical activity, expressed as
mean counts of photobeam interruptions, and total distance travelled (in
centimeters) in SHAM (n = 6) and OBX (n = 7) Lister Hooded rats. Data are
shown as means (±s.e.m.) at two time-points (5 and 10 min of
measurement). Student t-test, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.
14, α < 0.001 from day 15 to 28). Speciﬁcally, on the 1st day
of extinction, OBX and SHAM rats reacted to saline substitution
by increasing their mean active responding from 39 to
78.5 and from 16.86 to 45.17, respectively, which corresponds to
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Amchova et al. Enhanced WIN55,212-2 self-administration in depressive-like rats
FIGURE 3 | Cannabinoid self-administration training in OBX and SHAM
rats. Data are shown as means (±s.e.m.). (A) Acquisition phase: mean
active lever presses in SHAM (n = 6) and OBX (n = 7) Lister Hooded rats
during WIN55,212-2 self-administration. ∗∗∗α < 0.001, repeated measures
ANOVA. (B) Maintenance phase: mean WIN intake over the last seven
training sessions in SHAM (n = 6) and OBX (n = 7) Lister Hooded rats
during WIN self-administration. ∗∗∗α < 0.001, repeated measures ANOVA.
(C) Extinction phase: mean active lever presses in SHAM (n = 6) and OBX
(n = 7) Lister Hooded rats during extinction of WIN self-administration.
∗α < 0.05, ∗∗α < 0.01, and ∗∗∗α < 0.001, repeated measures ANOVA.
+101 and +168% with respect to the last (30th) day of WIN
self-administration training. Following 1-week extinction, OBX
and SHAM rats reduced their active responding of −41 and
−65%, respectively, with respect to day 1 extinction. Differences
between OBX and SHAM groups in the responding on the active
lever slightly reduced after 2 and 3 weeks of extinction training
(OBX: −65 and −79%, SHAM: −78 and −90%, respectively).
Finally, response latency (deﬁned as time elapsed from commencement
of the experimental session until the ﬁrst active lever
press) was signiﬁcantly different between the two groups. More
speciﬁcally, response latency was shorter in OBX than SHAM
rats from day 9 onward (repeated measures ANOVA: α = 0.029
from day 9 to 16; α < 0.001 from day 17 to 30) (Figure 4A),
which suggests that after initial exposure to the CB1 receptor
agonist the bulbectomized rats may be more motivated than control
animals to obtain it. Moreover, analysis of temporal patterns
of responses revealed quantitative but not qualitative differences
between OBX and SHAM rats during self-administration training
since the response rate was typically slow and evenly distributed
throughout the 2 h test session in both groups (Figure 4B).
EFFECT OF ACUTE PRE-TREATMENT OF CGS-12066B ON DRUG
SELF-ADMINISTRATION
The effect of an acute administration of the serotonin 5-HT1B
receptor agonist CGS-12066B (CGS) was tested only after acquiring
reliable WIN self-administration, i.e., once rats stabilized
daily drug intake. Overall, we did not ﬁnd changes in WIN selfadministration
after acute pre-treatment with the CGS in Lister
Hooded OBX or SHAM rats. Figure 5 illustrates the percentage
changes of active lever presses from the baseline after an acute
challenge with CGS (2.5, 5, and 10 mg/kg) and saline control
as compared to previous 6-day mean responding (i.e., baseline).
The repeated measures ANOVA with OBX/SHAM group
as cofactor did not detect a signiﬁcant effect of drug treatment
within each group nor between groups. The post-hoc p-values
for each drug dose were as follows for SHAM: 2.5 mg/kg: p =
0.241; 5 mg/kg: p = 0.071; 10 mg/kg: p = 0.128, and for OBX:
2.5 mg/kg: p = 0.963; 5 mg/kg: p = 0.652; 10 mg/kg: p = 0.523.
It was reported that the effects of serotonergic drugs may
differ signiﬁcantly depending on the animal strain and the experimental
conditions used (Horowitz et al., 1997; Uphouse et al.,
2002; Miryala et al., 2013), and that not all rat strains do selfadminister
WIN spontaneously (Deiana et al., 2007). Moreover,
the CGS compound was found to reduce amphetamine (Fletcher
and Korth, 1999) and alcohol (Tomkins and O’Neill, 2000;
Czachowski, 2005), but not cocaine (Parsons et al., 1996), selfadministration.
Thus, we decided to test the CGS compound
on the self-administration of a pharmacologically different drug,
such as METH, which is known to be strongly self-administered
by OBX rats. We therefore tested CGS in OBX and SHAM Sprague
Dawley rats self-administering METH using different responselike
operandum, i.e., nose-poking instead of lever-pressing, and
a slightly higher schedule of reinforcement (FR-2). Figure 6A
illustrates responses of SHAM and OBX rats on the active hole
during the acquisition and maintenance phases of METH selfadministration.
In line with previous ﬁndings (Kucerova et al.,
2009, 2012), rats stabilized METH self-administration behavior
within 14 days of training intake with a mean daily drug intake of
1.8 mg in SHAM and 3 mg in OBX animals (METH intake data
shown in Supplementary Figure 2). Repeated measures ANOVA
revealed no signiﬁcant effects over the ﬁrst 6 days of training,
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Amchova et al. Enhanced WIN55,212-2 self-administration in depressive-like rats
FIGURE 4 | Latency to the ﬁrst active response (A) and patterns of
responding (B) in SHAM and OBX Lister Hooded rats during WIN
self-administration. (A) Latency (seconds) to the ﬁrst active lever presses
in SHAM (n = 6) and OBX (n = 7) rats. Data are shown as means (±s.e.m.).
∗α < 0.05 and ∗∗∗α < 0.001, repeated measures ANOVA. (B) Quantitative,
but not qualitative, differences in the active responding patterns between
OBX and SHAM rats. Individual representative records illustrating
responding patterns of OBX (ﬁrst three patterns) and SHAM (last three
patterns) rats on the active lever on the last day of the self-administration
training (day 30). Each tick denotes the time of every response on the
active lever. Cumulative numbers of active responses made over the 2 h
test sessions are illustrated on the right side of each pattern.
whereas from day 7 onward a signiﬁcantly higher active nosepoking
rate was observed in OBX compared with SHAM rats
(repeated measures ANOVA: α < 0.05). However, while during
WIN self-administration the numbers of inactive lever-presses
was constantly below 5 during the maintenance phase, inactive
nose-pokes during METH self-administration were higher in
both OBX and SHAM rats (see Supplementary Figure 1B), an
effect likely due to the activational motor effects of METH. Yet,
the mean number of active nose-pokes was substantially higher
than the inactive ones, which supports the speciﬁcity of animal
responding for METH (preference of the active operandum during
the maintenance phase was higher than 70% in all animals).
Figure 6B reports the percentages of active nose pokes for METH
after acute pre-treatment with saline control, 10 and 15 mg/kg of
CGS-12066B compared to previous 6-day mean responding (i.e.,
baseline) in SHAM and OBX Sprague Dawley rats. The repeated
measures ANOVA with OBX/SHAM group as cofactor did not
detect a signiﬁcant effect of drug treatment within each group nor
between groups. The post-hoc p-values for each drug dose were
as follows for SHAM: 10 mg/kg: p = 0.508; 15 mg/kg: p = 0.550,
and for OBX: 10 mg/kg: p = 0.232; 15 mg/kg: p = 0.319. These
ﬁndings indicate that, as for WIN self-administration, acute pretreatment
with the 5-HT1B receptor agonist did not modify the
voluntary intake of METH.
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Amchova et al. Enhanced WIN55,212-2 self-administration in depressive-like rats
FIGURE 5 | Acute pre-treatment with CGS does not affect WIN
self-administration. Effect of acute pre-treatment with CGS-12066B on
WIN self-administration in SHAM (n = 6) and OBX (n = 7) Lister Hooded
rats. Data are expressed as percentage changes of active lever pressing
compared to 6-day baseline (assumed as 100%). The repeated measures
ANOVA did not detect a signiﬁcant effect of drug treatment.
IN VIVO MICRODIALYSIS OF DOPAMINE LEVEL IN THE NUCLEUS
ACCUMBENS SHELL OF LISTER HOODED RATS
Figure 7 shows results from microdialysis experiment aimed
at measuring the release of DA in the NAc shell of Lister
Hooded SHAM and OBX rats following an intravenous injection
of 0.3 mg/kg of WIN, a dose mimicking the mean amount
of drug typically self-administered by naive Lister Hooded rats
(Deiana et al., 2007; Fattore et al., 2007; Spano et al., 2010),
and known to increase DA level in the rat NAc shell (Tanda
et al., 1997). During the pre-treatment period, basal extracellular
values of DA in the NAc shell did not differ signiﬁcantly
between the two groups (Figure 7A). As shown in Figure 7B,
after WIN administration, we found a signiﬁcantly increased
(about +40%) extracellular DA level in SHAM rats compared
to their basal level during the ﬁrst 40 min after drug injection
[One-Way ANOVA F(8, 24) = 4.997, p = 0.0010]. However,
the WIN challenge did not increase DA levels in OBX rats, in
which DA levels did not signiﬁcantly differ from to their previous
baseline during the 2-h measurement [One-Way ANOVA
F(8, 24) = 0.3730, p = n.s.]. Data are expressed as mean ± s.e.m.
percentage variation of basal levels. Two-Way ANOVA revealed
a signiﬁcant effect of treatment × time interaction [F(8, 48) =
3.07, p = 0.0071; ∗∗p < 0.01 and ∗p < 0.05, Bonferroni post-
test].
DISCUSSION
Findings of the present study demonstrated that bulbectomized
rats: (i) do self-administer higher amount of the cannabinoid
CB1 receptor agonist WIN55,212-2 than SHAM control
rats, (ii) do not alter voluntary intake of the CB1
receptor agonist after acute pre-treatment with the serotonergic
5-HT1B receptor agonist CGS12066B, and (iii) do
not increase DA level in the NAc shell in response to an
acute challenge with a dose of WIN (0.3 mg/kg), as SHAM
rats do.
WIN SELF-ADMINISTRATION IN OBX AND SHAM RATS
Bulbectomized rats have been previously reported to
self-administer more nicotine (Vieyra-Reyes et al., 2008),
amphetamine (Holmes et al., 2002), and METH (Kucerova et al.,
2012) than SHAM control rats. Yet, despite clinical evidence
for a signiﬁcant association between smoking cannabis and
major depression (Horwood et al., 2012; Lev-Ran et al., 2013),
cannabimimetic drug-taking behavior was never investigated
in an animal model of depression. Cannabinoid CB1 receptor
agonists were shown to be readily self-administered by mice,
rats, and monkeys (Martellotta et al., 1998; Fattore et al., 2001;
Justinova et al., 2003). Notably, rate of responses was critically
dependent on a variety of experimental conditions including
drug unitary dose (Martellotta et al., 1998), food restriction
regimen (Fattore et al., 2001), and type of operandum (Deiana
et al., 2007). In this study, we adopted all parameters and
experimental conditions that support a robust cannabinoid
drug-taking behavior in the Lister Hooded rat strain (Deiana
et al., 2007).
Before starting self-administration training and microdialysis
experiments, we veriﬁed the development of a depressive-like
phenotype in OBX lesioned animals by assessing the presence of
anhedonia and hyperactive locomotor response to novel environment,
which are two of the major hallmarks of this animal model
of depression (Kelly et al., 1997; Song and Leonard, 2005; Romeas
et al., 2009).
WIN self-administration by OBX rats signiﬁcantly differed
from SHAM controls as OBX animals showed higher rates of
drug-associated operant responses during the maintenance phase,
i.e., after initial acquisition. Indeed, although both OBX and
SHAM rats needed a similar number of training sessions to
acquire self-administration behavior, rates of active lever-pressing
during the maintenance sessions were remarkably higher in OBX
than SHAM rats. Therefore, the amount of WIN consumed by
OBX rats over the 30 test sessions resulted signiﬁcantly higher
than in SHAM rats (mean cumulative amount of WIN over the
30-day training: 12.71 vs. 7.9 mg for OBX and SHAM, respectively).
Similar rates of acquisition indicate that OBX rats required
the same time of SHAM rats to stabilize their drug intake and
suggest that development of a depressive-like phenotype is not
associated with learning or memory deﬁcits able to affect the
acquisition of the operant task, although OBX animals have been
reported to display impaired spatial learning (Song and Leonard,
2005). On the other hand, higher drug intake in OBX rats during
the maintenance phase suggests that bulbectomized animals are
differently responsive than SHAM controls to the cannabinoid,
which might lend some support to the proposed self-medication
theory of smoking cannabis to alleviate symptoms of depression
(Gruber et al., 1996; Ogborne et al., 2000).
Our results are in line with the notion that OBX rats differ
in the behavioral responses to acute and repeated exposure to
other addictive drugs including METH (Kucerova et al., 2012),
alcohol (Chiang et al., 2008), nicotine (Vieyra-Reyes et al., 2008),
cocaine (Calcagnetti et al., 1996; Chambers et al., 2004), and
amphetamine (Holmes et al., 2002). Notably, enhanced WIN
intake by OBX rats recorded in this study was unlikely due to
changes in locomotor activity since motor activity during the
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Amchova et al. Enhanced WIN55,212-2 self-administration in depressive-like rats
FIGURE 6 | Acute pre-treatment with CGS does not affect METH
self-administration. (A) METH self-administration in OBX and SHAM rats.
Data expressed as daily means (±s.e.m.) of active nose-pokes in SHAM (n = 7)
and OBX (n = 7) Sprague Dawley rats during methamphetamine
self-administration training. Signiﬁcant difference was recorded from the day 7
onwards, ∗α < 0.05 (day 7–12: ∗α = 0.041, day 13–18: ∗α = 0.027, repeated
measures ANOVA). (B) Effect of acute pre-treatment with CGS-12066B on
METH self-administration. Data are expressed as percentage changes of active
lever pressing compared to six-day baseline (assumed as 100%). The repeated
measures ANOVA did not detect a signiﬁcant effect of drug treatment.
daily training session was not dissimilar between OBX and SHAM
animals as conﬁrmed by their similar numbers of photocell beams
breaks measured within the operant boxes during daily training
sessions.
Differences in the response rates on the active lever were also
observed when vehicle was substituted for the CB1 receptor agonist.
In fact, rates of responses in OBX rats were consistently
higher than in SHAM rats not only when WIN was contingently
available but also when it was absent, as during extinction
training. A neurobiological mechanism that may contribute to
the resilience of OBX rats to extinguish not-rewarded operant
responses is a dysfunction of the front-cortical neuronal circuits
critically involved in the inhibition of on-going activity upon
withdrawal of the reinforcers (Jentsch and Taylor, 1999). This
hypothesis is corroborated by the ﬁnding that OBX animals (i)
are unable to adapt to environmental changes and show hyperemotional
responses (Van Riezen and Leonard, 1990), (ii) exhibit
impulsive-like traits (Kamei et al., 2007), and (iii) display significant
increases in both CB1 receptor density and functionality in
the prefrontal cortex (Rodriguez-Gaztelumendi et al., 2009).
EFFECT OF 5-HT1B RECEPTOR ACUTE STIMULATION ON DRUG
SELF-ADMINISTRATION
In an attempt to evaluate possible mechanisms underlying the
observed differences in WIN self-administration between OBX
and SHAM rats, we tested the effect of a serotonin 5-HT1B
receptor agonist on the cannabinoid agonist intake. This choice
was based on the ﬁnding that cortical and hippocampal 5-HT1B
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Amchova et al. Enhanced WIN55,212-2 self-administration in depressive-like rats
FIGURE 7 | OBX rats do not show enhanced dopamine level following
acute WIN challenge. (A) Basal extracellular levels (fmol/μl of dialysate,
mean ±s.e.m.) of DA in the NAc shell of SHAM (n = 4) and OBX (n = 4)
Lister Hooded rats. No signiﬁcant difference was found between the two
groups (One-Way ANOVA, p = 0.45). (B) Effect of an intravenous
administration of WIN 0.3 mg/kg on DA release in the nucleus accumbens
shell of SHAM (n = 4) and OBX (n = 4) Lister Hooded rats. ∗p < 0.05 and
∗p < 0.01, Two-Way ANOVA followed by Bonferroni’s post-hoc test.
receptors are critically involved in ethanol dependence and that
their activation in limbic areas may attenuate amphetamine
self-administration (Miszkiel et al., 2012). Moreover, a hypofunctionality
of 5-HT1B receptors was observed in depressed
patients (Murrough et al., 2011), and a polymorphism at the 5HT1B
receptor gene was found to be associated with alcoholism
(Lappalainen et al., 1998). The 5-HT1B receptor agonist CGS-
12066B was shown to selectively decrease operant responses for
ethanol (Czachowski, 2005). This compound is a full agonist with
high selectivity to the 5-HT1B receptor (Neale et al., 1987) and,
to minor extend, to the 5-HT1A receptors. The range of doses
of the 5-HT1B receptor agonist CGS-12066B used in this study
was shown to be effective in acute in altering aggressive (De Boer
and Koolhaas, 2005) and sexual behavior (Maciag et al., 2006) as
well as some reward-related behaviors, such as DA-mediated reinforcement
(Parsons et al., 1996). However, it did not affect cocaine
self-administration in rats (Parsons et al., 1996), similarly to our
ﬁndings on WIN self-administration.
Importantly, both the effects of serotonergic drugs (Horowitz
et al., 1997; Uphouse et al., 2002; Miryala et al., 2013) and
WIN self-administration behavior (Deiana et al., 2007) have
been reported to greatly vary depending on rat strain and/or
experimental parameters and procedures adopted. Moreover, the
CGS compound was found to affect drug self-administration
selectively, as it decreases alcohol (Grant et al., 1997; Maurel
et al., 1999; Tomkins and O’Neill, 2000; Czachowski, 2005) and
d-amphetamine (Fletcher and Korth, 1999), but not cocaine
(Parsons et al., 1996) intake. Thus, we decided to test CGS-
12066B on the self-administration of METH for which OBX rats
are known to display higher responding level than SHAM rats
(Kucerova et al., 2012), as for WIN. Yet, acute pre-treatment with
the 5-HT1B receptor agonist CGS-12066B did not signiﬁcantly
alter the intake of METH neither in OBX and SHAM Sprague
Dawley rats nor in intact Wistar rats as recorded in our earlier
unpublished pilot experiment (data available as Supplementary
Figure 3).
Thus, the present ﬁndings indicate that WIN and METH, like
cocaine (Parsons et al., 1996) self-administration, are not affected
by acute stimulation of the 5-HT1B receptor. Serotonin 5-HT1B
receptors are expressed throughout the brain of rodents. They
are located in the axon terminals of both 5-HTergic and non-5HTergic
neurons where they act as inhibitory autoreceptors or
heteroreceptors, respectively, (Barnes and Sharp, 1999; Moret and
Briley, 2000; Pytliak et al., 2011; Cai et al., 2013), and have been
difﬁcult to study because of the diversity of their localization and
the absence of highly selective receptor antagonists. Findings from
the present study do not allow excluding the possibility that a
chronic rather than an acute stimulation of 5-HT1B receptors
might alter WIN and METH self-administration. Thus, future
studies will evaluate the effects of chronic stimulation of 5-HT1B
receptor by CGS-12066B, administered systemically or locally, on
WIN and METH self-administrations.
REDUCED SENSITIVITY OF OBX RATS TO THE WIN STIMULATION
EFFECT ON DOPAMINE RELEASE IN THE NUCLEUS ACCUMBENS SHELL
Enhanced drug self-administration can be linked to a dysfunction
in the reward system, which is very likely to occur in OBX
animals given the chemical and molecular changes that bulbectomy
induces in several neurotransmitter systems, including the
dopaminergic one (Masini et al., 2004; Sato et al., 2010), a major
component of the brain reward system.
In intact animals, acute administration of WIN55,212-2 is
known to increase extracellular DA levels in the NAc of freely
moving rats (Gardner and Lowinson, 1991; Cheer et al., 2004;
Polissidis et al., 2013). Moreover, DA content in the rat NAc shell
was shown to increase appreciably in respect to basal values during
WIN self-administration (Fadda et al., 2006). According to
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Amchova et al. Enhanced WIN55,212-2 self-administration in depressive-like rats
this, the SHAM rats in this study signiﬁcantly increased accumbal
DA levels in response to an intravenous administration of a dose
of WIN, 0.3 mg/kg, very similar to that daily self-administered by
rats. Notably, the same dose of WIN enhances DA levels in the
NAc shell of drug-naïve Sprague Dawley rats (Tanda et al., 1997).
However, in our experiment WIN did not enhance DA levels in
bulbectomized rats.
To explain this ﬁnding it could be of help to consider the
multiple dysregulations that OBX induce in the endocannabinoid
brain system. Cannabinoid CB1 receptor density is signiﬁcantly
increased in OBX rats in the medial prefrontal cortex (mPFC) and
amygdala while it does not change in the caudate-putamen, hippocampus,
and dorsal raphe nucleus (Rodriguez-Gaztelumendi
et al., 2009). CB1 receptor function is signiﬁcantly enhanced in
the mPFC of OBX animals with respect to SHAM controls, but
not in other brain regions with the exception of a slight, not
signiﬁcant, increase in the amygdala (Rodriguez-Gaztelumendi
et al., 2009). Moreover, OBX does not affect DA D1- and D2like
receptors in the NAc (Sato et al., 2010). Thus, potential
changes in the number or function of either the CB1 or the
dopaminergic receptors following OBX are unlikely to account
for the absence of WIN-induced effect on DA release in the
NAc of OBX rats. On the other hand, there is no clear evidence
that DA release in the reward pathway depends on DA receptors.
Instead, it is known that midbrain DA neurons produce
endocannabinoids which retrogradely inﬂuence the glutamate
and GABA projections and thus regulate the inhibitory and
excitatory inputs to the reward circuit (Melis and Pistis, 2007).
Glutamatergic and GABAergic systems are both dysregulated in
the OBX model leading, among others, to a hyperactive response
to novel environment (Song and Leonard, 2005). These dysregulations
may contribute to the differential reactivity of the
mesolimbic dopaminergic system in OBX rats. Thus, future studies
will be performed to assess whether chronic treatment with
this dose of WIN (0.3 mg/kg) as well as acute challenges with
higher WIN doses may elicit an increase in DA levels in the
NAc shell of OBX rats, and to evaluate CB1 and DA receptor
densities.
To summarize, this study demonstrated that OBX rats selfadminister
more cannabinoid agonist than SHAM control rats,
and that WIN taking behavior is not signiﬁcantly affected by
acute stimulation of 5-HT1B receptors. The close anatomical and
functional association between the olfactory bulbs and the limbic
system may help to understand why OBX rats differ from
SHAM rats in drug self-administration behavior. The neurons of
the olfactory bulbs are widely interconnected with other brain
regions including cortical areas and limbic nuclei (Song and
Leonard, 2005). The projections to these nuclei may be particularly
relevant to changes in emotional and reward-related
behavior in bulbectomized rats. Removal of the olfactory bulbs
may alter, if not disrupt, the activity in brain circuits, particularly
those inﬂuencing the dopaminergic system which is critical for
processing drug taking and seeking behaviors. As OBX rats, contrary
to SHAM, did not display a signiﬁcant increase of DA levels
in the NAc shell after an acute WIN challenge, we hypothesize
that a depressive-like state may alter the rewarding effects of the
drugs.
In conclusion, our ﬁndings showed that OBX markedly affects
self-administration of cannabinoid CB1 receptor agonist, possibly
through a reduction of its rewarding effects to which animals
compensate by increasing WIN intake. A decreased DA
neurotransmission in the NAc shell might contribute to this
compensatory behavior. Thus, a follow-up study will evaluate (i)
a dose-response effect of acute and chronic WIN and METH
administration on NAc shell DA levels in OBX and SHAM rats,
and (ii) DA levels after immediate (24 h), short-term (1 and 2
weeks), and long-term (4-weeks) cessation from chronic drug
exposure. Future studies will also evaluate whether OBX and
SHAM rats also differ in the reinstatement of cannabinoidseeking
behavior trigger by drug, cue, or stress primings.
AUTHOR CONTRIBUTIONS
Petra Amchova was responsible for the induction of OBX model
in the Czech Republic and its transfer to the Italian laboratory;
she collected the data in the Czech Republic and processed them
for analysis, and wrote a substantial part of the introduction and
methods sections of the manuscript. Jana Kucerova developed
the original idea and organized the experimental work in the
Czech Republic, and wrote a substantial part of the introduction,
methods, results and discussion sections of the manuscript.
Valentina Giugliano was responsible for the behavioral testing
and performance of the OBX surgery in Italy and collection
of data, and she cross-checked the materials and methods section
of the manuscript. Zuzana Babinska was responsible for
the behavioral testing in the Czech Republic and contributed
to microdialysis experiments in Italy, and she cross-checked the
materials and methods section of the manuscript. Mary Tresa
Zanda contributed substantially to behavioral testing and OBX
surgery in Italy and collection of data, and she cross-checked the
materials and methods section of the manuscript. Maria Scherma
was responsible for the microdialysis experiment in Italy and for
the related statistical data analysis and graphical presentation of
data. Ladislav Dusek performed the statistical data analysis and
contributed substantially to the results section and graphical presentation
of the results. Paola Fadda organized and supervised the
microdialysis experiment in Italy and was involved in the analysis
and discussion of the data, and contributed to the ﬁnal version
of the manuscript. Vincenzo Micale established the collaboration
of the two departments, was involved in discussion of the data
and contributed to the ﬁnal version of the manuscript. Alexandra
Sulcova was involved in the design of the study and discussion of
the data, and contributed to the ﬁnal version of the manuscript.
Walter Fratta was involved in the analysis and discussion of the
data, and contributed to the ﬁnal version of the manuscript. Liana
Fattore organized and supervised the experimental work in Italy
and enabled the transfer of the OBX technique; she wrote a substantial
part of the introduction, methods, results and discussion
sections of the manuscript.
ACKNOWLEDGMENTS
This work was supported by the project “CEITEC—Central
European Institute of Technology” (CZ.1.05/1.1.00/02.0068)
from European Regional Development Fund and the Internal
project of the Faculty of Medicine at Masaryk University:
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Amchova et al. Enhanced WIN55,212-2 self-administration in depressive-like rats
MUNI/11/InGA09/2012. The authors are grateful to Tony Fong
(Toronto, Canada) for kind help with manuscript preparation
and proof reading, to Barbara and Marta Tuveri (Cagliari,
Italy), Marcela Kucirkova, and Petra Kamenikova (Brno, Czech
Republic) for animal care.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online
at: http://journal.frontiersin.org/journal/10.3389/fphar.2014.
00044/abstract
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Conﬂict of Interest Statement: The authors declare that the research was conducted
in the absence of any commercial or ﬁnancial relationships that could be
construed as a potential conﬂict of interest.
Received: 19 November 2013; paper pending published: 13 December 2013; accepted:
25 February 2014; published online: 20 March 2014.
Citation: Amchova P, Kucerova J, Giugliano V, Babinska Z, Zanda MT, Scherma M,
Dusek L, Fadda P, Micale V, Sulcova A, Fratta W and Fattore L (2014) Enhanced selfadministration
of the CB1 receptor agonist WIN55,212-2 in olfactory bulbectomized
rats: evaluation of possible serotonergic and dopaminergic underlying mechanisms.
Front. Pharmacol. 5:44. doi: 10.3389/fphar.2014.00044
This article was submitted to Neuropharmacology, a section of the journal Frontiers in
Pharmacology.
Copyright © 2014 Amchova, Kucerova, Giugliano, Babinska, Zanda, Scherma,
Dusek, Fadda, Micale, Sulcova, Fratta and Fattore. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). The
use, distribution or reproduction in other forums is permitted, provided the original
author(s) or licensor are credited and that the original publication in this journal
is cited, in accordance with accepted academic practice. No use, distribution or
reproduction is permitted which does not comply with these terms.
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70
SUPPLEMENTARY MATERIAL FOR PAPER:
Enhanced self-administration of the CB1 receptor agonist WIN55,212-2 in olfactory
bulbectomized rats: evaluation of possible serotonergic and dopaminergic underlying
mechanisms
Authors: Petra Amchova, Jana Kucerova, Valentina Giugliano, Zuzana Babinska, Mary
Tresa Zanda, Maria Scherma, Ladislav Dusek, Paola Fadda, Vincenzo Micale, Alexandra
Sulcova, Walter Fratta, Liana Fattore
Frontiers in Pharmacology
Category: Original research
Research Topic: Addictive drugs targeting GPCRs: new cross-talk mechanisms
SUPPLEMENTARY FIGURE LEGENDS:
Supplementary Figure 1. OBX and SHAM rats do not differ in inactive operandum
responding. A) Mean number of inactive lever presses over the acquisition and
maintenance period (30 days) in SHAM (n=7) and OBX (n=7) Lister Hooded rats during
WIN self-administration. Data are shown as means (±SEM). Not significant differences,
repeated measures ANOVA, α = 0.768. B) Mean number of inactive nosepokes over the
acquisition and maintenance period (18 days) in SHAM (n=7) and OBX (n=7) Sprague
Dawley rats during METH self-administration. Data are shown as means (±SEM). The
repeated measures ANOVA did not detect a significant effect of drug treatment.
Supplementary Figure 2. OBX rats display enhanced METH self-administration
behaviour. Mean number of infusions in SHAM (n=6) and OBX (n=7) Sprague Dawley
rats during METH self-administration. Each infusion contains 0.08 mg/kg METH. The
mean number of infusions during the whole self-administration training was 23 in SHAM
and 35.8 in OBX (approx. 1.8 and 3 mg/kg METH respectively). Data are shown as daily
means (±SEM) and start to differ significantly from day 7 onwards, * α < 0.05, repeated
measures ANOVA.
Supplementary Figure 3. Acute pre-treatment with CGS does not affect METH selfadministration
in intact Wistar rats. Effect of acute pre-treatment with CGS-12066B on
methamphetamine self-administration in intact Wistar rats (n=5). Data are expressed as
percent of active lever pressing compared to six-day baseline (assumed as 100%). The
repeated measures ANOVA did not detect a significant effect of drug treatment.
71
Supplementary Figure 1: OBX and SHAM rats do not differ in inactive operandum
responding.
72
Supplementary Figure 2: OBX rats display enhanced METH self-administration
behaviour.
73
Supplementary Figure 3: Acute pre-treatment with CGS does not affect METH selfadministration
in intact Wistar rats.
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2.4.5. Differential characteristics of ketamine self-administration in the
olfactory bulbectomy model of depression in rats
Ketamine is studied for its rapid antidepressant effect with promising results in both
preclinical experiments (Scheuing et al., 2015) and clinical studies have yield promising
results demonstrating the antidepressant potential of ketamine (Newport et al., 2015, Xu et
al., 2015). Therefore, this study assessed the characteristics of operant ketamine selfadministration
and relapse-like behaviour in the OBX model of depression following a
previously validated approaches (De Luca and Badiani, 2011, Caffino et al., 2016)coll. We
hypothesized increased ketamine taking behaviour in the OBX model and increased
relapse-like behaviour in the self-administration and reinstatement paradigms as in
analogous studies on methamphetamine (Babinska et al., 2016, Kucerova et al., 2012).
This study supports the validity of the animal model of dual disorder of depression and
drug abuse. Chronic ketamine intake reversed the depressive-like phenotype. In
accordance with previous studies, OBX animals showed increased operant intake of the
drug. However, ketamine-seeking behaviour in the model of relapse was lower in the OBX
animals compared to SHAM animals. This finding contradicts previous studies reporting
increased methamphetamine (Babinska et al., 2016) and cocaine (Frankowska et al., 2014)
seeking behaviour in the reinstatement paradigm. This indicates substantially different
underlying neuroadaptation changes between chronic ketamine vs. psychostimulant
exposure.
Babinska Z, Ruda-Kucerova J. Differential characteristics of ketamine addiction in the
olfactory bulbectomy model of depression in rats. Eur J Pharmacol, 2016, under review.
IF (2015) 2.730
Citations (WOS): N/A
75
76
Differential characteristics of ketamine self-administration in the olfactory
bulbectomy model of depression in rats
Authors: Zuzana Babinska, Jana Ruda-Kucerova*
Author’s Affiliation:
Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czech
Republic
*Corresponding Author:
Jana Ruda-Kucerova, Department of Pharmacology, Faculty of Medicine, Masaryk
University, Kamenice 5, 625 00 Brno, Czech Republic
E-mail: jkucer@med.muni.cz, Phone: +420 549 494 238, Fax: N/A
Short Title: Ketamine abuse in the OBX model
77
Abstract
Ketamine has been extensively studied for its antidepressant potentials with promising
results in both preclinical and clinical studies. The main concern to clinical ketamine uses
is its potential for abuse. Therefore, the aim of this study was to assess the characteristics
of operant intravenous (IV) ketamine self-administration and relapse-like behaviour in the
olfactory bulbectomy (OBX) model of depression.
Twenty-five male Wistar rats were divided randomly into two groups; in one group the
bilateral olfactory bulbectomy was performed while the other group was sham operated.
Intravenous self-administration procedure was conducted under a fixed ratio 1 schedule of
reinforcement, ketamine was available at 0.5 mg/kg/infusion. After stable drug intake was
established, rats underwent a 14-day period of forced abstinence. A drug-free
reinstatement session was then conducted in operant boxes. Forced swim test took place
before the self-administration protocol and on the first day of abstinence.
Consistent with previous studies with other substances, OBX animals showed increased
operant IV ketamine self-administration. In contrast ketamine-seeking behaviour in the
OBX group was no different from SHAM animals during the reinstatement session;
whereas, previous studies on other psychostimulants like methamphetamine and cocaine
reported increases. Our findings suggests substantially different underlying
neuroadaptations between chronic ketamine vs. psychostimulant exposure.
Keywords: ketamine; self-administration; reinstatement; olfactory bulbectomy;
depression; Wistar rats
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1. Introduction
Ketamine at sub-anaesthetic doses is now extensively studied for its antidepressant
potential in both preclinical experiments (Scheuing et al., 2015) and clinical studies with
promising results (Xu et al., 2015). However, potential clinical use of ketamine raises
questions of its abuse potential. Intake of the drug leads to psychological addiction but
probably not physical dependence (Bokor and Anderson, 2014). Notably, there is clear
evidence suggesting a higher ketamine abuse rate in patients with clinical depressive
symptoms (Fan et al., 2016) in accordance with the self-medication hypothesis (Hall and
Queener, 2007). Notably, increased availability of ketamine on the market may also lead to
increase in its abuse rate (Arunogiri et al., 2016). The fact that incidence of fatal poisoning
from ketamine dramatically increased in the recent years (Hamani and Nobrega, 2012)
demonstrates the need of prudence when using ketamine clinically.
In preclinical setting ketamine was shown to possess strong reinforcing potential in both
conditioned place preference and self-administration paradigms (Mutti et al., 2016; van der
Kam et al., 2009). A self-administration study in rats described a clear relationship
between environmental conditions and development of ketamine addiction (De Luca and
Badiani, 2011). This relation has also been confirmed in humans (De Luca et al., 2012). A
recent report aimed to distinguish the effect of one dose and chronic ketamine selfadministration
and reported differences in brain derived neurotrophic factor (BDNF)
levels, as a key variable distinguishing the antidepressant and reinforcing properties of the
drug. Acute ketamine dose increased BDNF in hippocampus while chronic operant intake
has an opposite effect (Caffino et al., 2016). This finding is in accordance with a clinical
study showing increased serum BDNF levels in ketamine abusers (Ricci et al., 2011).
Ketamine was also shown to dose-dependently increase dopamine levels in the nucleus
79
accumbens, which is a shared mechanism of all abused substances (Masuzawa et al.,
2003).
Currently, there is no study evaluating operant ketamine intake in animals with depressivelike
phenotype. Such study would benefit future development of new antidepressants with
glutamatergic mechanism of actions. For this study, we used the bilateral olfactory
bulbectomy model of depression. This model closely mimics neurochemical,
neuroanatomical, behavioural and endocrine changes in patients with major depression
(Song and Leonard, 2005). Our team has developed a rat model of depression and
addiction dual disorder where olfactory bulbectomized animals showed higher selfadministration
of methamphetamine (Kucerova et al., 2012), increased relapse-like
behaviour (Babinska et al., 2016) and differential dopamine and serotonin release in
nucleus accumbens shell after a methamphetamine challenge (Ruda-Kucerova et al.,
2015b). Similar findings were also reported earlier in self-administration of amphetamine
(Holmes et al., 2002) and of CB1 receptor agonist WIN55,212-2 (Amchova et al., 2014).
The overall aim of this study is to assess the characteristics of operant ketamine selfadministration
and relapse-like behaviour in the OBX model of depression. We
hypothesized increased ketamine taking behaviour in the OBX model and increased
relapse-like behaviour in the self-administration and reinstatement paradigms as in
analogous studies on methamphetamine (Babinska et al., 2016; Kucerova et al., 2012).
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2. Material and methods
2.1. Animals
Twenty five male albino Wistar rats (8 weeks old, with weight range of 250 to 300 g at the
beginning of the experiment) were purchased from the Masaryk University breeding
facility (Brno, Czech Republic). The rats were housed individually in standard rodent
plastic cages. Environmental conditions during the whole study were constant: relative
humidity 50-60 %, room temperature 23 ºC ± 1 ºC, inverted 12-hour light-dark cycle
(6 a.m. to 6 p.m. darkness). Food and water were available ad libitum. There were two
experimental groups: SHAM = sham operated rats (n=10 at the beginning of the study) and
OBX = olfactory bulbectomized rats (n=15 at the beginning of the study). The final
number of animals was n=9 in the sham operated group and n=6 in the OBX group. The
reasons for exclusion of the animals were as follows: death after surgery (n=3),
behavioural deficits (n=1), death during the study (n=2), incomplete OBX (n=4). All
procedures were performed in accordance with EU Directive no. 2010/63/EU and
approved by the Animal Care Committee of the Faculty of Medicine, Masaryk University,
Czech Republic and Czech Governmental Animal Care Committee, in compliance with
Czech Animal Protection Act No. 246/1992.
2.2. Drugs and treatments
Ketamine (KET) solution was prepared by diluting a ready-made preparation Calypsol®
inj. sol. (50 mg in 1 ml) with saline to obtain desired concentration. For IV selfadministration
a solution of 0.5 mg/kg per infusion was used. The solutions were prepared
for specific animals depending on their body weights rounded to the closest category of
250 g, 300 g, 350 g, etc. This paradigm is adapted from Emmett-Oglesby MW (Fort
Worth, USA) [31] and routinely used in our laboratory [26, 30, 32, 33]. For the locomotor
81
activity testing concentrations of the solutions ranged from 5 to 15 mg/kg and were
prepared in the same manner.
2.3. Olfactory bulbectomy surgery
At the beginning of the study the rats were randomly divided into two groups and the
bilateral ablation of the olfactory bulbs was performed in accordance with the standard
method as described earlier [27, 30]. In brief, animals were anaesthetized with ketamine 50
mg/kg and xylazine 8 mg/kg given intraperitoneally. The top of the skull was shaved,
swabbed with an antiseptic solution, after which a midline frontal incision was made
through the skin on the skull. After exposure of the skull, 2 burr holes were drilled at the
points 7 mm anterior to the bregma and 2 mm lateral to bregma suture. Both olfactory
bulbs were aspirated while paying particular attention not to damage the frontal cortex.
Prevention of blood loss was achieved by filling the dead space with a haemostatic sponge.
The skin above the lesion was closed with suture and the antibacterial neomycin and
bacitracin powder was applied. Sham operated rats underwent the identical anaesthetic and
drilling procedures as OBX animals, but their bulbs were left intact. A period of 14 days
was allowed for the recovery from the surgical procedure and the development of the
depressive-like phenotype. During this period, animals were handled daily for few minutes
to eliminate aggression, which could otherwise arise [25, 34]. At the end of the
experiment, rats were euthanized by an anaesthetic overdose and the brains were dissected
for confirmation of the successful removal of the olfactory bulbs. Animals with incomplete
removal of the olfactory bulbs were eliminated from the analysis (n=4).
2.4. Food self-administration protocol
Food self-administration was employed to develop self-administration operant behaviour
in the animals. The training was conducted as already described [35] in 10 operant boxes
82
(30x25x30 cm, Coulbourn Instruments, USA) using nose-poke operandi under a fixed ratio
1 (FR-1) schedule of reinforcement, i.e. animal had to make 1 nose-poke to the active
operandum to obtain a single palatable pellet (BioServ, sweet dustless rodent pellets,
F0021-Purified Casein Based Formula - 45mg). Each cage was provided with two nosepoke
holes allocated on one side and programmed by software Graphic State Notation 3.03
(Coulbourn Instruments, USA). The cage was illuminated by a house light during the
whole session. Self-administration sessions lasted 30 minutes during the dark period of the
inverted light-dark cycle. The length of the training was 5 days. All animals ate the vast
majority of the gained pellets.
2.5. Intravenous drug self-administration surgery
The IV self-administration catheter was implanted after completion of the food selfadministration
training following standard procedure described earlier [35, 36]. In brief,
animals were deeply anesthetized with IP injections of 50 mg/kg ketamine plus 8 mg/kg
xylazine. Catheter was inserted 3.7 cm into the right external jugular vein to the right
atrium and securely sutured. A subcutaneous tunnel was made and the catheter exited the
skin in the midscapular area. Since the implantation, the catheters were flushed daily by
enrofloxacine (17 mg/kg) solution followed by 0.1 ml of a heparinized (1%) sterile saline
solution to prevent infection and occlusion. When a catheter was found to be blocked or
damaged, the animal was excluded from the analysis.
2.6. Intravenous self-administration protocol
Ketamine self-administration was conducted as previously described [18, 37] in 10
standard experimental boxes (30x25x30 cm, Coulbourn Instruments, USA) using nosepoking
as operandum. Each cage was provided with two nose-poke holes allocated on one
side and programmed by software Graphic State Notation 3.03 (Coulbourn Instruments,
83
USA). Nose-pokes in the active hole led to the activation of the infusion pump and
administration of a single infusion followed by a 10 sec timeout, while nose-poke
stimulation was recorded but not rewarded, i.e. fixed ratio 1 (FR-1) schedule of
reinforcement. Infusions were delivered by a syringe within an automatic infusion pump
located outside the chamber. The infusion pumps were connected to liquid swivels which
were fixed to the catheters via polyethylene tubing withinside a metal spring tether. The
cage was illuminated by a house light during the session. The light was flashing when
infusion was being administered (5 sec) and off during the time-out period. Selfadministration
sessions lasted 120 minutes and took place 7 days/week between 8 a.m. and
3 p.m. during the dark period of the cycle.
After 21 days of daily ketamine intake at FR-1 the maintenance phase was terminated and
rats were kept in their home cages for the 14 days of the forced abstinence period. On the
day of reinstatement, rats were placed into self-administration chambers for the last session
taking 120 minutes and the numbers of responses on the active drug paired nose-poke and
the inactive nose-poke were recorded but the drug was not delivered. Responses on the
active nose-poke are considered to reflect reinstatement of drug seeking behaviour, while
responses on inactive nose-poke are interpreted to reflect general locomotor and
exploratory activity.
2.7. Forced swim test (FST)
A modified FST [38] was used to measure immobility of the rats, as described previously
[5, 39]. Briefly, the rats were individually placed into a plexi-glass cylinder filled with 30
cm of water (24±1 °C). The sessions were video-taped for later scoring and the water was
changed after every animal. A time-sampling scoring technique was used, whereby the
predominant behaviour, i.e. immobility, swimming or climbing, in each 5-s period of the 5
84
minutes test was recorded. OBX rats acquire the depressive-like phenotype by surgery,
therefore they should exhibit spontaneous immobility in the forced swim test. Furthermore,
the aim of the test was to assess spontaneous behaviour, i.e. not a drug effect. Hence, there
is no need to have a pre-test exposure to forced swimming the day before to induce
helplessness [5, 40]. The test was performed twice: in order to assess the basal depressivelike
phenotype the first test took place before the IVSA surgery, the second test aimed to
assess the potential change in the depressive-like profile after ketamine self-administration
– first day of forced abstinence period. There were in total four weeks between the tests.
2.8. Statistical Data analysis
Primary data were summarized using arithmetic mean and standard error of the mean
estimate (SEM). Food self-administration data were analysed by repeated measures (RM)
ANOVA model (factor: group, repeated variable: day) and Bonferroni post-test for the
group*time-point interaction. IV self-administration behaviour showed different dynamics
every week. Therefore, the data were summarized as weekly means for each animal and
then analysed by repeated measures ANOVA model (factor: group, repeated variable: day)
and Bonferroni post-test. Data from the reinstatement session and forced swim test were
analysed by t-test or Mann-Whitney U test (MWU test) depending on the result of
Kolmogorov-Smirnov test of normality. The analyses were calculated using Statistica 12
(StatSoft, USA). A value p<0.05 was recognized as boundary of statistical significance in
all applied tests.
85
3. Results
3.1. Food self-administration in SHAM and OBX rats
The acquisition of food taking behaviour (sweet pellets) was used in order to train the
operant behaviour. Figure 1 shows daily mean numbers of active nose-poking, inactive
nose-poking, number of pellets eaten and the mean day of reaching the acquisition
criterion. There was no difference in the active nose-poking (RM ANOVA, F=1.5888,
p=0.18806, n.s.) and pellet intake between the groups (RM ANOVA, F=1.3493, p=0.2615,
n.s.). However, inactive nose-poking reveals highly significantly increased activity in the
OBX group on the first two days (RM ANOVA, F=16.218, p=0.0000, Bonferroni posttest,
p<0.001). All rats acquired operant behaviour during 5 days of training; however, the
OBX group met the acquisition criteria (day when the animals started to prefer the active
nose-poke more than 75 %) significantly later: SHAM animals 1.2 day and OBX rats 2.2
days, T-test, p=0.006.
3.2. Ketamine IV self-administration in SHAM and OBX rats
Ketamine IV self-administration data are presented as the daily mean values of number of
active nose-pokes, inactive nose-pokes, infusions and dose of ketamine (mg/kg). Our
system uses nose-poke operandi, therefore, the number of nose-pokes and infusions does
not correspond (as in retractable lever systems). Furthermore, ketamine solution used for
self-administration was prepared for body weight categories with 50 g resolution. To
assess even this potential source of inaccuracy, we convert the number of infusions to drug
dose using the exact body weight. As visible on the Figure 2, the number of active and
inactive nose-pokes did not differ throughout the whole 3 weeks of the protocol (RM
ANOVA, effect of group: F=1.361, p=0.264 and F=0.015, p=0.904 respectively).
However, both number of infusions and ketamine dose showed development towards
86
higher drug intake in the OBX group as compared to SHAM controls. Infusions: RM
ANOVA, effect of group: F=11.188, p=0.005, effect of group*time-point interaction:
F=3.940, p=0.032. Bonferroni post-test for the group*time-point interaction: week 1, n.s.;
week 2, n.s.; week 3, p=0.009. Ketamine dose: RM ANOVA, effect of group: F=10.692,
p=0.006, effect of group*time-point interaction: F=4.068, p=0.029. Bonferroni post-test
for the group*time-point interaction: week 1, n.s.; week 2, p=0.822; week 3, p=0.0104.
Furthermore, the characteristics of the ketamine intake were different between the groups.
Figure 3 shows individual data on self-administered dose over 3 weeks of the study to
provide better overview of the findings.
3.3. Reinstatement of ketamine seeking behaviour
In the reinstatement session only, active and inactive nose-poking can be assessed (no drug
delivery). Figure 4 compares the mean number of both types of nose-pokes in this session
together with mean nose-poking in the last week of the maintenance phase in SHAM and
OBX rats. There was no difference between the performance in the reinstatement session
and last maintenance week in SHAM rats (MWU test, n.s.) but OBX rats exhibited lower
drug seeking behaviour in the reinstatement session that in the maintenance phase (T-test,
p=0.016). However, when the active responding in the reinstatement session was
converted to a percent of mean nose-poking in the week 3 (reinstatement / mean week 3 x
100) a robust difference was shown: SHAM did mean 252 % of nose-poking in the week 3
while OBX animals only 60 %. This difference does not reach full statistical significance
with 5% threshold (T-test, p=0.059) but the result may deserve some attention despite of
comparing reinforced and non-reinforced responding. In order to understand the finding
better we prepared the Table 1 showing individual data. Lastly, there was no difference in
87
the numbers of inactive nose-pokes neither within groups or in SHAM vs. OBX
comparison.
3.4. Forced swim test
The first test assessing the basal condition of the OBX and SHAM rats revealed significant
decrease of swimming behaviour and increase of immobility in the OBX group (t test,
p=0.0003, p=0.0386 respectively). Chronic ketamine self-administration lead to important
changes in the behaviour of the OBX animals, in the second FST OBX rats showed
increased climbing and decreased immobility scores as compared to SHAM controls (t
test, p=0.0155, p=0.050 respectively) suggesting alleviation of the depressive-like
condition (Figure 5).
88
4. Discussion
This study observed different characteristics of ketamine intake behaviour in the IV selfadministration
model between OBX rats and SHAM rats. These differences cannot be
explained by changes in natural reward seeking behaviour as active nose-poking during
training was similar between the groups. The higher inactive nose-poking in the OBX
group on the first two days of the training which led to a delay in meeting acquisition
criteria (i.e. 75% preference of active operandum) could be explained by hyperactivity of
the OBX group. Typical novelty induced hyperactivity is commonly found in OBX rats
during the initial phases of behavioural assessments, typically in open-field test (Song and
Leonard, 2005).
In accordance with our hypothesis, OBX rats exhibited a higher drug intake during the
maintenance phase of IV ketamine self-administration. Similar behaviour was reported
previously in different drugs of abuse such as amphetamine (Holmes et al., 2002),
methamphetamine (Kucerova et al., 2012) and CB1 agonist WIN55,212-2 (Amchova et
al., 2014) with a similar time-course, i.e. higher drug intake in the OBX rats is developed
approximately ten days after initiation of IVSA. This behaviour mimics the enhanced rate
of drug addiction in depressive humans, which is believed to be an attempt of selfmedication
(Hall and Queener, 2007; Khantzian, 1985).
In this study the depressive-like behaviour was demonstrated in the first (basal) forcedswim
test (FST) where OBX animals showed increased immobility scores. Consequently,
this phenotype was reversed after chronic ketamine exposure, i.e. decreased immobility
scores and increased climbing behaviour similarly as shown earlier (Fraga et al., 2014).
This is in accordance with clinical case reports of patients abusing ketamine to relieve their
depression (Bonnet, 2015; Liu et al., 2015). However, it seems that in preclinical
89
experiments even a single dose leads to protracted antidepressant-like effect and longer
exposure to the drug has a similar outcome (Browne and Lucki, 2013). So far, there is just
one report on acute ketamine in the OBX model showing positive results (Holubova et al.,
2016). Therefore, we cannot conclude that the antidepressant-like effect detected in this
study was exclusively owing to chronic ketamine exposure.
Interestingly, in this study OBX and SHAM rats exhibited quite different intake dynamics.
While approximately one half of the SHAM animals kept low intake, the other half
exhibited an escalation of ketamine intake in the second week of maintenance phase.
Majority of the OBX rats escalated their drug intake during first week and kept it for the
rest of the study. This may be a result of different neurochemical adaptations in the OBX
model to chronic ketamine exposure, which yet to be fully explained. The most probable
mechanism underlying ketamine effectiveness in reversing depressive (and depressivelike)
symptoms would be its ability to modulate glutamatergic signalling which
consequently influences synaptic plasticity (Vasquez et al., 2014).
Ketamine acts in the brain mainly by inhibition of NMDA (N-methyl-D-aspartate) and
activation of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors
while ketamine also stimulates glutamate synthesis (Browne and Lucki, 2013; Tizabi et al.,
2012). The overall result of these actions is an increase in glutamate signalling. Aside from
its actions on the glutamatergic system, ketamine acts to some extent also as a muscarinic
receptor antagonist, acetylcholinesterase inhibitor, dopamine D2 receptor partial agonist
and serotonin 5-HT2A receptor agonist, monoaminergic transporters’ inhibitor and opioid
receptor agonist (Browne and Lucki, 2013; Kapur and Seeman, 2002).
Based on clinical findings, glutamatergic dysregulation is considered to be one of the core
characteristics of major depression as suggested by the glutamate hypothesis of depression
90
(Sanacora et al., 2012). Numerous animal models of depression (surgical, behavioural and
genetic) exhibit impaired glutamate signalling in which glutamate enhancing treatments
rescued depressive phenotypes (Tokita et al., 2012). In the OBX model, the glutamatergic
system is generally considered to be hypoactive. Studies have shown decreased
extracellular glutamate levels in the olfactory cortex along with increased glutamate
decarboxylase activity. Lower NMDA receptor density was also found in several brain
regions, e.g. prefrontal and piriform cortexes, lateral amygdaloid nucleus and thalamic
nucleus (Harkin et al., 2003; Song and Leonard, 2005). However, the exact nature of
glutamatergic dysregulation seems to be much more complex as some studies showed
increased NMDA density in the prefrontal cortex (Webster et al., 2000) and amygdala
(Nakanishi et al., 1990). Moreover, our previous results showed increased extracellular
glutamate levels in the nucleus accumbens shell in OBX rats (Ruda-Kucerova et al.,
2015b). Nevertheless, novelty induced cortical glutamate release in the OBX model is
believed that to be responsible for some of the typical depressive phenotypes, especially
irritability, hyperactivity in unknown environment and maladaptation to stress (Ho et al.,
2000). This is in accordance with our previous results showing increased locomotor
activity corresponding to enhanced glutamate levels measured using in vivo microdialysis
in OBX rats (Ruda-Kucerova et al., 2015b).
At the same time, increased ketamine self-administration seen here in OBX rats may also
be explained by the effects of ketamine on the dopaminergic system. Chronic
administration of ketamine increases dopamine levels in many brain regions together with
decreased expression of D2 receptors (Li et al., 2015; Tan et al., 2012). Ketamine is also
known to act as a D2 partial agonist (Kapur and Seeman, 2002). These combined effects
lead to a moderate enhancement of dopaminergic tone resulting in a positive reinforcing
91
effect. Furthermore, the OBX model is potentially more susceptible to dopaminergic
drugs. Our previous study reported that OBX rats have decreased basal dopamine levels in
the nucleus accumbens shell (Ruda-Kucerova et al., 2015b) which may explain the higher
operant self-administration of addictive substances such as synthetic cannabimimetic drug
(Amchova et al., 2014) and methamphetamine (Kucerova et al., 2012).
The glutamate hypothesis of depression implies that impaired production of BDNF
following glutamatergic dysregulation leads to an abnormal neuroplasticity (Sanacora et
al., 2012). This notion has been supported by clinical studies reporting reduced serum
BDNF levels in patients with major depression, which can be normalized upon
antidepressant treatments (Teche et al., 2013). Preclinical research demonstrated that
ketamine increases BDNF levels in several brain regions (Duman et al., 2012). Clinical
trials have also shown that plasma BDNF levels increased upon both acute (Duncan et al.,
2013) and sub-chronic ketamine treatments (Haile et al., 2014) in patients with depression.
However, different ketamine abusers were shown to have increased (Ricci et al., 2011) as
well as decreased (Ke et al., 2014) BDNF levels. Preclinical evidence in OBX animals
shows robust decrease of BDNF levels in brain tissue (Rinwa et al., 2013), which is in
accordance with clinical findings. Rapid increase of BDNF is hypothesized as one of the
critical components of the antidepressant mechanism of ketamine (Haile et al., 2014).
Therefore, this mechanism could explain our results showing antidepressant effect upon
chronic ketamine self-administration. However, the exact mechanisms of ketamine
induced NMDA receptor blockade-mediated BDNF changes are yet to be elucidated.
During the drug-free reinstatement phase, there was no difference in the ketamine-seeking
behaviour between OBX and SHAM rats in terms of active and inactive nose-poking. This
confirms no effect of memory in the test, the preference of the active operandum remained
92
equal in both groups. OBX rats were less active during the reinstatement session compared
to their behaviour in the last week of maintenance phase while there was no such
difference in the SHAM rats. As previously suggested [35] we performed conversion of
the active nose-poking in the reinstatement session to a percent of mean nose-poking in the
week 3 and this attenuation of active operandum stimulation in the OBX group was more
evident. However, usually only operandum performance is evaluated, therefore we
summarize that there is apparently no difference between the groups. This unexpected
result is contradictory to previous findings acquired in a study of methamphetamine
relapse. In the same model, OBX rats were shown to increase methamphetamine-seeking
behaviour in the reinstatement session (Babinska et al., 2016).
In conclusion, this study supports the validity of the self-medication hypothesis explaining
the dual disorder of depression and drug abuse as shown by a reversal of the depressivelike
phenotype upon ketamine self-administration. In accordance with previous studies,
OBX animals show increased operant intake of the drug. However, ketamine-seeking
behaviour in the model of relapse was lower in the OBX animals compared to SHAM
animals. This finding contradicts previous studies reporting increased methamphetamine
(Babinska et al., 2016) and cocaine (Frankowska et al., 2014) seeking behaviour in the
reinstatement paradigm. More importantly, this indicates substantially different underlying
neuroadaptation changes between chronic ketamine vs. psychostimulant exposure.
93
5. Acknowledgements
This study was written at Masaryk university as part of the project „Experimental
pharmacological development in neuropsychopharmacology and oncology” number
MUNI/A/1284/2015 with the support of the Specific University Research Grant, as
provided by the Ministry of Education, Youth and Sports of the Czech Republic in the year
2016 and funds from the Faculty of Medicine MU to junior researcher Jana Ruda-
Kucerova.
The authors are grateful for support in behavioural testing and excellent animal care to
Jaroslav Nadenicek and for the proof-reading to Tony Fong (Toronto, ON).
94
6. Statement of interest
None to declare.
95
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102
Figures:
Figure 1: operant self-administration of sweet pellets
The line graphs present the mean ±SEM of daily numbers of active nose-pokes, selfadministered
pellets and inactive nose-pokes in SHAM and OBX animals. RM ANOVA
detected a significant effect of group only in number of inactive nose-pokes and
Bonferroni post-test identified significant differences between the groups on the first two
days (p<0.001***). This indicates the hyperactive response to novel environment typical
for the OBX model. Furthermore, T-test revealed significant difference in the mean day
when animals reached 75 % preference of the active operandum (p=0.006**).
103
Figure 2: maintenance of ketamine self-administration
All data are presented as the daily mean ±SEM values of number of active nose-pokes,
inactive nose-pokes, infusions and dose of ketamine (mg/kg). The number of active and
inactive nose-pokes did not differ throughout the whole 3 weeks of the protocol (RM
ANOVA, effect of group: n.s.). Number of infusion was found to be significantly
increased in the OBX group: RM ANOVA, Bonferroni post-test for the group*time-point
interaction: week 1, n.s.; week 2, n.s.; week 3, **p=0.009. Similarly, self-administered
ketamine dose was found to be higher: RM ANOVA, Bonferroni post-test for the
group*time-point interaction: week 1, n.s.; week 2, p=0.822; week 3, **p=0.0104.
104
Figure 3: individual data on self-administered ketamine dose
SHAM animals show either low intake in all sessions (n=5) or escalation of intake in the
second week (n=4). OBX rats show escalation of intake in the first week and then keep the
same trend till the end (only 2 animals show low intake, yet higher than SHAM). The
interrupted lines indicate excluded data (technical reasons).
105
Figure 4: reinstatement of ketamine seeking behaviour
The bar graphs indicate the mean ±SEM number of both types of nose-pokes in the
reinstatement session together with mean ±SEM nose-poking in the last week of the
maintenance phase in SHAM and OBX rats. There was no difference between the
performance in the reinstatement session and last maintenance week in SHAM rats (MWU
test, n.s.) but OBX rats exhibited lower drug seeking behaviour in the reinstatement
session that in the maintenance phase (T-test, *p=0.016). However, when the active
responding in the reinstatement session was converted to a percent of mean nose-poking in
the week 3 (reinstatement / mean week 3 x 100) a robust difference was shown: SHAM
did mean 252 % of nose-poking in the week 3 while OBX animals only 60 % (T-test,
p=0.059). There was no difference in the numbers of inactive nose-pokes neither within
groups nor in SHAM vs. OBX comparison (MWU test n.s., T-test n.s., respectively).
106
Figure 5: Forced swim test
All data are presented as the mean ±SEM values. The first – basal – test revealed
significant decrease of swimming behaviour and increase of immobility in the OBX group
(t test, ***p=0.0003, *p=0.0386 respectively). Chronic ketamine intake lead to important
changes in the behaviour of the OBX animals, in the second FST OBX rats showed
increased climbing and decreased immobility scores (t test, *p=0.0155, *p=0.050
respectively).
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Table 1: reinstatement individual data
The table provides a detailed overview of individual drug seeking behaviours (active nosepokes).
The column “week 3 min“ and “week 3 max“ show the minimum and maximum
number of active nose-pokes in the last week of maintenance phase. Column “week 3
mean” shows the weekly mean and “reinstatement” the number of active nose-pokes in the
reinstatement session. Last “%” column indicates the calculation: reinstatement / mean
week 3 x 100. As clearly visible, the high variability in the SHAM group attributes to only
two animals with very high responding in the last maintenance week (rendering low
percentage of the reinstatement activity). On the other hand the OBX group shows
relatively consistent % of responding with only one exception of 188 %.
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3. Schizophrenia and addiction comorbidity
3.1. Background
The “self-medication hypothesis” was developed to explain not only the comorbidity of
depression and drug abuse but also for other psychiatric disorders (Hall and Queener,
2007, Khantzian, 1985, Khantzian, 2013, Khantzian and Albanese, 2009). The clinical
evidence indicates, that the incidence of drug abuse is higher in the population with
psychiatric morbidity (Wedekind et al., 2010), including schizophrenia (Koskinen et al.,
2009, Mesholam-Gately et al., 2014). Almost 50 % of patients with schizophrenia suffer
comorbid addiction (Lybrand and Caroff, 2009) and this comorbidity is associated with
substantially higher burden of the disease (Schmidt et al., 2011, Hartz et al., 2014), higher
suicide attempt rate (McLean et al., 2011, Melle et al., 2010) and also non-adherence
(Wilk et al., 2006)to antipsychotic therapy. All drug classes were shown to be abused by
the patients with schizophrenia. The most common was nicotine (Wing et al., 2012,
Chambers et al., 2001, Mackowick et al., 2014) and alcohol (Krystal et al., 2006, Kalyoncu
et al., 2005, Kerner, 2015, Regier et al., 1990) but abuse of opiates (Kern et al., 2014),
amphetamines (Grant et al., 2012) and Cannabis drugs (McLoughlin et al., 2014) is
reported as well.
There is certain association between development of schizophrenia and drug addiction
(Volkow, 2009). A typical example is a risk of schizophrenia onset in young people
smoking Cannabis (Kucerova et al., 2014, Caspi et al., 2005, Hall and Degenhardt, 2015,
Semple et al., 2005). Methamphetamine induced psychosis is a well-known condition in
drug addicts (Yui et al., 2000, Gururajan et al., 2012).
Therefore, similarly as in the depression – addiction dual disorder, a common distortion of
neurobiological mechanisms underlying schizophrenia and substance abuse is expected.
The most obvious is the dopaminergic system, which is dysregulated in both psychiatric
disorders. It was already proposed as the main factor increasing the vulnerability of
patients with schizophrenia to drug abuse (Chambers et al., 2001). Conformable with the
self-medication hypothesis, patients with schizophrenia might relieve their negative and
cognitive symptoms (Mackowick et al., 2014, Ng et al., 2013).
109
There are only several preclinical studies examining drug abuse behaviours in
schizophrenia-like phenotype, mostly using different neurodevelopmental models (Micale
et al., 2013) This approach to model schizophrenia seems to be highly valid (Kucerova et
al., 2014, Micale et al., 2013). Most pre-clinical studies on this dual diagnosis used
neonatal ventral hippocampal lesion (NVHL) model of schizophrenia (Tseng et al., 2009).
Rats in this model were subjected to cocaine operant self-administration where they
responded more for the drug and needed more days to extinguish the drug-seeking
behaviour. This indicates higher motivation to obtain the drug and also showed higher
drug-induced (Chambers and Self, 2002) and cue-induced (Karlsson et al., 2013)
reinstatement. Similarly in methamphetamine self-administration study NVHL rats
achieved higher break-points in progressive ratio paradigm confirming higher motivation
while there was no difference in responding at fixed ratio for either the drug or food
(Brady et al., 2008). Alcohol drinking was repeatedly tested in the NVHL model as well
showing rather inconclusively increased vulnerability of the schizophrenia-like phenotype
(Berg et al., 2011, Jeanblanc et al., 2014).
Prenatal immune activation models have demonstrated certain validity as well. Prenatal
lipopolysaccharide exposure lead to increased alcohol intake in adulthood (Liu et al.,
2004). Rats in the poly I:C model were reported to show enhanced amphetamine induced
reinstatement of conditioned place preference (Richtand et al., 2012).
A recently developed neurodevelopmental model of schizophrenia induced by prenatal
treatment with DNA-alkylating mitotoxin methylazoxymethanol acetate (MAM) has been
(Lodge and Grace, 2009) seems to be suitable for studying schizophrenia-addiction
comorbidity. MAM-treated animals show higher behavioural response to amphetamine
challenge dose resembling sensitization to the drug (Lodge and Grace, 2012). However,
despite the proven aberrant dopaminergic functioning, the influence of the MAM
phenotype on addictive behaviour was not confirmed in a cocaine self-administration study
(Featherstone et al., 2009).
Our team has only recently developed and validated the MAM model. We have recorded a
complex behavioural profile indicating the schizophrenia-like phenotype is present in these
animals (Stark et al., 2015). We have performed two studies on drug taking behaviour in
this animal model: one with methamphetamine and another with alcohol drinking
110
paradigm. The findings from the experiment with methamphetamine are mostly in
accordance with the negative data previously shown with cocaine (Featherstone et al.,
2009). However, the alcohol drinking study showed some promising data (Ruda-Kucerova
et al., 2016).
3.2. Aims
The research on the animal model of the schizophrenia and addiction comorbidity aimed
to:
1. Establish the rat model of the dual disorder using prenatal methylazoxymethanol
treatment as a model of schizophrenia while methamphetamine abuse was
modelled by intravenous self-administration
2. Establish the rat model of the dual disorder using prenatal methylazoxymethanol
treatment as a model of schizophrenia while alcohol abuse was modelled by
drinking in the dark paradigm with sucrose fading procedure
Both studies were published together, Ruda-Kucerova et al., 2016.
111
3.3. Methods
3.3.1. Animals
Time mated female albino Sprague-Dawley rats were purchased from Charles River
(Germany) at gestational day 13 and housed individually. The rats were housed
individually in standard rodent plastic cages. Environmental conditions during the whole
study were constant: relative humidity 50-60 %, room temperature 23◦C ± 1◦C, inverted
12-hour light-dark cycle. Food and water were available ad libitum. All experiments were
conducted in accordance with all relevant laws and regulations of animal care and welfare.
The experimental protocol was approved by the Animal Care Committee of the Masaryk
University, Faculty of Medicine, Czech Republic, and carried out under the European
Community guidelines for the use of experimental animals.
3.3.2. Methylazoxymethanol (MAM) model of schizophrenia
Methylazoxymethanol acetate (MAM) was administered intraperitoneally to the dams on
gestational day (GD) 17, saline was used as vehicle (Lodge, 2013, Lodge and Grace,
2009). The average surviving litter size was n=9.6 in control and n=11.5 in MAM treated
mothers. The average proportion of male and female offspring was 52 % of males and
48 % of females. No cross-fostering was used, the mothers were regularly weighted and no
differences were observed between control and MAM treated mothers. The offspring were
weaned on the postnatal day (PND) 22 and housed in sections of 5 and later individually
during the drug addiction studies initiated at age of 9 weeks.
3.3.1. Alcohol drinking paradigm
Alcohol intake was assessed by using the drinking in dark paradigm with the sucrose
fading procedure (Samson, 1986, Czachowski, 2005). The drinking sessions lasted 90
minutes daily and started at 10 a.m. (3 hours after start of the dark period of the day). For
the session the water bottle was switched for another one containing the alcohol solution.
At the end of the daily session alcohol bottles were removed and standard water bottles
were returned to the home cage. All solutions were presented at room temperature. The
sucrose fading training phase was performed as follows: 10% alcohol and 5% sucrose (3
112
days), 15% alcohol and 5% sucrose (3 days), 20% alcohol and 5% sucrose (4 days), 20%
alcohol and 2% sucrose (3 days), 20% alcohol and 1% sucrose (4 days). The training lasted
17 days in total. From day 18 onward the animals were given under the same conditions
20% alcohol only. This phase of stable alcohol intake lasted 18 days (maintenance of the
alcohol drinking). In continuation the rats were subjected to 14 days of forced abstinence
when the alcohol solution was not available. After this period 20% alcohol was given
again at the same time for 5 more days to model relapse of the alcohol drinking behaviour
after abstinence. Rats were not food or water deprived throughout the study. Ethanol intake
was calculated as grams of ethanol per kg of body weight (animals were weighed daily).
3.3.2. IV self-administration (IVSA) surgery and procedures
The IV self-administration study was performed in the same manner as described in the
section 2.3.3.
3.3.3. Locomotor activity
Locomotor activity was assessed as described in the section 2.3.5.
3.3.4. Sucrose preference
Sucrose preference test was performed as described in the section 2.3.7.
113
3.4. Results
3.4.1. Reactivity to addictive drugs in the methylazoxymethanol (MAM)
model of schizophrenia in male and female rats
This study validated the rat model of the dual disorder of schizophrenia and addiction
using prenatal methylazoxymethanol treatment as a neurodevelopmental model of
schizophrenia (Lodge, 2013, Lodge and Grace, 2009) while methamphetamine abuse was
modelled by intravenous self-administration and alcohol abuse was modelled by drinking
in the dark paradigm with sucrose fading procedure (Czachowski, 2005, Samson et al.,
1988, Thiele and Navarro, 2014). Furthermore, both studies (alcohol and
methamphetamine) were designed to cover maintenance, forced abstinence and
reinstatement to the respective drug in order to provide data on all stages of drug addiction
modelling. Both male and female rats were used to address the potential sex dependent
differences.
The study suggests that the female sex and schizophrenia-like phenotype induced by the
prenatal MAM exposure may work synergistically to enhance alcohol consumption.
Different models of schizophrenia were used in alcohol studies which all have
demonstrated to have some merit in modelling escalation of alcohol consumption (Berg et
al., 2011, Jeanblanc et al., 2014). However, there was only a minor alteration of addictive
behaviours towards methamphetamine in the MAM animals. This is in accordance with a
methodologically similar study with cocaine (Featherstone et al., 2009).
At this stage, the NVHL model seems to be of more interest but the application of MAM
model to study this type of substance abuse remains understudied. Therefore, it is not
possible to conclude that the reward related processes in MAM model are intact.
Ruda-Kucerova J, Babinska Z, Amchova P, Stark T, Drago F, Sulcova A, Micale V.
Reactivity to addictive drugs in the methylazoxymethanol (MAM) model of schizophrenia
in male and female rats. World J Biol Psychiatry. 2016, doi:
10.1080/15622975.2016.1190032, in press.
IF (2015) 4.159
Citations (WOS): 0
ORIGINAL INVESTIGATION
Reactivity to addictive drugs in the methylazoxymethanol (MAM) model of
schizophrenia in male and female rats
Jana Ruda-Kucerovaa
, Zuzana Babinskaa
, Petra Amchovaa
, Tibor Starka
, Filippo Dragob
, Alexandra Sulcovac
and Vincenzo Micaleb,c
a
Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czech Republic; b
Department of Biomedical and
Biotechnological Sciences, Section of Pharmacology, School of Medicine, University of Catania, Catania, Italy; c
Behavioral and Social
Neuroscience Group, CEITEC – Central European Institute of Technology, Masaryk University, Brno, Czech Republic
ABSTRACT
Objectives: Patients with schizophrenia often suffer comorbid substance abuse regardless of gender.
However, the vast majority of studies are only conducted in male subjects. Therefore, the
aim of these experiments is to assess addictive behaviors of both sexes in a neurodevelopmental
model of schizophrenia induced by prenatal methylazoxymethanol (MAM) acetate exposure.
Methods: MAM (22 mg/kg) was administered intraperitoneally on gestational day 17. Two studies
were performed in the offspring: (1) an alcohol-drinking procedure to assess daily intake of 20%
alcohol and relapse-like behavior after a period of forced abstinence; (2) Methamphetamine
(METH) intravenous self administration (IVSA) followed by forced abstinence and reinstatement
phases.
Results: MAM exposure during the prenatal period did not change alcohol drinking regardless of
sex. However, MAM females showed higher alcohol consumption in comparison to MAM males.
The METH IVSA study revealed only a modest increase of drug consumption in MAM males, while
there was no difference between the female groups. Reinstatement data showed no effect of the
MAM model in either sex, but suggested increased responding in female rats.
Conclusions: This study suggests that female sex and schizophrenia-like phenotype may work
synergistically to enhance alcohol consumption. However, future research is needed to establish
paradigms in which these findings would be readily assessed to test anti-addiction treatments.
ARTICLE HISTORY
Received 10 December 2015
Revised 19 April 2016
Accepted 9 May 2016
KEYWORDS
Abuse; alcohol;
methamphetamine; sex
differences; schizophrenia
Introduction
Drug addiction is known to be more prevalent in
patients with psychiatric morbidity than in the general
population (Wedekind et al. 2010). This is particularly
well documented in affective disorders such as anxiety,
depression (Volkow 2004) and schizophrenia (SCZ;
Koskinen et al. 2009; Mesholam-Gately et al. 2014).
Almost 50% of SCZ patients suffer comorbid addiction
(Lybrand and Caroff 2009), which is linked with a substantially
higher burden of the disease as these
patients have a higher rate of hospitalizations, a
shorter life expectancy (Schmidt et al. 2011; Hartz et al.
2014) and a higher suicide attempt rate (Melle et al.
2010; McLean et al. 2011). The most common drug
addiction is nicotine, with a prevalence of 70–90% in
SCZ patients compared to 26% in the general population
(Chambers et al. 2001; Wing et al. 2012;
Mackowick et al. 2014). The high prevalence of other
substances use, such as alcohol (Regier et al. 1990;
Kalyoncu et al. 2005; Krystal et al. 2006; Kerner 2015),
opiates (Kern et al. 2014), amphetamine psychostimulants
(Grant et al. 2012) and cannabis (McLoughlin
et al. 2014) is also alarming. Recently, emerging clinical
studies have suggested differential abuse patterns in
men and women to different substances in a variety of
research paradigms (Johnson et al. 2010; Bahorik et al.
2013), indicating a growing research interest (Mendrek
2015).
Several lines of evidence support the association
between SCZ and addiction (Volkow 2009). There is a
known risk of triggering SCZ by cannabis use especially
during adolescence (Caspi et al. 2005; Semple et al.
2005; Kucerova et al. 2014; Hall and Degenhardt 2015),
and by psychostimulants, e.g., methamphetamine
(METH; Yui et al. 2000; Gururajan et al. 2012) and
cocaine (Malave and Broderick 2014). This suggests a
common distortion of neurobiological mechanisms
underlying both SCZ and substance abuse, namely the
dopaminergic (DAergic) system.
CONTACT Dr. Jana Ruda-Kucerova jkucer@med.muni.cz Masaryk University, Faculty of Medicine, Department of Pharmacology, Kamenice 5, 625 00
Brno, Czech Republic.
ß 2016 Informa UK Limited, trading as Taylor & Francis Group
THE WORLD JOURNAL OF BIOLOGICAL PSYCHIATRY, 2016
http://dx.doi.org/10.1080/15622975.2016.1190032
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DAergic abnormalities have been well described in
both SCZ and substance abuse. It is possible that the
DAergic dysfunction in SCZ patients disrupts normal
reward pathways predisposing individuals to higher
risks for drug abuses (Chambers et al. 2001).
Dopamine (DA) dysregulation in SCZ is complex.
Positive symptoms of SCZ are associated with
increased DA signaling from enhanced subcortical DA
release (mostly via D2 receptors). In contrast, negative
symptoms are believed to be due to decreased DA signaling
as a result of decreased D1 receptor activation
in the prefrontal cortex and alterations of the D3 signaling
(Brisch et al. 2014). Due to this decreased DA
signaling, SCZ patients rarely ever experience reward
feelings (Brisch et al. 2014; Mesholam-Gately et al.
2014). Substance abuse is thought of as a self attempt
by SCZ patients to relieve this symptom, and this
abuse is essentially a drug-induced release of DA in
the prefrontal cortex. In SCZ, it is probably to relieve
negative and cognitive symptoms. Similar notions
about substance abuse in psychiatric morbidity have
led to the proposal of the self-medication hypothesis
(Khantzian 1985). Currently, there is a discussion about
potential positive effects of nicotine in SCZ patients
(Mackowick et al. 2014), which is well supported by
pre-clinical data (Ng et al. 2013). This, however,
remains controversial due to the efficacy of nicotine
and in contrast the harmful effects of smoking (Hahn
et al. 2013). Also, this self-medication strategy could
potentially be secondary to DA-suppressing antipsychotic
treatment (Samaha 2014).
Despite the high prevalence of drug addiction and
SCZ comorbidity, there are few pre-clinical studies
examining drug-abuse behaviors in the SCZ-like
phenotype. A composite translational animal model
could provide a basis for investigation of the mechanisms
underlying the interaction between the two disorders.
Valid assessment of substance-abuse
characteristics in the SCZ-like phenotype requires
chronic animal models of SCZ, i.e., neurodevelopmental
models seem to be the most useful (Micale et al.
2013). Furthermore, operant approaches to model drug
abuse are considered the most relevant for the human
disorder (O’Connor et al. 2011).
Until recently, most pre-clinical studies on the dual
diagnosis used a neonatal ventral hippocampal lesion
(NVHL) model of SCZ (Tseng et al. 2009). Rat males in
this model were subjected to cocaine-operant selfadministration
testing and the animals were found to
respond more for the drug. The animals also took longer
to extinguish drug-seeking behaviors, indicating a
higher motivation to obtain the drug which can be
interpreted as higher drug-induced (Chambers and Self
2002) and cue-induced (Karlsson et al. 2013) reinstatement.
Similarly, in a METH self-administration study,
NVHL rats achieved higher break-points in a progressive
ratio paradigm confirming higher motivation,
while there was no difference in responding at a fixed
ratio for either the drug or food (Brady et al. 2008).
Alcohol drinking was repeatedly tested in the NVHL
model, showing rather inconclusively an increased vulnerability
in the SCZ-like phenotype (Berg et al. 2011;
Jeanblanc et al. 2014). Furthermore, prenatal immune
activation models have also demonstrated certain validity.
Prenatal lipopolysaccharide exposure led to
increased alcohol intake in adulthood (Liu et al. 2004).
Rats in the poly I:C model were reported to show
enhanced amphetamine-induced reinstatement of
conditioned place preference (Richtand et al. 2012).
DNA-alkylating mitotoxin methylazoxymethanol (MAM)
acetate prenatal treatment has been established as a
well validated neurodevelopmental model of SCZ
(Lodge and Grace 2009). This model seems suitable for
studying SCZ-addiction comorbidity given its chronic
nature (as opposed to acute pharmacological models)
as well as its high face and construct validity described
by the behavioral (i.e., augmented locomotor response
to amphetamine, social deficits and cognitive impairments)
and neurochemical (i.e., enhanced activity in
the mesolimbic DAergic system and decreased parvalbumin
interneuron density in medial prefrontal cortex
and hippocampus) changes (Micale et al. 2013).
Moreover, prenatally MAM-treated animals show a
higher behavioral response to an amphetamine challenge
dose resembling sensitization to the drug
(Lodge and Grace 2012). Interestingly, despite the proven
aberrant dopaminergic functioning, the influence
of this phenotype on addictive behavior has not been
identified in a cocaine self-administration study assessing
both fixed and progressive schedules of reinforcement,
extinction and drug-induced reinstatement
(Featherstone et al. 2009). However, this finding still
does not rule out differential reactivity to drugs of
abuse because only one psychostimulant drug was
tested in a single paradigm.
More importantly, in all these studies only male offspring
were used and the vast majority of both preclinical
and clinical studies are conducted on male
subjects only. Despite the fact that the absolute number
of women suffering SCZ is less than men, this issue
should be properly addressed because women also
suffer from comorbid substance abuse (Abel et al.
2010) and, unlike men, do not improve after hospitalization
intervention (Bahorik et al. 2013). The validity of
the MAM model in female offspring was already shown
in a previous study reporting similar behavioral
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alterations to MAM exposure regardless of gender
(Hazane et al. 2009).
Therefore, the aim of this study is to assess possible
differences in: (1) alcohol drinking, one of the most
commonly abused substances (Kalyoncu et al. 2005;
Krystal et al. 2006), and (2) operant METH intravenous
self administration (IVSA) between MAM-exposed and
control rats of both sexes. Furthermore, both studies
were designed to cover maintenance, forced abstinence
and reinstatement to the respective drug to provide
data on all stages of drug addiction modelling.
A forced abstinence model where the animal does not
have access to the operant box was used because it
mimics human behaviour. In rehabilitation centres,
patients usually discontinue drug use and protected
from exposure to drug-related environments. A preclinical
paradigm based on this approach is readily
used (Fuchs et al. 2006; Reichel and Bevins 2009;
Yahyavi-Firouz-Abadi and See 2009; Ruda-Kucerova
et al. 2015a). It provides perhaps a more translational
alternative to extinction procedures because it measures
drug-seeking behavior following a period of involuntary
drug withdrawal, when the motivation of
drug-response behavior is not influenced by any training
procedures. Furthermore, this paradigm can be
used in both alcohol drinking and METH IVSA studies
allowing a better comparison.
Material and methods
Animals
Time mated female albino Sprague-Dawley rats were
purchased from Charles River (Germany) at gestational
day (GD) 13 and housed individually. MAM acetate was
administered intraperitoneally on GD 17 to 37 rats,
while the vehicle was administered to 13 control rats.
The average surviving litter size was n ¼ 9.6 in control
and n ¼ 11.5 in MAM-treated mothers. Two litters were
lost (killed by the mother), one control and one MAM
treated. The average proportion of male to female
offspring was 52% male and 48% female. No crossfostering
was used, the mothers were regularly
weighed and no differences were observed between
control and MAM-treated mothers. The offspring were
weaned on postnatal day 22 and housed in sections of
five and later individually, during the drug addiction
studies, initiated at the age of 9 weeks. All females
were left with intact gonads to assess addictive behavior
in a population with natural estrous cycle (RudaKucerova
et al. 2015a). For the alcohol drinking study,
20 male (10 vehicle and 10 MAM treated) and 20
female (10 vehicle and 10 MAM treated) offspring
were used. For the METH IVSA study different groups
of 20 male (10 vehicle and 10 MAM treated) and 30
female (11 vehicle and 19 MAM treated) offspring were
used. The alcohol study was finished by all animals,
but the final numbers in the IVSA study were lower
due to surgery or catheter patency. At the end of the
study, there were n ¼ 9 male vehicle (M VEH), n ¼ 8
male MAM (M MAM), n ¼ 9 female vehicle (F VEH) and
n ¼ 16 female MAM (F MAM) included in the analysis.
All experimental groups were composed of offspring
from four to five different mothers. Environmental conditions
during the whole study were constant: relative
humidity 50–60%, temperature 23 ± 1 
C, inverted 12-h
light–dark cycle (from 07:00 to 19:00 h). Food and
water were available ad libitum throughout the study.
All experiments were conducted in accordance with all
relevant laws and regulations of animal care and welfare.
The experimental protocol was approved by the
Animal Care Committee of the Masaryk University
Faculty of Medicine, Czech Republic, and carried out
under the European Community guidelines for the use
of experimental animals.
Drugs and treatments
MAM acetate (Midwest Research Institute, Kansas City,
USA) was dissolved in saline and administered intraperitoneally
at dose 22 mg/kg in a volume of 1 ml/kg on
GD 17, as previously described (Moore et al. 2006).
Saline was administered to the control group as
vehicle.
Ethanol 96% was purchased from a local pharmacy
and dissolved using distilled water to the desired concentration
(from 10 to 20%, see alcohol-drinking
protocol).
METH (Sigma, St Louis, MO, USA), available in the
operant cage for IVSA was 0.08 mg/kg per infusion
with the maximum number of infusions obtainable in
one session set to 50 as previously described and validated
(Kucerova et al. 2009, 2012; Amchova et al.
2014; Ruda-Kucerova et al. 2015a).
Alcohol-drinking study
Sucrose preference test
A two-bottle choice procedure was used to determine
the sucrose intake at two timepoints during the alcohol
study. Test 1 was conducted to assess possible
anhedonia before alcohol training. Test 2 was performed
on the first day of alcohol abstinence to reveal
the possible development of alcohol-induced anhedonia
(Kalejaiye et al. 2013). During a 24-h training phase,
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each rat was provided with two water bottles in their
home cage in order to adapt the rats to drinking from
two bottles. After training, one bottle (counterbalanced
across rats) was randomly switched to contain 1%
sucrose solution. After 24-h, both bottles were
removed and the amount of liquid remaining in each
bottle was measured. The sucrose preference score
was calculated as the percentage of sucrose solution
ingested relative to the total amount of liquid
consumed.
Alcohol-drinking procedure
The drinking-in-the-dark paradigm was used along
with the sucrose-fading procedure for training and
adapted from published protocols (Samson et al. 1988;
Czachowski 2005). The drinking sessions lasted 90 min
daily and started at 10:00 h (3 h after the lights went
off). During this time the water bottle was switched for
another one containing the alcohol solution. At the
end of the daily session alcohol/sucrose bottles were
removed and standard water bottles were returned to
the home cage. The alcohol/sucrose solutions were
presented at room temperature. The sucrose-fading
training phase was organized as follows: 10% alcohol
and 5% sucrose (3 days), 15% alcohol and 5% sucrose
(3 days), 20% alcohol and 5% sucrose (4 days), 20%
alcohol and 2% sucrose (3 days), 20% alcohol and 1%
sucrose (4 days). The training lasted 17 days in total.
From day 18 onward the animals were given 20% alcohol
only under the same conditions. This phase of stable
alcohol intake lasted 18 days (maintenance of the
alcohol drinking). In continuation, the rats were subjected
to 14 days of forced abstinence when the alcohol
solution was not available. After this period 20%
alcohol was given again at the same time for a further
5 days to model the relapse of the alcohol-drinking
behavior after abstinence. Rats were not food or water
deprived throughout the study. Ethanol intake was calculated
as grams of ethanol per kg of body weight
(animals were weighed daily). Blood alcohol levels
were not assayed in the study as it was shown previously
that the alcohol levels do not differ in male and
female rats (Murawski and Stanton 2011).
METH IVSA study
Locomotor activity test
Before starting the METH IVSA study, the baseline
behavioural profile was assessed in all animals. In
brightly lit room, rats were individually tested for locomotor
activity using the Actitrack system (Panlab,
Spain) as previously described (Pistovcakova et al.
2008; Ruda-Kucerova et al. 2015a). Each Plexiglas arena
(45 Â 45 Â 30 cm) was surrounded by two frames
equipped with photocells located one above another
at 2 and 12 cm over the cage floor. Animals were
placed in the centre of arena and the spontaneous
behavior was tracked for 10 min. In the test, horizontal
locomotor activity (the trajectory calculated by the system
as beam interruptions that occurred in the horizontal
sensors) and vertical activity (number of rearing
episodes breaking the photocell beams of the upper
frame) were recorded. At the end of the session, animals
were returned to their home cage, and the arenas
were wiped with 1% acetic acid to avoid olfactory
cues. The test was carried out during 1 day (morning
hours) in all animals starting with males and continuing
with females. The animals were brought to the test
room individually.
Food self-administration protocol
Food self administration was employed to develop
self-administration operant behaviour in the animals.
The training was conducted as already described
(Ruda-Kucerova et al. 2015a) in 10 operant boxes
(30 Â 25 Â 30 cm, Coulbourn Instruments, USA) using
nose-poke operandi under a fixed ratio 1 (FR-1) schedule
of reinforcement, i.e., animal had to make one
nose-poke to the active operandum to obtain a single
palatable pellet (BioServ, sweet dustless rodent pellets,
F0021-Purified Casein Based Formula – 45 mg, sweet
taste attributed by 276 g/kg of monosaccharides and
310 g/kg of sucrose). Each cage was provided with two
nose-poke holes allocated on one side and programmed
by software Graphic State Notation 3.03
(Coulbourn Instruments). The cage was illuminated by
a house light during the whole session. Self-administration
sessions lasted 30 min during the dark period of
the inverted light–dark cycle 7 days/week, and at the
end the rats were returned to their home cages.
IVSA protocol
Animals were deeply anesthetized with an intraperitoneal
injection of 50 mg/kg ketamine plus 8 mg/kg xylazine.
Under aseptic conditions, a permanent
intracardiac silastic catheter was implanted through
the external jugular vein to the right atrium. The outer
part of the catheter exited the skin in the midscapular
area. After surgery, a 1-week recovery was allowed.
The catheters were flushed daily using enrofloxacine
(17 mg/kg) solution followed by 0.1 ml of a heparinized
(1%) sterile saline solution to prevent infection and
occlusion of the catheter. METH IVSA was conducted
as previously described (Kucerova et al. 2009; Kucerova
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et al. 2012; Amchova et al. 2014; Ruda-Kucerova et al.
2015a) in the same operant boxes (Coulbourn
Instruments) using nose-poke operandi under a FR-1.
Nose-poking in the active hole led to the activation of
the infusion pump and administration of an infusion
followed by a 10-s timeout, when nose-poking was
recorded but not rewarded. The cage was illuminated
by a house light during the session. The light was
flashing when the system was administering infusion
(5 s), and was off during the timeout period to provide
an environmental cue associated with METH infusion.
IVSA sessions lasted 90 min and took place 7 days/
week between 08:00 and 15:00 h during the dark
period of the inverted light–dark cycle, and at the end
rats were returned to their home cages. After 14 days
of METH intake the maintenance phase was terminated
and rats were kept in their home cages for the 14
days of the forced abstinence period. On day 15 of
abstinence, rats were placed into IVSA chambers for
the last 90-min reinstatement session which was, apart
from drug delivery, identical to the maintenance sessions.
The numbers of responses on the active drugpaired
nose-poke and the inactive nose-poke were
recorded but the drug was not delivered.
Statistical data analysis
Primary data were summarized using arithmetic mean
and standard error of the mean (SEM) estimates.
Sucrose preference and open-field data were analyzed
using two-way analysis of variance (ANOVA) (factors:
sex, MAM model, repeated factor: day) and Bonferroni
post-hoc test for multiple comparisons. For evaluation
of alcohol intake, food intake and maintenance variables
in the METH study, repeated measures ANOVA
with the same factors and Bonferroni post-hoc test
was employed. Two-way ANOVA (factors: sex, MAM
model) and Bonferroni post-hoc test were also used
for analysis of METH reinstatement and alcohol-relapse
data. The data on the percentage of alcohol intake
during the relapse phase were non-parametric
(Kolmogorov-Smirnov test of normality), therefore the
Kruskal-Wallis test was used. The analyses were calculated
using Statistica 12 (StatSoft, USA). A value
P < 0.05 was recognized as boundary of statistical significance
in all applied tests.
Results
Alcohol-drinking study
Sucrose preference test
As shown in Figure 1, all rats consumed the same proportion
of sucrose solution (approximately 80%) both
before the beginning of the alcohol study and during
abstinence. Two-way ANOVA (two factors: sex and
MAM model) did not reveal any significant differences
induced by sex, MAM model or their interaction
(F ¼ 1.144, F ¼ 0.363 and F ¼ 0.811, respectively).
Alcohol drinking
Figure 2 shows mean daily intake of pure (theoretical
100%) ethanol per kg of body weight in all groups during
both the maintenance phase and the relapse after
abstinence. Repeated measures ANOVA (two factors:
sex and MAM model) detected a significant effect of
sex–model interaction: F ¼ 2.931, P ¼ 0.0241, but not
sex or MAM model alone. Bonferroni post-hoc test
indicated that the F MAM group consumed significantly
more alcohol than the M MAM group in both
the maintenance and relapse phases of the study
(P values indicated in the graph by * symbols).
However, control animals (M VEH vs. F VEH) showed
Figure 1. The sucrose preference test. The test was conducted in male and female control and MAM animals. Data are percentages
(±SEM) of sucrose solution consumed in 24 h at two timepoints, i.e., Test 1 (A) was conducted before alcohol drinking training and
Test 2 (B) on the first day of forced abstinence from maintenance of 20% alcohol drinking. Two-way ANOVA (two factors: sex and
MAM model), not signficant.
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only few significant data points during the relapse
phase (P values indicated in the graph by # symbols).
Therefore, these data indicate a trend to
increased alcohol intake in the females, which is
strongly enhanced by the MAM model. For better
visualization this is also shown in the Figure 3, which
depicts the summarized maintenance (A) and relapse
(B). Furthermore, these numbers were converted to a
percent of mean baseline alcohol intake (mean
intake in relapse sessions divided by mean intake in
maintenance) to assess the effect of the period of
abstinence on the drug-intake behaviour (C).
However, due to a very high variability and the nonGaussian
distribution of the data no difference was
detected (Kruskal-Wallis non-parametric test), despite
a trend to higher intake in relapse in the control
female rats (140%).
METH IVSA study
Baseline locomotor characteristics
Before starting the IVSA protocol, baseline locomotor
and exploratory activity was assessed in all groups to
exclude the possibility that these characteristics would
lead to different drug-taking behaviour. Figure 4 illustrates
the results on total horizontal (A) and vertical (B)
activity (number of rearing episodes) and the number
of faecal droppings during the session (C). Two-way
ANOVA (two factors: sex and MAM model) revealed no
significant differences in the distance travelled but
detected the main effect of sex in the numbers of rearing
episodes (F ¼ 11.099, P ¼ 0.002) and droppings
(F ¼ 9.894, P ¼ 0.003). The Bonferroni post-hoc test
added one significant difference in the number of
droppings between the M VEH and F VEH groups
Figure 2. Alcohol drinking. Data are shown as mean (±SEM) daily intake of pure (theoretical 100%) ethanol per kg of body weight
during both maintenance and the relapse phase. The F MAM group consumed more alcohol than the M MAM in both maintenance
and relapse phases of the study: *P < 0.05, **P < 0.01, ***P < 0.001, repeated measures ANOVA followed by Bonferroni post-hoc
test. On days 52 and 53 the F VEH group consumed more alcohol than the M VEH: #P < 0.05, repeated measures ANOVA followed
by Bonferroni post-hoc test.
Figure 3. Alcohol drinking summary. (A) The overall (17 days) mean (±SEM) values of (theoretical 100%) alcohol intake during the
maintenance phase of the study. Repeated measures ANOVA followed by Bonferroni post-hoc test has revealed significant difference
between the M MAM and F MAM groups (P < 0.05). (B) The overall (5 days) mean (±SEM) values of (theoretical 100%) alcohol
intake during the relapse phase of the study. Repeated measures ANOVA followed by Bonferroni post-hoc test revealed significant
difference in the M VEH vs. F VEH (P < 0.05) and M MAM vs. F MAM (P < 0.05) groups. (C) The mean (±SEM) percent of baseline
alcohol intake (mean intake in relapse sessions divided by mean intake in maintenance in each animal) in all groups. Kruskal-Wallis
non-parametric test, not significant.
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(P ¼ 0.015), showing a lower number in females. This
baseline behavioural profile indicates only sex-dependent
differences which are unlikely to contribute to dissimilar
behaviour in the operant cage. Males exhibited
rather higher anxiety-related behaviours visible mainly
in the number of droppings as a measure of general
emotionality.
Food self administration
Food-taking behaviour was assessed as a mean number
of self-administered pellets during the last 5 days
of training when the intake was stable. Figure 5
depicts the significantly higher pellet intake in both
groups of female rats compared to the respective male
groups as indicated by repeated measures ANOVA
(two factors: sex and MAM model), main effect of sex,
F ¼ 5.250, P ¼ 0.002. The Bonferroni post-hoc test further
detected a significant increase of pellet intake in
both F VEH and F MAM groups as compared with M
VEH and M MAM rats, respectively (detailed P values
are indicated in the graph). Generally, all the pellets
delivered were also eaten by the animals during the
session, rarely were a few of them (several, never more
than 10) left intact on the cage floor or in the feeder.
Furthermore, this happened only on the first days of
the training and was not repeated on subsequent days
in the same animals.
Maintenance of METH IVSA
The acquisition and maintenance of METH-taking
behavior was assessed firstly in terms of mean number
of nose-pokes, infusions self administered per session,
and secondly by the mean METH dose per session in
mg/kg. As shown in Figure 6, there was no difference
between the groups in the active (A) or inactive (B)
nose-poking as well as number of infusions (C),
repeated measures ANOVA with two factors: sex and
MAM model. However, when the number of infusions
was converted to a METH dose per kg of body weight,
ANOVA detected a main effect of the model: F ¼ 3.059,
P ¼ 0.022 and Bonferroni post-hoc test indicated a significant
difference between the M VEH and M MAM
groups, with the MAM model leading to a decrease of
METH intake (day 10, P ¼ 0.023). This effect was no longer
significant in the following days.
Reinstatement of METH IVSA
After the 2-week period of forced abstinence, one last
reinstatement session was performed with no drug
availability. The only measure of the drug-seeking
behaviour was the responding operandi. Figure 7
shows the mean number of active (A) and inactive (B)
Figure 4. Baseline behavioural profile assessed by open-field test. (A) Total distance travelled (in cm), (B) number of rearing episodes
(vertical activity) and (C) mean number of faecal droppings. All data are shown as means (±SEM). Two-way ANOVA revealed
the main effect of sex (marked by dotted line frames) in the number of rearings and droppings. Bonferroni post-hoc test identified
only a significant difference in number of droppings between M VEH and F VEH (P < 0.05).
Figure 5. Maintenance of food self administration. The graph
shows the mean (±SEM) number of pellets self administered in
all groups (t-test, *P 0.05, ***P 0.001). The F VEH group
consumed more pellets than the M VEH group: #P < 0.05,
##P < 0.01. Similarly F MAM animals consumed more pellets
than M MAM animals: *P < 0.05, **P < 0.01, repeated measures
ANOVA followed by Bonferroni post-hoc test.
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nose-pokes during this session, the number of active
responses as a percent of mean baseline nose-poking
in maintenance (C) and the percentage of active nosepoke
preferences calculated as the number of active
nose-pokes in the reinstatement session/mean nosepoking
during the 14 days of maintenances phase (D)
as previously described (Ruda-Kucerova et al. 2015a).
Two-way ANOVA (two factors: sex and MAM model)
has indicated only one difference in the percent of
mean baseline nose-poking (C) with the main effect of
sex, F ¼ 6.890, P ¼ 0.012.
Discussion
Alcohol-drinking study
This study has provided evidence that the differential
addictive behaviour in the MAM model can be partially
sex-dependent. There were no differences in the
sucrose preference test in either phase of the study
(i.e., naive and abstinent animals). All animals naturally
preferred the sucrose solution. This indicates that the
animals did not have impaired behaviour related to a
natural reward such as anhedonia or other depressivelike
phenotype. Furthermore, prenatal MAM exposure
did not lead to changes of 20% alcohol-drinking
behaviour in either gender. However, alcohol consumption
was significantly higher in females compared
to males in the MAM-treated groups (F MAM vs. M
MAM). This suggests that the prenatal MAM exposure
may increase vulnerability for alcohol drinking in
female rats. During the relapse phase, this phenomenon
was still present when sex difference in control
animals reached significance, i.e., control females consumed
more alcohol than control males. To our knowledge,
this is the first report on alcohol-drinking
behaviour in the MAM model.
Regarding sex differences, Piano et al. (2005)
showed Sprague-Dawley female rats to have a higher
consumption of a liquid ethanol at ethanol concentrations
from 5 to 8% in the diet available 24 h/day. This
effect can be, to a large extent, explained by presence
of female gonadal hormones because the ovariectomized
group showed similar consumption as males.
Interestingly, blood ethanol levels were similar in all
groups (Piano et al. 2005). In a later analogous study,
the same team found no significant differences in alcohol
drinking (Piano et al. 2007). Long-Evans female rats
were shown to reduce 10% alcohol drinking after
Figure 6. Maintenance of METH intake. (A) The mean numbers of active (drug-paired) nose-pokes over the 14 days of the METH
IVSA in all groups; analogously (B) depicts mean numbers of inactive (non-drug-paired) nose-pokes, (C) The mean numbers of METH
infusions and (D) the mean METH dose in mg/kg. All data are shown as means (±SEM). The only significant difference between the
M VEH and M MAM groups was identified in the METH dose (day 10, $P ¼ 0.023), repeated measures ANOVA followed by
Bonferroni post-hoc test.
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ovariectomy, which was counteracted by oestrogen
supplementation (Ford et al. 2002). Furthermore, intact
Wistar female rats were shown to have a higher intake
of 6% alcohol than a male group (Juarez and Barrios
de Tomasi 1999). However, these sex specificities are
not easy to replicate in all designs. Moreover, there are
also studies showing no differences between genders
in different strains (Piano et al. 2007; Anderson et al.
2012). In addition, behavioural traits other than absolute
alcohol consumption or blood level may differ
between the sexes, such as memory test performances
or depressive- and anxiety-like phenotype. This suggests
a higher vulnerability of females to the same
level of alcohol exposure (Gomez and Luine 2014).
Regarding the SCZ-like phenotype, most studies
were performed using the NVHL neurodevelopmental
model in male rats. These studies have that shown animals
can easily develop behavioural sensitization to
ethanol compared with sham rats (Conroy et al. 2007).
NVHL rats were found to have higher operant self
administration of sweet alcohol solution, but not
sucrose or alcohol alone (Berg et al. 2011). A new
study evaluating intermittent drinking in home cage
and operant self administration of alcohol identified a
loss of control of over 20% alcohol drinking in adulthood
after animals had been exposed to light drinking
in adolescence (10% solution). These animals also
reached higher break-points in a progressive ratio
schedule of alcohol self administration, and a higher
intake in a drug-primed reinstatement session after
extinction training. Interestingly, there were no differences
between control and NVHL rats in either adolescence
or adulthood drinking alone or sucrose
consumption (Jeanblanc et al. 2014). Another neurodevelopmental
model of SCZ-like phenotype induced by
prenatal lipopolysaccharide administration in male offspring
showed enhanced alcohol drinking (Liu et al.
2004). Therefore, it seems that the SCZ-like phenotype
induced by different neurodevelopmental models
enhances vulnerability to alcohol addiction and allows
further study of the dual disorder in pre-clinical setting.
METH self-administration study
This set of experiments has shown rather sex-dependent
differences that significantly altered addictive
Figure 7. Reinstatement of METH-seeking behaviour. (A) The mean numbers of active (drug-paired) nose-pokes in the reinstatement
session in all groups; analogously (B) the mean numbers of inactive nose-pokes, (C) mean percent of baseline active nose-poking
(mean number of nose-pokes during maintenance phase/number of nose-pokes Â100 in each animal) and (D) the mean active
nose-poke preference in the reinstatement session. All data are shown as means (±SEM). Two-way ANOVA revealed the main effect
of sex (marked by dotted line frames) in the percent of mean baseline nose-poking (C), while Bonferroni post-hoc test did not show
any other significances.
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behaviour in the MAM model. Consistent with our previous
data, female sex leads to significant increases in
operant self administration of sweet pellets (RudaKucerova
et al. 2015a), which was also apparent in the
MAM animals. Food self administration was very different
from the METH-taking behaviour, suggesting a
higher motivation for natural reward in females. This
was shown as approximately three times more selfadministered
pellets in females than in males,
corresponding with earlier results showing fewer unreinforced
responses of female rats when testing for
food-reward-lever holding (van Hest et al. 1987).
However, the natural reward-oriented outcome is very
different from METH-related operant behaviour which
rules out the possibility of a general gender-specific
difference in the reward processes.
Furthermore, MAM exposure in male rats induced
only sporadic differences in self-administered doses
of METH, suggesting a trend to lower consumption
of the drug in the model. This effect was not apparent
in female rats which showed a lower intake than
males in the maintenance of METH self administration
(Ruda-Kucerova et al. 2015a). In the reinstatement
session, the only sex-dependent difference was
identified in accordance with increased relapse-like
behaviour in females (Ruda-Kucerova et al. 2015a).
To our knowledge, this is the first study evaluating
potential interactions of sex and the SCZ-like phenotype.
However, there are published papers on sex
differences in psychostimulant addiction and relapse,
and also studies on addiction in neurodevelopmental
models of SCZ.
There is a large body of pre-clinical evidence on sex
differences to psychostimulants showing female rats
to be more vulnerable to their behavioural effects
(Robinson et al. 1982; Stohr et al. 1998; Becker et al.
2012), readily developing behavioural sensitization
after repeated treatment (Robinson 1984; van Haaren
and Meyer 1991; Harrod et al. 2005). However, the
results on METH addiction in male and intact (not
gonadectomized) female rats are quite contradictory. It
was shown that there is lower self administration of
METH in females (Ruda-Kucerova et al. 2015a), no differences
in METH Conditioned Place Preference
(Schindler et al. 2002), higher METH intake in both
short and 6-h long IVSA sessions or progressive IVSA
paradigm (Roth and Carroll 2004; Reichel et al. 2012).
These contradictions might originate in different
experimental paradigms and dose ranges. Furthermore,
a higher reinstatement (relapse-like behaviour) in intact
female rats was also reported with both METH (Holtz
et al. 2012; Cox et al. 2013; Ruda-Kucerova et al. 2015a)
and cocaine (Lynch and Carroll 2000; Lynch and Taylor
2004). This study is consistent with the current results
confirming a higher trend of reinstatement to METH
seeking in females as shown by the percentage of
basal responding.
Other addiction studies of the SCZ-like phenotype
have shown quite contradictory data on the
differential psychostimulant intake in male rats. The
NVHL rats were demonstrated to reach higher
break-points and gained more METH infusions in a
progressive ratio schedule of reinforcement (Brady
et al. 2008). In a similar study with cocaine, NVHL
males were also reported to meet later extinction
criteria and dose-dependently increased drug-primed
reinstatement (Chambers and Self 2002). However, a
cocaine self-administration study in the MAM model
did not show any differences, either in drug taking
under fixed or progressive schedules at several
doses or in extinction and drug-induced reinstatement
(Featherstone et al. 2009). The authors of the
study stated that while MAM treatment is known to
increase reactivity of the mesocorticolimbic DAergic
system, this effect might not be sufficient to alter
the reinforcing properties of cocaine. It is important
to keep in mind that increased behavioural response
to amphetamine is apparently not fully reproducible.
The same laboratory has even reported once an
increased response of MAM rats to an amphetamine
dose of 0.5 mg/kg and not 2 mg/kg (Perez et al.
2013) in a subsequent study with the same design
otherwise (Perez et al. 2014). Therefore, the result of
no effect reported in the cocaine IVSA study
(Featherstone et al. 2009) or a trend of lower METH
intake in MAM males in the current study could be
a characteristic hyperactive response of the MAMexposed
animals to amphetamine due to basal
hyperdopaminergia (Lodge and Grace 2012). If true,
this could indicate that MAM-exposed rats may
need lower doses for the same DA release in the
reward pathway and consequently to experience a
similar level of pleasure. This hypothesis could be
proven by an in vivo microdialysis study. However,
a similar behavioural effect to psychostimulant challenge
was demonstrated in the NVHL model
(Chambers and Taylor 2004), while reports on IVSA
studies were positive (Chambers and Self 2002;
Brady et al. 2008).
Another explanation could be based on the
pharmacology of amphetamine-like drugs. Given the
fact that psychostimulant administration leads to very
strong dose-dependent dopamine release in the
reward circuit reaching up to several hundreds of percent
of the basal level (Di Chiara et al. 2004), a ceiling
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effect of the DA release could play a role (RudaKucerova
et al. 2015b).
Conclusions
Our study suggests that the female sex and the SCZlike
phenotype induced by prenatal MAM exposure
may work synergistically to enhance alcohol consumption.
Different SCZ models were used in alcohol studies
which have all been demonstrated to have some merit
in modelling escalation of alcohol consumption. Future
research will be needed to establish paradigms that
would be readily assessed to test anti-addiction treatments.
Furthermore, adolescence in the prenatally
MAM-exposed animals seems to be a vulnerable period
during which the MAM-induced phenotype can be
pharmacologically changed or modified including
drugs of abuse as proven in the case of alcohol
(Jeanblanc et al. 2014) and diazepam treatment (Du
and Grace 2013).
The usefulness of prenatal MAM exposure for modelling
psychostimulant addiction can be questioned. At
this stage, the NVHL model seems to be of more interest,
but the application of the MAM model to study
this type of substance abuse remains under studied.
Therefore, it is not possible to conclude that the
reward-related processes in the MAM model are intact.
Furthermore, sex-dependent variables are not readily
assessed in dual-disorder pre-clinical studies; therefore,
any conclusion on the matter would not go beyond
speculation.
Acknowledgements
This work was supported by project No. 3SGA5789 financed
from the SoMoPro II Programme that has acquired a financial
grant from the People Programme (Marie Curie Action) of
the Seventh Framework Programme of EU according to the
REA Grant Agreement No. 291782 and was further cofinanced
by the South-Moravian Region. This publication
reflects only the authors’ views and the Union is not liable
for any use that may be made of the information contained
therein. This research was also carried out under the project
CEITEC 2020 (LQ1601) with financial support from the
Ministry of Education, Youth and Sports of the Czech
Republic under the National Sustainability Programme II. This
study was written at Masaryk university with co-financing of
the project ‘‘Experimental pharmacological development in
neuropsychopharmacology and oncology’’ number MUNI/A/
1284/2015 with the support of the Specific University
Research Grant, as provided by the Ministry of Education,
Youth and Sports of the Czech Republic in the year 2016 and
funds from the Faculty of Medicine MU to junior researcher
Jana Ruda-Kucerova.
The authors are grateful for support in behavioural testing
and excellent animal care by Jaroslav Nadenicek, and for
proof-reading by Heejae Chung and Tony Fong (Toronto,
ON).
Statement of interest
None to declare.
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4. Sex-dependent specificities in the drug abuse
There is a large body of clinical evidence suggesting differential characteristics of the
disorder in men and women. Despite the absolute number of female methamphetamine
abusers being lower than the male ones, women usually appear more dependent, show
higher escalation rates (Dluzen and Liu, 2008, Becker and Hu, 2008) and most importantly
tend to experience more frequent relapses (Bobzean et al., 2014, Fattore et al., 2014). The
abuse of psychostimulant drugs (cocaine, methamphetamine, etc.) is currently on the rise
among women, and it has been shown that women experience higher cravings and suffer
more relapses than men (Becker and Hu, 2008). These gender specific differences require
specific treatment strategies for men and women (Brecht et al., 2004, Munro et al., 2006,
Terner and de Wit, 2006). This particularly applies to relapse-prevention which represents
a key treatment challenge especially for women (Brecht and Herbeck, 2014). Preclinical
studies of drug addiction were carried out with male subject only for a long time because
significant influence of the oestrous cycle is well known in terms of behavioural and
neurochemical effects but recently this approach has been abandoned on order to identify
the gender differences and develop new more specific treatments.
There are four main biological factors accounting for gender differences in the drug
addiction:
1) Different levels of sex hormones
2) Gender dependent dimorphism in the brain reward system (unrelated to actual
hormonal levels)
3) Different pharmacokinetics and pharmacodynamics of the drugs in men and women
4) Genetic differences linked to sex chromosomes
Levels of male and female gonadal hormones strongly affect the behaviour of people
which concerns the addictive one as well (Becker et al., 2012). However, there have been
reported also gender dependent differences in the brain structure in both humans and
animals (Carroll and Anker, 2010). Another source of the gender differences comprise
metabolic adjustments leading to pharmacokinetic changes of the drugs including different
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fat deposition, amount of water in the body or proportion of skeletal muscles (Graziani and
Nistico, 2015, Fattore and Fratta, 2010) as confirmed in animal models (Milesi-Halle et al.,
2015, Milesi-Halle et al., 2007) and clinical experience (Frackiewicz et al., 2000).
Pharmacodynamical changes could be underlined by changes in connectivity of the
neuronal tracts and neurotransmitter systems which are modified prenatally by gonadal
hormones or chromosomes (Graziani and Nistico, 2015, Fattore et al., 2008). These
pharmacodynamical specificities are the major source of subjective differences in the
effects of the abused drugs and to the variable tendency to develop the addiction, tolerance
or sensitization to the drug. Namely dopaminergic system was suggested to be sexually
dimorphic, which contributes to the differential reactivity of men and women towards
drugs of abuse (Melis et al., 2005). However, there is apparently also a strong hormonal
effect on pharmacokinetics of abused substances as shown in cocaine (Niyomchai et al.,
2006).
We have focused primarily on the effect of oestrogens and we found an increased
methamphetamine intake in females with high levels of oestrogens (Kucerova et al., 2009).
This is in accordance with other preclinical and also clinical experience. Clinical studies
have shown increased subjective rewarding properties of amphetamine during follicular
phase of the menstrual cycle (Justice and De Wit, 2000, Justice and de Wit, 1999) and also
higher craving (Carpenter et al., 2006). Preclinical evidence suggests a strong relationship
between oestrogens and enhanced addictive behaviour (Becker et al., 2001) while
progesterone has opposite effect (Justin et al., 2010, Quinones-Jenab and Jenab, 2010).
This approach was already evaluated as a treatment option for cocaine addiction in clinical
trials (Evans and Foltin, 2006, De Wit, 2011).
One of the main factors of drug addictions faced by clinicians is the relapse. The hormonal
effects on relapse in humans or relapse-like behaviour in animal models were mostly
conclusively described showing once again enhancement by oestrogens (Larson and
Carroll, 2007, Anker and Carroll, 2011) and suppression by progestins (Lynch and
Sofuoglu, 2010, Anker et al., 2007). However, there is a lack of more naturalistic studies
which should evaluate the behaviour of female abusers without simplifying the matter to
just hormonal levels. Therefore, we designed a simple study to assess relapse-like
behaviour in male rats and their female counterparts with a free oestrous cycle and we
confirmed increased vulnerability of the female group towards methamphetamine abuse
(Ruda-Kucerova et al., 2015a).
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4.1. Aims
The research on the sex differences in addictive behaviours aimed to:
1. Investigate the effect of sex and oestrogen levels on intravenous self-administration
of methamphetamine
 Section 4.3.1., Kucerova et al., 2009
2. Extend the approach to a potentially differential influence of behavioural
sensitization to methamphetamine in both sexes
 Section 4.3.1., Kucerova et al., 2009
3. Further validate the model by assessing the relapse-like behaviour towards
methamphetamine in both sexes
 Section 4.3.2., Ruda-Kucerova et al., 2015
131
4.2. Methods
4.2.1. Animals
Adult rats of Wistar or Sprague-Dawley strain of both sexes were used in the studies. The
rats were housed individually in standard rodent plastic cages. Environmental conditions
during the whole study were constant: relative humidity 50-60 %, room temperature 23◦C
± 1◦C, inverted 12-hour light-dark cycle. Food and water were available ad libitum. All
experiments were conducted in accordance with all relevant laws and regulations of animal
care and welfare. The experimental protocol was approved by the Animal Care Committee
of the Masaryk University, Faculty of Medicine, Czech Republic, and carried out under the
European Community guidelines for the use of experimental animals.
4.2.1. Ovariectomy and oestrogen supplementation
In one study female animals were gonadectomized during the IVSA surgery. The ovaries
were removed and the uterus below was ligated. Access to the ventral cavity was allowed
by one central incision (Caine et al., 2004).
Oestrogen (Estradiol benzoate salt suspension in AGOFOLLIN DEPOT®, Biotika a.s.,
Slovak Republic dissolved in saline) was administered once a week intramuscularly as a
depot formulation. The dose of 0.28 mg/kg used is expected to maintain the hormone
plasma levels in the physiological range of rat oestrous cycle (Mendoza-Rodriguez et al.,
2003). The control group of ovariectomized rats received the same volume of saline
solution instead.
4.2.2. IV self-administration (IVSA) surgery and procedures
The IV self-administration study was performed in the same manner as described in the
section 2.3.3.
4.2.3. Locomotor activity
Locomotor activity was assessed as described in the section 2.3.5.
132
4.3. Results
4.3.1. Impact of repeated methamphetamine pretreatment on intravenous
self-administration of the drug in males and estrogenized or non–
estrogenized ovariectomized female rats
The present study was designed to evaluate the effects of gender, oestrogen, and potential
behavioural sensitization (Robinson and Berridge, 1993, Vanderschuren and Pierce, 2010)
expected to be associated with repeated methamphetamine pretreatment on
methamphetamine intake in the intravenous self-administration paradigm in male rats and
ovariectomized female rats with and without oestrogen substitution.
The highest spontaneous methamphetamine intake in the IV self- administration was
demonstrated in estrogenized ovariectomized females, with lower intake in males, and the
lowest intake in non-estrogenized ovariectomized females. Repeated pre-exposure to
methamphetamine produced a significant decrease in the mean number of
methamphetamine infusions self-administered per sessions in males, as well as, in
estrogenized ovariectomized females, but not in non-estrogenized ones. This may indicate
that methamphetamine infusions self-administered during the sessions produced stronger
reinforcing effects in rats previously exposed to methamphetamine than in drug naïve
animals and that the lack of oestrogen in ovariectomized females may provide protection
from the development of such changes in drug effects. In humans, it has been
demonstrated that higher levels of oestrogen are associated with greater subjective
stimulation after amphetamine in women (White et al., 2002), but amphetamine-stimulated
dopamine release can be greater in men (Dluzen and Liu, 2008), which could perhaps
increase vulnerability of men to neurotoxic effects of amphetamines (Munro et al., 2006).
The findings from the pre-clinical and clinical studies should be taken into an account
when creating specific prevention and treatment programs for men and women.
Kucerova J, Vrskova D, Sulcova A. Impact of repeated methamphetamine pretreatment
on intravenous self-administration of the drug in males and estrogenized or nonestrogenized
ovariectomized female rats. Neuro Endocrinol Lett. 2009, 30(5): 663-70.
IF 1.047
Citations (WOS): 13
To cite this article: Neuroendocrinol Lett 2009; 30(5):663–670
ORIGINALARTICLE
Neuroendocrinology Letters  Volume 30  No. 5  2009
Impact of repeated methamphetamine
pretreatment on intravenous self-administration
of the drug in males and estrogenized or non–
estrogenized ovariectomized female rats
Jana Kucerova, Dagmar Vrskova, Alexandra Sulcova
Masaryk University, Faculty of Medicine, Department of Pharmacology, Brno, Czech Republic.
Correspondence to: Jana Kucerova, PharmD.
Department of Pharmacology, Faculty of Medicine, Masaryk University Brno
Tomesova 12, 662 43 Brno, Czech Republic.
phone: +420 549 494 238; fax: +420 549 492 364; e-mail: jkucer@med.muni.cz
Submitted: 2009-06-18	 Accepted: 2009-10-25	 Published online: 2009-11-12
Key words: methamphetamine; IV self-administration; methamphetamine intermittent
pretreatment; gender differences; ovariectomy; estrogen; wistar rats
Neuroendocrinol Lett 2009; 30(5):663–670  PMID: 20035259   NEL300509A05  © 2009 Neuroendocrinology Letters • www.nel.edu
Abstract OBJECTIVE: The female animals were already recorded to respond differently to
methamphetamine (MET) abuse than males. This gender dissimilarity may be
caused by the influence of estral cycles and different susceptibility to behavioural
sensitization.
METHODS: Influences of gender and pre-exposure to MET were studied in the
rat model of MET intravenous self-administration (IVSA). The fixed ratio (FR)
paradigm was employed in male rats (M) and estrogenized (F-ESTR) and nonestrogenized
ovariectomized female rats (F-OVX) either pre-exposed or notexposed
to MET pretreatment.
RESULTS: In rats that were not pre-exposed to MET, F-ESTR self-administered
more MET infusions than each of the other groups, but F-OVX self-administered
less than each of the other groups; the same trend was apparent in the MET pretreated
groups. MET pre-exposure decreased subsequent MET IVSA in all groups
except F-OVX.
CONCLUSION: Thus, pre-exposure to MET and the loss of inherent estrogen in
females notably decreased the intake of MET by rats, suggesting that abuse liability
was reduced. Estrogen’s effects on MET self-administration here correspond
with accumulating evidence of stronger behavioural responses of females to drugs
of abuse.
Abbreviations :
MET	 - methamphetamine
SAL 	 - saline
M 	 - males
OVX 	 - ovariectomy
F-OVX 	 - ovariectomized females
F-ESTR 	 - estrogenized ovariectomized females
IVSA 	 - IV self-administration
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Jana Kucerova, Dagmar Vrskova, Alexandra Sulcova
Introduction
Although the role of gender in the mechanisms of drug
action remains unclear, both preclinical and clinical
studies indicate that ovarian hormones, particularly
estrogen, play a role in producing sex differences in
drug abuse (Lynch et al. 2002). These differences in
the behavioural reactivity to drugs in men and women
(Brecht et al. 2004, Munro et al. 2006) will probably
require different strategies in the prevention and treatment
of addiction depending on gender. So far, most
experimental studies have been conducted with male
subjects; many researchers have chosen to ignore the
influences of the estrous cycle, which is more difficult
to study, but has an important impact on animal behaviour.
The reasons for differences in female reactivity to
drugs can be due to pharmacokinetic specifics (MilesiHalle
et al. 2007) such as distinct metabolising enzyme
activities, distribution volume and other parameters.
Pharmacodynamic effects of drugs can also be dependent
on structural changes induced in early life by
the physiological hormonal levels in the brain or on
particular receptor gene expression modulated by sex
hormones (Hu et al. 2004). Experiments in laboratory
rodents showed that estrogen levels can regulate behavioural
responses to drugs of abuse, especially psychomotor
stimulants, including methamphetamine (MET).
During estrus, the effects of abused drugs are more pronounced,
and this is reflected in gender differences in
general patterns of drug abuse (Sell et al. 2002, Becker
& Hu, 2008)
Behavioural sensitization to drugs and the adaptations
in striatal neurotransmission that are associated
with this sensitization are thought to play an important
role in certain aspects of addiction (Ohmori et al. 2000).
Dopaminergic activity in the brain is enhanced by
estrogen in positive correlation with behavioural effects
(Thompson & Moss, 1994, Thompson et al. 2000, White
et al. 2002). However, positron emission tomography
(PET) scans performed after amphetamine administration
in humans showed increased reactivity of the striatal
dopamine system in men compared with women
(Munro et al. 2006). The interactions between ovarian
hormones, dopamine, and drugs of abuse are not clear
yet, and more studies are necessary for further elucidation.
Nevertheless, in a number of studies females were
more prone to develop sensitization than males (Phillips
et al. 1997, Becker et al. 2001, Sell et al. 2002, Hu
& Becker, 2003, Kawakami et al. 2007, Kucerova et al.
2008).
Intravenous drug self-administration by laboratory
animals is a model for testing dependence potential and
abuse liability of drugs (Collins et al. 1984). Compared
to males, females are reported to become addicted more
rapidly (and subsequently to relapse more readily following
abstinence) when drugs of abuse are offered
in the drug self-administration model at lower doses
(Becker & Hu, 2008). The acquisition rate was found to
be faster in female compared to male rats self-administering
nicotine, alcohol, heroin, cocaine or MET (Lynch
et al. 2002), presumably indicating that the reinforcing
effects of these drugs are stronger in females. Rates of
acquisition are also dependent on variables such as
drug dose, circadian variability in access to drug and
previous drug exposure (potentially leading to behavioural
sensitization) (Roth & Carroll, 2004).
Understanding the influences of gender and sex hormones
on drug self-administration in animals may lead
to improved strategies for treatment and prevention
of drug abuse in humans. While progress is beginning
to be made in this area, much remains to be done. In
particular, methamphetamine is a widely-abused and
highly-addictive drug that has serious health consequences,
yet has received less research attention than
other major drugs of abuse. Therefore, the present study
was designed to evaluate the effects of gender, estrogen,
and potential behavioural sensitization expected to be
associated with repeated MET pretreatment on MET
intake in the intravenous self-administration (IVSA)
paradigm in male rats (M) and ovariectomized female
rats with (F-ESTR) and without estrogen substitution
(F-OVX).
Material and methods
Animals
The present study with Wistar rats (purchased in the
Laboratory Animal Breeding and Experimental Facility,
Masaryk University Brno, Czech Republic) consisted
of 6 experiments (2 with males – M: n=36, 2 with
ovariectomized females – F-OVX: n=36, 2 with ovariectomized
females substituted regularly with estrogen
– F-ESTR: n=36). Adult male rats weighing 350–400 g
and female rats weighing 250–300 g at the beginning
of experiment (in order to assure the catheter position
stability in animals not growing much to the length
anymore) were housed in sections of five in standardized
rat plastic cages during the first two weeks of the
experiment. After the catheter implantation surgery
was performed, rats were housed individually in plastic
cages standardized for separate stabling. During the
whole experiment the environmental conditions were
constant: relative humidity 50%, temperature 23 °C ±
1 °C, inverted 12-hour light-dark cycle (5 a.m. to 5 p.m.
darkness). Food and water were available ad libitum. All
experiments were conducted in accordance with all relevant
laws and regulations of animal care and welfare.
The animal study protocol was approved by the Animal
Care Committee of the Masaryk University Faculty of
Medicine, Brno, Czech Republic, and carried out under
the European Community guidelines for the use of
experimental animals.
Surgery
Under general anaesthesia (xylazine 8 mg/kg + ketamine
50 mg/kg intraperitoneally in combination with
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Impact of repeated methamphetamine pretreatment on intravenous self-administration of the drug
isoflurane inhalation for induction to anaesthesia) a
permanent intracardiac silastic catheter (our own production)
was implanted through the external jugular
vein into the right atrium. The outer part of the catheter
exited the skin in the midscapular area. A small
nylon bolt was fixed on the skull with dental acrylic to
stainless-steel screws embedded in the skull; this served
as a tether to prevent the catheter from being pulled
out while the rat was in the self-administration chamber.
During the surgery all the female animals were
gonadectomized. The ovaries were removed and the
uterus below was ligated. Access to the ventral cavity
was permitted by one central incision. The catheters
were flushed daily before all the sessions with 0.2 ml
of heparinized cephalosporine (Vulmizolin 1.0 inj
sic, Biotika a.s., Slovak Republic) solution (0.05  mg/
kg in saline with 2.5 I.U./kg) and 0.05 ml of heparin
(HEPARIN LECIVA inj. sol. 1x10ml/50  I.U.) solution
(5 i.u.) to prevent infection and occlusion of the
catheter. During this procedure the blood was aspired
daily to assess the patency of the catheter, and changes
in general behaviour, weight and other circumstances
were recorded. When a catheter was found blocked the
animal was excluded immediately from the analysis.
Drugs and treatments
Methamphetamine (MET) from Sigma Chemical, Co.,
St Louis, MO, USA was used for both intraperitoneal
drug pretreatment and IVSA. The administration of
MET prior to IVSA was according to the following
dosing regimen, which was successfully used in our
previous studies (Landa et al. 2006, Landa et al. 2008)
to induce behavioural sensitization: 0.5 mg/kg/day,
intraperitoneally, for 14 days, administered in home
cages. The identical volume (1  ml/kg/day) and route
of administration of saline solution (SAL) were used
for all control treatments. The MET dose available for
IVSA was 0.08 mg/kg per single infusion with the maximum
number of infusions during one session set to 50
(Vinklerova et al. 2002).
Estrogen (Estradiol benzoate salt suspension in
AGOFOLLIN DEPOT®, Biotika a.s., Slovak Republic
dissolved in saline) was administered to ovariectomized
females (F-ESTR) once a week intramuscularly
as a depot (Shansky et al. 2004). The dose of 0.28 mg/kg
used is expected to maintain the hormone plasma levels
in the physiological range of rat estrous cycle (Mendoza-Rodriguez
et al. 2003). The other group of ovariectomized
rats (F-OVX) received the same volume of
saline solution instead.
Each animal group (M, F-ESTR, and F-OVX) was
randomly divided into four subgroups (n1,2,3,4) for the
following treatments: a) n1=6: 14 days of daily pretreatment
with saline (SAL), 1.0 ml/kg, intraperitoneally +
the 15th day surgery procedure + 14 days of recovery
and drug washout + 21 days of IVSA of SAL; b) n2=12:
14 days of daily pretreatment with SAL, 10.0  ml/kg,
intraperitoneally + the 15th day surgery procedure + 14
days of recovery and drug washout + 21 days of IVSA
of methamphetamine (MET); c) n3=6: 14 days of daily
pretreatment with MET, 0.5 mg/kg, intraperitoneally +
the 15th day surgery procedure + 14 days of recovery
and drug washout + 21 days of IVSA of SAL, 1.0 ml/kg,
intraperitoneally; d) n4=12: 14 days of daily pretreatment
with MET, 0.5 mg/kg, intraperitoneally + the 15th
day surgery procedure + 14 days of recovery and drug
washout + 21 days of IVSA of MET, 0.5 mg/kg, intraperitoneally.
The MET or SAL pretreatment was given
in the home-cage daily at the same time within the dark
period of the light cycle.
Self-administration apparatus and procedure
Standard experimental chambers (with all accessories
provided by Coulbourn Instruments, USA) with two
nose-poke holes allocated on one side of the cage were
programmed by software L2T2 (Coulbourn Instruments,
USA). IVSA sessions were initially conducted
under the fixed ratio (FR) schedule of reinforcement
starting at FR1 (each correct response reinforced).
Fixed-ratio requirements were raised (e.g. FR2 –
two correct responses required, FR3 – three correct
responses required, etc.) when the animal fulfilled the
following conditions for three consecutive sessions:
a) at least 70% preference for the active nose-poke; b)
minimum intake of 10 infusions per session; c) stable
intake of the drug (maximum 10% deviation). Active
nose-pokes led to the activation of the infusion pump
and administration of a single infusion paired with a
2-s light cue, followed by a 30-sec time-out. Nose-pokes
in the other (non-active) hole were recorded but had no
programmed consequences. The cage was illuminated
by a house light, which was off during the time-out.
There were 21 daily sessions in 21 consecutive days,
each lasting 120  minutes and taking place regularly
between 7 a.m. and 4 p.m. during the dark period of
the reversed light cycle. After the session the animals
were returned to the home-cage.
Statistical Data analysis
For statistical analysis of differences in either saline
or MET IVSA the Mann-Whitney U test was applied
(comparing nose-poke responses on the active lever to
those on the inactive lever), and for evaluation of the
IVSA acquisition rates a Survival Data Analysis (PetoPeto-Wilcoxon
test) was used. Level of statistical significance
was determined to p<0.05.
Results
Table 1 demonstrates the reinforcing properties of the
dosing IVSA MET schedule as all groups of rats (M,
F-ESTR, F-OVX) regardless of repeated pretreatment
(SAL or MET) exhibited preference for active (reinforced)
nose-poke over the inactive (non-reinforced)
nose-poke when nose-poking was reinforced by MET
(0.08 mg) infusions. In each of these groups the number
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Jana Kucerova, Dagmar Vrskova, Alexandra Sulcova
of active nose-pokes reinforced by MET was significantly
higher than number of inactive nose-pokes. On
the other hand, the number of nose-pokes into active vs.
inactive hole was not significantly different in groups
that were allowed to self-administer saline, regardless
of repeated pretreatment (SAL or MET).
Figure 1 shows the percentage of rats from each
group (M, F-ESTR, F-OVX) acquiring MET selfadministration
after 14 days of withdrawal after 14 day
pre-treatment with saline (A) or methamphetamine (B)
that met the criteria for increasing FR from initial FR1
to FR2. Differences between acquisition rates of MET
IVSA of individual rat groups are shown in both parts
of the figure (A and B) were not significant according
to a Survival Data Analysis (Peto-Peto-Wilcoxon test).
However, there is an apparent trend in both conditions
(A-absence or B-presence of MET pre-exposure) showing
that non-estrogenized female castrates (F-OVX)
exhibited the slowest rate in reaching higher FR conditions
of all three groups, and the lowest incidence of
meeting this criterion.
This trend is also apparent in Figures 2A and B: at
the end of experiment (21st day of consecutive sessions)
the highest cumulative percentage of animals staying on
FR1 conditions were non-estrogenized female castrates
(F-OVX). In contrast, the highest FR7 requirement for
MET IVSA was performed only by the group of male
rats (M) after MET pre-exposure (Figure 2B). However,
there was no significant difference in acquisition rate
of MET self-administration between SAL and MET
pretreated animals, although some of the latter animals
were able to reach one-step higher FR as a more
demanding IVSA condition (more nose-pokes needed
to obtain one MET infusion) compared to saline pretreated
rats (Figure 2).
Figure 3 shows acquisition of MET self-administration
over 21 consecutive sessions in all three groups
of rats (M, F-ESTR, F-OVX) with (Figure 3B) or without
(Figure 3A) repeated pretreatment with MET.
The F-OVX group self-administered the lowest while
F-ESTR group the highest number of MET infusions
over the course of the experiment under both pretreatment
conditions (MET or SAL). In the groups that
received repeated SAL pretreatment (Figure 3A), the
number of MET infusions received was significantly
higher in F-ESTR group compared to M (p=0.005) and
F-OVX (p=0.0001) groups, and F-OVX animals were
consuming significantly (p=0.0001) lower number of
MET infusions than both M and F-ESTR animals. The
same trend was apparent in the MET pretreated groups
(Figure 3B). The number of infusions self-administered
by F-ESTR group was significantly higher than in the M
(p=0.0001) and F-OVX (p=0.0001) groups while F-OVX
animals self-administered significantly (p=0.0001)
lower number of MET infusions (Figure 3B).
Figure 4B shows that M and F-ESTR groups repeatedly
exposed to MET self-administered a significantly
lower number of MET infusions than those to
saline (M: p<0.0005 and F-ESTR: p<0.001). The MET
pretreatment had no effect on number of infusions
self-administered in the F-OVX group (p=0.0849).
Figure 4A shows that there were no significant differences
in the SAL IVSA in any of experimental groups
after both, pretreatment with SAL and MET. The only
exception was the group F-OVX which self-administered
higher number of SAL infusions after repeated
Tab. 1. The table shows the mean number of nose-pokes in the 21 IVSA sessions (non-active: not associated with
IVSA; active: associated with IVSA) exhibited during the whole experiment by rat males (M) and ovariectomized
females with presence (F-ESTR) or absence (F-OVX) of estrogen substitution (depot suspension of estradiol
benzoate, 0.28 mg/kg/week) after 14 days of withdrawal from 14 day intraperitoneal pretreatment with either
saline (SAL) or methamphetamine (MET – 0.5 mg/kg/day). Statistical evaluation was processed by the MannWhitney
U test.
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Impact of repeated methamphetamine pretreatment on intravenous self-administration of the drug
Fig. 1. The cumulative percentage of males (M) and ovariectomized
females either with (F-ESTR) or without (F-OVX) estrogen
substitution (depot suspension of estradiol benzoate,
0.28 mg/kg/week) after 14 days of withdrawal from 14-day
intraperitoneal pretreatment with either (a) saline (SAL) or (b)
methamphetamine (MET – 0.5 mg/kg/day) that met the criteria
for MET IVSA for switching from FR1 to FR2 conditions in 21
consecutive sessions of the experiment. Evaluation: Survival
Data Analysis (Peto-Peto-Wilcoxon test, non-significant).
Fig. 2. The percentage of rat males (M) and ovariectomized females
either with (F-ESTR) or without (F-OVX) estrogen substitution
(depot suspension of estradiol benzoate, 0.28 mg/kg/week)
meeting the criteria for MET IVSA under gradually increasing
FR requirements after 14 days of withdrawal from 14-day
intraperitoneal pretreatment with either (a) saline (SAL) or (b)
methamphetamine (MET – 0.5 mg/kg/day).
pretreatment with MET. This difference was significant
when compared with matching M and F-ESTR groups
(M: p<0.001 and F-ESTR: p<0.001). In all groups the
mean intake was notably lower than 10 infusion/session
criterion used as an indicator of reinforcing effects.
Discussion
This study demonstrates that Wistar rats repeatedly
exposed to MET (14 daily doses of 0.5 mg/kg)
self-administered a lower number of MET infusions
under a fixed ratio schedule (FR) of MET infusions
(0.8 mg/infusion) compared to animals pretreated
with saline. The same trend was observed to some
extent in all groups of rats (M, F-ESTR, and F-OVX)
but differentially depending on the gender. Both
estrogenized ovariectomized female groups (F-ESTR)
regardless of prior repeated MET or SAL pretreatment
self-administered higher numbers of MET infusions
than corresponding male groups. The while non-estrogenized
ovariectomized (F-OVX) female groups were
self-administering the lowest number of MET infusions
regardless of prior MET exposure, and also their
acquisition rates were the lowest. Though there was no
statistically significant difference, an apparent trend of
facilitation of MET IVSA acquisition was present in rats
repeatedly pre-exposed to MET. This increased drugseeking
behaviour in this model could be considered a
sign of behavioural sensitization (Lorrain et al. 2000) ,
which however can be influenced by the IVSA experimental
paradigm itself. A similar trend was described
by Lorrain et al. (2000) in the rats self-administering
amphetamine under a progressive ratio schedule but
not under a fixed ratio schedule. The acquisition of
IVSA behavior might also be influenced by a previous
association of the drug effect and the experimental
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668 Copyright © 2009  Neuroendocrinology Letters  ISSN 0172–780X  •  www.nel.edu
Jana Kucerova, Dagmar Vrskova, Alexandra Sulcova
cage environment (Reid et al. 1998). In this study, the
animals were pre-exposed to MET in their home-cages.
In a parallel experiment a different group of male rats
were placed for 30 minutes after the intraperitoneal
administration of MET pre-treatment into the operant
cage used later for IVSA sessions. No significant differences
in acquisition rates of MET IVSA were found
when compared with the experimental design used in
the present study (unpublished data).
The susceptibility of the female organism to effects
of drugs of abuse, including the induction of behavioural
sensitization, has mostly been reported by both
pharmacological experimental studies and clinical trials
as being higher and enhanced further by increased
estrogen levels: (Lynch et al. 2002, Becker & Hu, 2008).
The pharmacokinetic and metabolic profiles of a drug
have also been suggested as playing significant roles in
the differential pharmacological response to MET in
male and female rats. Slower MET clearance and lower
metabolite (amphetamine) formation were reported
in the Sprague-Dawley female rats (Milesi-Halle et
al. 2005, Milesi-Halle et al. 2007). The development
of behavioural sensitization has also been reported to
vary between rat strains, possibly due to different brain
penetration of MET. In Wistar rats, the brain penetration
was found to be increased in repeatedly and behavioural
sensitization to the effects of MET were observed
in MET-treated animals, but these effects were not
found to occur in Long-Evans strain (Fujimoto et al.
2007). In the present experiment using Wistar rats with
the IVSA method, which is the most widely used model
for assessing the relative abuse liability of drugs of
abuse, we confirmed that estrogen levels can influence
intake of MET. The repeated pre-exposure to MET,
which was proven to induce behavioural sensitization
to stimulatory effects on locomotion in the same rat
Fig. 3. The acquisition of methamphetamine (MET) infusions
(0.08 mg/infusion) in all 21 IVSA daily sessions in the male rats
(M - filled squares, A: n1=11, B: n2=12), ovariectomized female
rats with estrogen substitution (F-ESTR - open circles, a: n1=11,
b: n2=12), and ovariectomized female rats with no hormonal
substitution (F-OVX - open squares, a: n1=11, b: n2=11) after
14-day withdrawal from 14 days of intraperitoneal pretreatment
with either (a) saline (SAL) or (b) methamphetamine (MET – 0.5
mg/kg/day). Data are shown as daily means ± SEM.
Fig. 4. The effect of methamphetamine (MET) pretreatment
(0.5 mg/kg/day, for 14 days, intraperitoneally) on either saline
(SAL) (a) or MET (b) self-administration after 14-day withdrawal
from the pretreatment. The graphs show the mean number of
IVSA infusions received during the whole experiment in male
rats (M) and ovariectomized females rats either with (F-ESTR)
or without (F-OVX) estrogen substitution (depot suspension
of estradiol benzoate, 0.28 mg/kg/week). Groups of rats after
control SAL pretreatment = open bars, groups of rats after
MET pretreatment = filled bars. Data shown as means ± SEM.
Statistical evaluation was done by using the Mann-Whitney U
test (* p<0.001, ** p<0.0005).
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Impact of repeated methamphetamine pretreatment on intravenous self-administration of the drug
strain (Landa et al. 2008), lowered the number of MET
infusions self-administered during consecutive 21 daily
sessions in all groups (M, F-OVX and F-ESTR). This
effect is not likely to have been due to habituation as
the control SAL-pretreated rats were allowed to selfadminister
MET at the same IVSA paradigm. It is also
unlikely that the lower rates of IVSA seen after MET
pre-exposure were actually due to sensitization (comparable
to increasing the dose per infusion), since there
was evidence that MET exposed rats were less likely to
acquire the self-administration response. Under the
fixed-ratio procedure used here, the decreased IVSA
after MET pre-exposure as well as after ovariectomy
were likely due to a reduced motivation to obtain MET
or a reduced reinforcing effect of MET.
Reports in the existing literature on the relationship
between stimulatory effects of drugs on rodent
locomotion and their IVSA are not consistent. Nevertheless,
the neurobiological basis of the brain systems
underlying both locomotor activation (Schindler et al.
2002) and reward are believed to be sexually dimorphic
(for review see: (Dluzen & Liu, 2008, Becker et
al. 2001). The pharmacological mechanism of action
of amphetamine and its derivatives (such as MET)
involves indirect adrenergic action inducing a massive
release of biogenic amines, particularly dopamine and
noradrenaline, from the storage sites in nerve terminals
to the synapses, and blockade of their reuptake
(Kish, 2008). There are confirmed sex differences in
changes of dopamine extracellular levels and turnover
induced by methamphetamine in rodents (for review
see: (Becker & Hu, 2008), as well as in dopamine
release in humans (Munro et al. 2006). However, in
rats, structural differences caused by the sex hormones
were also found in the early brain ontogenesis, which
was not dependent on the actual hormonal level (Hu
et al. 2004). According to binding studies, there are sex
differences reported in densities of dopamine receptor
subtypes in the rat striatum and the nucleus accumbens
fluctuating dependently on estrous cycle (Becker
& Hu, 2008). The number of dopamine D1 (and to
some extent also of D2) binding sites is higher in male
rats compared to females and estradiol administration
is shown to downregulate D2 receptors in dorsolateral
striatum while enhancing basal dopamine extracellular
concentrations (“dopaminergic tone”) (Xiao & Becker,
1994). Thus, estradiol can elicit changes in dopamine
release and dopamine receptor activity leading to
greater behavioural response to psychostimulant drugs
in intact females in estrus or estrogenized ovariectomized
females. This correlates well with a report that
under IVSA with FR conditions female rats obtained
significantly more MET infusions (0.02 mg/infusion)
compared to males (Roth & Carroll, 2004), as well as
with the results of the present study in which the estrogenized
ovariectomized females (F-ESTR with and
without pre-exposure to MET) self-administered the
highest number of MET infusions (0.08 mg/infusion).
The higher number of MET infusions in M groups compared
to F-OVX groups in our experiment corresponds
with suggestion that due to higher basal dopamine tone
in the male striatum and the nucleus accumbens (Xiao
& Becker, 1994), a greater dopaminergic stimulation is
required to achieve a rewarding effect (Becker & Hu,
2008).
Our results also showed a significantly higher saline
intake in the F-OVX group repeatedly pre-exposed to
MET compared to the rest of the SAL self-administering
groups. Intravenous SAL is not usually found to
have reinforcing effects, but it is known that estrogen
can influence electrolyte homeostasis. In the rat model
of angiotensin II-induced thirst, the chronic administration
of estradiol attenuated water-seeking behaviour
(Fregly & Thrasher, 1978). This was confirmed further
by other rat experiments and evaluated as central
interaction mechanism between this peptide hormone
and estrogen on a genomic level (Kisley et al. 1999).
Estrogens also may influence body fluid regulation by
interacting with several neurotransmitters, including
serotonin, dopamine and noradrenaline (Kucharczyk,
1984). In rats it was proven that water drinking can
be initiated by administration of dopaminergic drugs
(Zabik et al. 1993). This could be reason for higher
IVSA saline intake in the F-OVX rat group lacking
estrogen influence and moreover being pretreated with
dopaminergically acting MET in the present study.
In summary, the highest spontaneous methamphetamine
intake in our model of MET IV self- administration
in rats was demonstrated in estrogenized
ovariectomized females, with lower intake in males,
and the lowest intake in non-estrogenized ovariectomized
females. Repeated pre-exposure to MET (potentially
inducing behavioural sensitization) produced
a significant decrease in the mean number of MET
infusions self-administered per sessions in males, as
well as, in estrogenized ovariectomized females, but
not in non-estrogenized ones. This may indicate that
MET infusions self-administered during the sessions
produced stronger reinforcing effects in rats previously
exposed to MET than in drug naïve animals (perhaps
due to behavioural sensitization) and that the lack
of estrogen in ovariectomized females may provide
protection from the development of such changes in
MET effects. Thus, preclinical studies indicate that
behavioural and neurobiological responses to psychostimulant
drugs are sexually dimorphic and point to a
particular role of estrogen, but all mechanisms underlying
this dimorphism are not completely clear yet. In
humans, it has been demonstrated that higher levels
of estrogen are associated with greater subjective stimulation
after amphetamine in women (White et al. 2002),
but amphetamine-stimulated dopamine release can
be greater in men (Dluzen & Liu, 2008), which could
perhaps increase vulnerability of men to neurotoxic
139
670 Copyright © 2009  Neuroendocrinology Letters  ISSN 0172–780X  •  www.nel.edu
Jana Kucerova, Dagmar Vrskova, Alexandra Sulcova
effects of amphetamines (Munro et al. 2006). The findings
from the pre-clinical and clinical studies should be
taken into an account when creating specific prevention
and treatment programs for men and women.
Acknowledgements
We would like to thank Drs. Zuzana Justinova and
Leigh Panlilio for their help with preparation of this
manuscript.
This work was supported by the Czech Ministry of
Education – research project: MSM0021622404 and
by the Masaryk University Rector’s grants: MUNI/11/
C0002/2006 and MUNI/11/C0001/2007.
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141
4.3.2. Sex differences in the reinstatement of methamphetamine seeking after
forced abstinence in Sprague-Dawley rats
The aim of this study was to assess gender differences in all stages of operant IV selfadministration
of methamphetamine in male and female rats (Fattore et al., 2014) while the
gonads of all animals were kept intact assuring physiological oestrous cycle in females.
Furthermore, possible gender differences in acquisition and maintenance of food selfadministration
were assessed in order to compare the operant behaviour towards natural
reward (food) and the drug of abuse.
The data showed a lower consummator methamphetamine intake during maintenance
phase of the self-administration together with higher vulnerability to the reinstatement of
methamphetamine seeking behaviour in female rats after forced abstinence. These effects
seem to be robust enough, thus relatively independent on the current hormonal level.
Therefore, we propose this paradigm for preclinical screening for potential new
medications specific for women.
Ruda-Kucerova J, Amchova P, Babinska Z, Dusek L, Micale V, Sulcova A. Sex
differences in the reinstatement of methamphetamine seeking after forced abstinence in
Sprague-Dawley rats. Front Psychiatry. 2015, 6: 91. doi: 10.3389/fpsyt.2015.00091.
IF N/A
Citations (WOS): 1
ORIGINAL RESEARCH
published: 06 July 2015
doi: 10.3389/fpsyt.2015.00091
Edited by:
Miriam Melis,
University of Cagliari, Italy
Reviewed by:
Angelo Giovanni Icro Maremmani,
University of Pisa, Italy
Cristina Cadoni,
National Research Council, Italy
*Correspondence:
Jana Ruda-Kucerova,
CEITEC – Central European Institute
of Technology, Masaryk University,
Kamenice 5, Brno 625 00,
Czech Republic
jkucer@med.muni.cz
Specialty section:
This article was submitted to
Addictive Disorders and Behavioral
Dyscontrol, a section of the journal
Frontiers in Psychiatry
Received: 03 April 2015
Accepted: 09 June 2015
Published: 06 July 2015
Citation:
Ruda-Kucerova J, Amchova P,
Babinska Z, Dusek L, Micale V and
Sulcova A (2015) Sex differences in
the reinstatement of
methamphetamine seeking
after forced abstinence in
Sprague-Dawley rats.
Front. Psychiatry 6:91.
doi: 10.3389/fpsyt.2015.00091
Sex differences in the reinstatement
of methamphetamine seeking
after forced abstinence in
Sprague-Dawley rats
Jana Ruda-Kucerova1,2
*, Petra Amchova1,2
, Zuzana Babinska1,2
, Ladislav Dusek3
,
Vincenzo Micale1,4
and Alexandra Sulcova1
1
Experimental and Applied Neuropsychopharmacology Group, Central European Institute of Technology (CEITEC), Masaryk
University, Brno, Czech Republic, 2
Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czech
Republic, 3
Institute of Biostatistics and Analyses, Faculty of Medicine, Masaryk University, Brno, Czech Republic, 4
Section
of Pharmacology, Department of Biomedical and Biotechnological Sciences, School of Medicine, University of Catania,
Catania, Italy
Preventing relapse to drug abuse is one of the struggles faced by clinicians in order to
treat patients with substance use disorders (DSM-5). There is a large body of clinical
evidence suggesting differential characteristics of the disorder in men and women, which
is in line with preclinical findings as well. The aim of this study was to assess differences
in relapse-like behavior in methamphetamine (METH) seeking after a period of forced
abstinence, which simulates the real clinical situation very well. Findings from such
study might add new insights in gender differences in relapse mechanisms to previous
studies, which employ a classical drug or cue-induced reinstatement procedure following
the extinction training. Adult male and female Sprague-Dawley rats were used in IV
self-administration procedure conducted in operant boxes using nose-poke operandi
(Coulborn Instruments, USA). Active nose-poke resulted in activation of the infusion pump
to deliver one intravenous infusion of METH (0.08 mg/kg). After baseline drug intake was
established (maintenance phase), a period of forced abstinence was initiated and rats
were kept singly in their home cages for 14 days. Finally, one reinstatement session
in operant boxes was conducted. Females were found to self-administer significantly
lower dose of METH. The relapse rate was assessed as a number of active nosepokes
during the reinstatement session, expressed as a percentage of active nose-poking
during the maintenance phase. Females displayed approximately 300% of active nosepokes
compared to 50% in males. This indicates higher vulnerability to relapse of METH
seeking behavior in female rats. This effect was detected in all females, independently
of current phase of their estrous cycle. Therefore, this paradigm using operant drug
self-administration and reinstatement of drug-seeking after forced abstinence model can
be used for preclinical screening for potential new anti-relapse medications specific for
women.
Keywords: methamphetamine, reinstatement of drug-seeking behavior, forced abstinence, sex/gender
differences, Sprague-Dawley rats
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Ruda-Kucerova et al. Sex differences in METH relapse model
Introduction
Methamphetamine (METH) addiction is a serious psychosocial
problem, which leads to organic harm of the body as well as
distortion of the normal functioning of affected people within
the society and family. There is a large body of clinical evidence
suggesting differential characteristics of the disorder in men and
women. Despite the absolute number of female METH abusers
being lower than the male ones, women usually appear more
dependent, show higher escalation rates (1, 2) and most importantly
tend to experience more frequent relapses (3, 4). These
gender specific differences require specific treatment strategies
for men and women (5–7). This particularly applies to relapseprevention,
which represents a key treatment challenge especially
for women (8).
The preclinical approach to model drug addiction with the
highest validity is usually considered as the operant drug self
administration. To mimic relapse in this paradigm, a period of
extinction procedure can be employed when the animal still has
a regular access to the operant box but the drug delivered by infusion
pump is replaced by vehicle. After certain number of sessions,
the subject stops to respond to the active operandum (e.g., lever or
nose-poke). After reaching a specific extinction criteria (number
of active/inactive responses lower than a set number), one last
session is conducted and the reinstatement of the drug-seeking
behavior is primed by an environmental factor (stress, cues) or a
drug dose. Such studies have repeatedly shown female rats to be
more vulnerable to drug-primed relapse of METH seeking behavior
at conditions of time limited sessions (2 h), which mimic rather
consummatory behavior, as well as prolonged self-administration
sessions. This is considered to provide a better model for loss of
control over drug taking, leading to escalation of drug consumption
(9) known from a clinical situation (3). Similarly, a higher
relapse-like behavior was found in female rats after priming by
conditioned cue and to even higher extent by METH dose (10).
Earlier, analogous results were reported in studies with cocaine
(11, 12) and fentanyl (13).
However, this paradigm does not mimic the human treatment
very well, because the patient usually discontinues the drug abuse
in the drug rehabilitation center and for some time does not
have access to the drug-related environments. Therefore, a forced
abstinence model was developed where the animal does not have
access to the operant box and is kept in the home cage for some
time (14–16); thus, the motivation of drug response behavior is
not influenced by any training procedures.
Furthermore, many preclinical studies, which assess sexdependent
differences, isolate the hormonal effect either by
ovariectomy and subsequent hormonal supplementation (17, 18)
or by constant tracking of the estrous cycle phase (10, 19). These
approaches already explained extensively the role of gonadal hormones
in the reward processes showing enhancement of drug
intake by estradiol (17, 18, 20–22) and attenuation of drug seeking
by progesterone (4, 23). However, the possibilities of clinical
applications of these findings are limited, so far only progesterone
was tested as a treatment for nicotine relapse in women (24) and
such treatment would have many undesirable side effects. Consequently,
an ideal animal model with high face, construct, and
predictive validity for testing new relapse-prevention treatments
should not be based on hormonal levels only.
The intact animals (males and freely cycling females) showed
no sex differences to effects of amphetamines in the animal model
of conditioned place preference (CPP) (25, 26). Interestingly, CPP
for METH did not occur in ovariectomized rats but developed in
females treated with estradiol (27). Therefore, gender differences
in the CPP paradigm might be biased by fluctuating hormonal
levels in intact females. However, results supporting higher vulnerability
to METH in intact female rats were reported too. Female
rats displayed higher increase of locomotor activity, which lasted
for longer time and had higher scores of stereotypies than male
rats (28). These results indicate the sex differences may depend,
besides hormonal influences, also on different pharmacokinetic
processes in females (29).
Therefore, the aim of this study was to assess gender differences
in all stages of operant IV self-administration of METH
in male and female rats while the gonads of all animals were
kept intact assuring physiological estrous cycle in females. We
expected a higher variability in the female group, especially in
the reinstatement of METH seeking behavior due to different
hormonal stages. However, we hypothesized that this variability
may be overpowered by all other significant gender differences.
Furthermore, we assessed possible gender differences in acquisition
and maintenance of food self-administration in order to
compare the operant behavior toward natural reward (food) and
the drug of abuse.
Materials and Methods
Animals
Eight-week-old male and female albino Sprague-Dawley rats
weighing 175–200 g (females) and 200–225 g (males) at the beginning
of the experiment were purchased from Charles River (Germany).
The rats were housed individually in standard rat plastic
cages, the experiments on males and females were performed
separately, to assure the self-administration room is dedicated
to one gender at a time only. Environmental conditions during
the whole study were constant: relative humidity 50–60%, temperature
23 ± 1°C, inverted 12-h light–dark cycle (6 a.m. to 6
p.m. darkness). Food and water were available ad libitum. All
experiments were conducted in accordance with all relevant laws
and regulations of animal care and welfare. The experimental
protocol was approved by the Animal Care Committee of the
Masaryk University, Faculty of Medicine, Czech Republic, and
carried out under the European Community guidelines for the use
of experimental animals.
Drugs and Treatments
Methamphetamine from Sigma Chemical, Co., St Louis, MO,
USA available in the operant cage for IV self-administration was
0.08 mg/kg per infusion with the maximum number of infusions
obtainable in one session set to 50. The solutions were prepared
for specific animals depending on their body weights rounded
to the closest category of 250, 300, 350 g, etc. This paradigm is
adapted from Emmett-Oglesby MW (Fort Worth, TX, USA) (30)
and routinely used in our laboratory (17, 31–33).
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Ruda-Kucerova et al. Sex differences in METH relapse model
Locomotor Activity Test
After adaptation period at the beginning of the study basal behavioral
profile was assessed in both males and females. In brightly
lit room, rats were individually tested for locomotor activity using
the Actitrack system (Panlab, Spain) as previously described (34,
35). Each Plexiglas arena (45 cm × 45 cm × 30 cm) was equipped
with 2 frames equipped with photocells located one above another
2 and 12 cm above the cage floor. Each animal was placed in the
center of arena and the spontaneous behavior was tracked for
10 min. During the test, the horizontal locomotor activity (the trajectory
as calculated by the system from beam interruptions that
occurred in the horizontal sensors) and vertical activity (number
of rearing episodes breaking the photocell beams of the upper
frame) were recorded. At the end of the session, animals were
returned to their home cage and arenas were wiped with 1% acetic
acid to avoid olfactory cues.
Intravenous Drug Self-Administration Surgery
Animals were deeply anesthetized with i.p. injections of 50 mg/kg
ketamine plus 8 mg/kg xylazine. Under aseptic conditions, a permanent
intracardiac silastic catheter was implanted through the
external jugular vein to the right atrium. The outer part of the
catheter exited the skin in the midscapular area. After surgery,
each animal was allowed for recovery, individually in its home
cage with food and water freely available. Since the implantation,
the catheters were flushed daily by heparinized cephazoline (Vulmizolin
1.0 g) solution followed by 0.1 ml of a heparinized (1%)
sterile saline solution to prevent infection and occlusion of the
catheter. During recovery, changes in general behavior and body
weight were monitored. When a catheter was found to be blocked
or damaged, the animal was excluded from the analysis. At the end
of the study, there were n = 6 male and n = 6 female rats included
to the analysis.
Intravenous Self-Administration Protocol
Methamphetamine self-administration was conducted as
previously described (17, 32) in 10 standard experimental
boxes (30 cm × 25 cm × 30 cm, Coulbourn Instruments, USA)
using nose-poking as operandum under a FR-1 schedule of
reinforcement, i.e., animal had to make 1 nose-poke on the active
hole to obtain a single drug infusion. Each cage was provided
with two nose-poke holes allocated on one side and programed by
software Graphic State Notation 3.03 (Coulbourn Instruments,
USA). Nose-pokes in the active hole led to the activation of the
infusion pump and administration of a single infusion followed
by a 10 s timeout, while nose-poke stimulation was recorded but
not rewarded. The cage was illuminated by a house light during
the session. The light was flashing when administering infusion
and off during the time-out period. Self-administration sessions
lasted 90 min and took place 7 days/week for 2 weeks in total
between 8 a.m. and 3 p.m. during the dark period of the inverted
light–dark cycle.
After 14 days of stable METH intake, the maintenance phase
was terminated and rats were returned to their home cages for
the 14 days of the forced abstinence period. On day 15, rats
were placed into self-administration chambers for the last 90 min
reinstatement session. The numbers of responses on the active
drug-paired nose-poke and the inactive nose-poke were recorded
but the drug was not delivered. Responses on the active nose-poke
are considered to reflect the reinstatement of drug-seeking behavior,
while responses on inactive nose-poke reflect non-specific
locomotor and exploratory activity.
Food Self-Administration Protocol
Food self-administration was conducted in the same experimental
boxes as METH study (Coulbourn Instruments, USA) in a
separate batch of animals. Under the FR-1 schedule of reinforcement
1 nose-poke lead to activation of a feeder and delivery
of a single palatable pellet (BioServ, sweet dustless rodent pellets,
F0021-Purified Casein Based Formula – 45 mg). The cage
was illuminated by a house light during the whole session. Selfadministration
sessions lasted 30 min during the dark period of
the inverted light–dark cycle.
Statistical Data Analysis
Primary data were summarized using arithmetic mean and SE
of the mean estimate. Behavioral data were analyzed by t-test.
IV METH self-administration data during the 14 days of maintenance
were analyzed at individual days by t-test and at 5-day
intervals by mixed ANOVA model with Greenhouse–Geisser correction.
Acquisition of food self-administration was evaluated by
comparison of mean day of reaching 70% preference of active
nose poke by Mann–Whitney U test. Maintenance of food selfadministration
was analyzed at individual days by t-test. Statistical
analyses were computed using SPSS 19.0.1 (IBM Corporation,
2010). A p-value <0.05 was recognized as boundary of statistical
significance in all applied tests.
Results
Basal Locomotor Characteristics
Before starting the IV self-administration protocol, basal locomotor
and exploratory activity was assessed in both males and
females to exclude the possibility that these characteristics would
lead to different drug taking behavior. Horizontal and vertical
locomotor activity was measured and a proportion of each in the
inner zone of the arena was calculated in order to evaluate differences
in the status of anxiety in male and female rats. Figure 1
illustrates the results on total distance traveled, vertical activity
(number of rearing episodes), and inner part of arena preference.
There were no basal behavioral differences between the sexes,
which could contribute to dissimilar behavior in the operant cage.
As expected, both sexes avoided the central part of the arena,
which represents normal rodent behavior and neither one shows
highly anxiogenic behavior or locomotor hyper- or hypo-activity.
Acquisition and Maintenance of
Methamphetamine Self-Administration in Male
and Female Rats
The acquisition and maintenance of METH taking behavior
were assessed, first, in terms of mean number of infusions
self-administered per session and, second, by the mean METH
dose per session in milligram per kilogram. Figure 2A shows
number of infusions obtained per daily session and mean number
of infusions during the entire acquisition phase in male and female
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Ruda-Kucerova et al. Sex differences in METH relapse model
FIGURE 1 | Male and female rats have the same basal behavioral
profile. Total distance traveled (in centimeters), number of rearing
episodes (vertical activity), and % of preference of the central part of the
arena did not reveal any gender difference. Data are shown as means
(±SEM), t-test, n.s.
rats during the acquisition phase of METH self-administration
training. ANOVA revealed no significant effects over the whole
period. However, when the number of infusions was converted
to a METH dose per kilogram of body weight, males were
found to self-administer higher dose at the end of the acquisition
phase as compared to females. More specifically, as depicted
in the Figure 2B, mean METH intake during the last 5 days
of training was significantly higher in males than in females,
i.e., 2.5 and 1.5 mg/kg, respectively (mixed ANOVA model:
p = 0.038).
Reinstatement of Methamphetamine
Self-Administration in Male and Female Rats
After the 2-week-long period of forced abstinence one last reinstatement
session was performed with no drug availability. The
only measure of the drug-seeking behavior is the number of
active operandum responses. This number was converted to
a percent of mean basal nose-poking (14 days of acquisition
and maintenance). There was a massive difference between the
sexes recorded: male rats showed mean percent of responding
48.3% whereas females showed 295.7% (mixed ANOVA model,
p = 0.001). Results are reported on the Figure 3.
Acquisition of Food Self-Administration in Male
and Female Rats
The acquisition of food taking behavior (sweet pellets) was
assessed in terms of day when the animals started to prefer the
active nose-poke more than 70%. Figure 4A shows the development
of active nose poke preference (%) over all sessions in male
and female rats. Figure 4B reports the mean day for reaching 70%
preference of the active operandum, which was 4.7 in males and
2.2 in females (Mann–Whitney U test, p = 0.014). The maintenance
phase of the food self-administration was evaluated as a
mean number of self-administered pellets during the last 5 days
when the intake was stable. Figure 5 depicts the significantly
higher pellet intake in female rats as compared to males (138–175
and 51–73, respectively, p ≤ 0.05).
Discussion
Findings of the present study demonstrated that male and female
rats had equal basal locomotor and exploratory activity. Thus,
differences in operant IV self-administration cannot be accounted
for differences in locomotor activity. Furthermore, the food selfadministration
has shown a very different dynamics than the
METH study, suggesting higher motivation to obtain natural
reward (sweet pellet) in females, which learned the operant procedure
faster (acquisition) and self-administered approximately
three times more pellets than males. This behavior toward natural
reward is very different from METH-related operant behavior,
which rules out the possibility of general gender specific difference
in the reward processes.
During the maintenance phase of the METH selfadministration,
female rats were found to self-administer
the same number of infusion, but their METH intake in terms of
dose per kilogram of body weight was found lower. This measure
is not widely used among the self-administration studies, usually
only the numbers of nose pokes (or lever presses) and infusions
are reported. However, we propose this measure to be considered
as highly valid for several reasons. Despite the solution of the
drug being available in the operant box matches the body weight
of the particular animal, the solutions are prepared for certain
body weight category, e.g., solution for animal weighting 300 g
can be used for rats reaching approximately 280–320 g (this fact
is usually not described exactly in the Section “Materials and
Methods” of the papers). This discrepancy, aggravated by the
fact that the body weight of the animal changes over the course
of the experiment, could be a source of significant differences
in the dose taken even at conditions of the same number of
infusions. This is a confounding factor, which complicates the
comparison of findings from different laboratories. Furthermore,
this approach should be used when the number of behavioral
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FIGURE 2 | Acquisition and maintenance of methamphetamine intake in
male and female rats. The (A) part shows number of infusions expressed as
daily means over the 14 days of acquisition and maintenance of the METH IV
self-administration. The bar graph depicts the mean number of infusions over
the whole 14 days period. There were no statistically significant differences in
this measure (mixed ANOVA model). The (B) part shows in an analogical way
the mean dose in milligram per kilogram of METH self-administered by male
(n = 6) and female (n = 6) rats. The groups start to differ significantly from the
day 10 with t-test results: day 10 (p = 0.021), day 11 (p = 0.049), and day 14
(p = 0.048). The bar graph shows the mean number of infusions over the last
5 days of the maintenance period (day 10–14) when the drug intake started to
be significantly higher in male rats (p = 0.038, mixed ANOVA model).
FIGURE 3 | Reinstatement of methamphetamine seeking behavior in
male and female rats. The graphs show a percent of mean basal
nose-poking (14 days of acquisition and maintenance) and number of
nose-pokes in the reinstatement session in male and female rats. There was
a statistically significant difference between the sexes in both measures:
male rats showed mean % of responding 48.3% and females 295.7%
(mixed ANOVA model, p = 0.001). The apparent difference between the
sexes is further confirmed by behavioral activity reflected in a mean number
of nose-pokes: 41.0 in males and 136.5 in females (mixed ANOVA model,
p = 0.006).
responses (nose pokes or lever presses) does not match the
number of infusions delivered. This is always the case when the
system uses nose poke operandi (and in some cases levers which
do not retract after infusion delivery).
Previous studies have shown that female rats to be more vulnerable
to behavioral effects of psychomotor stimulants including
cocaine (36–38) and, in particular, amphetamines (including
METH), which elicited a higher increase of locomotion in females
than males or reach the same behavior profile at lower dose (25,
28, 39, 40). Other studies have repeatedly shown that females
with intact gonads tend to develop readily behavioral sensitization
to psychostimulant drugs after repeated treatment (41–43).
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FIGURE 4 | Acquisition of food self-administration behavior in male
and female rats. The (A) part shows the course of the active nose poke
preference development during acquisition and maintenance of the food
self-administration. The bar graph (B) depicts the mean day the animals
reached and kept 70% preference of the active operandum: 4.7 in males and
2.2 in females (Mann–Whitney U test, p = 0.014).
FIGURE 5 | Maintenance of food self-administration in male and
female rats. The graph shows pellet intake in female rats as compared to
males (t-test, *p ≤ 0.05, ***p ≤ 0.001).
Furthermore, there is new evidence of specific pharmacokinetic
differences in METH self-administration studies, where males
were shown to have lower area under curve (AUC) of METH probably
due to rapid drug elimination (29). The apparent higher efficacy
of the amphetamines found in this and previously mentioned
studies in female rats could be explained by the pharmacokinetic
differences.
Similarly, in clinical studies, there has been shown that men
are more sensitive to the reinforcing effects of a high dose of
-amphetamine than women, who respond rather to low doses at
a random phase of the menstrual cycle (44). This is consistent with
our data, which showed that males developed higher stable intake
of METH than females (2.5 and 1.5 mg/kg daily, respectively).
Furthermore, women were shown to experience greater increases
in diastolic pressure and nausea than men at the same doses while
the ability to discriminate -amphetamine was equal in both sexes
(45). These lines of evidence further support translational validity
of our finding of lower METH intake in female rats.
However, in fixed ratio self-administration paradigms, the
reports on gender differences in the maintenance phase are
numerous and quite contradictory in both clinical (45, 46) and
preclinical studies, showing both higher and lower drug intake in
female subjects (21, 47).
Progressive ratio IV self-administration paradigm or prolonged
access to the drug might be better tools to unravel gender differences
as these may be linked to appetitive behavior (21). Female
rats have been repeatedly shown to achieve higher breaking points
in METH self-administration study suggesting higher motivation
to obtain the drug (10, 48). This is consistent with the robust gender
difference in the reinstatement found in this study, where the
motivation of animals for the drug-seeking was not abolished by
extinction training. At this point, active responses to the operandum
are the only measure to report because the session is performed
without delivering the drug. We found a highly significant
difference in the percent of mean basal nose-poking, as well as in
the absolute number of active operant responses. The enhancing
effect of estradiol and attenuating effects of progesterone on psychostimulant
(-amphetamine, METH, cocaine) intake in female
gender is repeatedly and consistently reported in both clinical
(49–52) and preclinical studies (17, 20–22). Therefore, the higher
variability in the reinstatement operant responding in the female
group detected in this study probably originated from different
hormonal stage. This conclusion can be supported by an earlier
study, which employed the extinction and both drug- and cueprimed
reinstatement, where females were found more vulnerable
in both reinstatement procedures and also exhibited higher variability
than males. Interestingly, the numbers of lever presses in
the conditioned cue-primed reinstatement session were approximately
40 in males and 120 in females (10). These absolute numbers
are similar to those reported in the present study: 41 and 136,
respectively. Therefore, this effect seems to be well reproducible
and strain independent (Long-Evans vs. Sprague-Dawley rats).
The forced abstinence model was proposed as a potentially
better tool to model a spontaneous relapse in rodents (15, 53).
To our knowledge, this is the first report of gender differences
in the paradigm of reinstatement after forced abstinence.
Extinction-based approach to study relapse-like behavior phase
in the preclinical setting show contradictory results – females
appear to meet the extinction criteria later than males (11), but
negative results have been reported as well (54). Both studies were
conducted with cocaine.
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Taken together, this study reports lower consummatory METH
intake during maintenance phase of the self-administration
together with higher vulnerability to the reinstatement of METH
seeking behavior in female rats after forced abstinence. These
effects seem to be robust enough, thus relatively independent on
the current hormonal level. Therefore, we propose this paradigm
for preclinical screening for potential new medications specific for
women. However, the main limitation for the translation of these
results to human medicine is the absence of psychosocial aspects,
which are impossible to reflect in animal studies.
Acknowledgments
The authors are grateful to Amy Chen and Heejae Chung
(Toronto, ON, Canada) for their kind help with manuscript
preparation and proof reading. This study was financed from
the SoMoPro II programme. The research leading to this
invention has acquired a financial grant from the People Programme
(Marie Curie action) of the Seventh Framework Programme
of EU according to the REA Grant Agreement No.
291782. The research is further co-financed by the SouthMoravian
Region. The study reflects only the author’s views
and that the Union is not liable for any use that may
be made of the information contained therein. Further cofinancing
was by the project “CEITEC – Central European
Institute of Technology” (CZ.1.05/1.1.00/02.0068) from European
Regional Development Fund, project of specific research
at the Masaryk University (MUNI/A/1116/2014), and the Internal
project of the Faculty of Medicine at Masaryk University
(MUNI/11/InGA09/2012).
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Conflict of Interest Statement: The authors declare that the research was conducted
in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Copyright © 2015 Ruda-Kucerova, Amchova, Babinska, Dusek, Micale and Sulcova.
This is an open-access article distributed under the terms of the Creative Commons
Attribution License (CC BY). The use, distribution or reproduction in other forums is
permitted, provided the original author(s) or licensor are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No
use, distribution or reproduction is permitted which does not comply with these terms.
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5. Discussion
5.1. Depression-addiction comorbidity
The self-medication hypothesis was recently challenged. The main reason is that different
types of psychiatric patients should opt for different drugs of abuse. For example, a patient
with attention deficit disorder would prefer amphetamines, while a person with elevated
anxiety would prefer alcohol known for its anxiolytic properties. However, this paradigm
does not reflect the real clinical situation where the population of psychiatric patients does
indeed have increased rate of addiction over general population but the drug choice does
not correlate with the disorder. Furthermore, the statement of resolving addiction issues
with adequate treatment of the underlying psychiatric disease was not confirmed either
(Lembke, 2012).
The scientific discussion was not concluded yet as there are new papers at least partially
confirming this theory. Despite the questioned validity of the self-medication hypothesis,
the existence of shared neurochemical and functional distortions in psychiatric disorders
and addiction (Goodkind et al., 2015) remains valid. This fact is widely supported by the
clinical experience showing a strong association of the psychiatric morbidity with drug
abuse (Conner et al., 2008a, Conner et al., 2008b, Nunes and Levin, 2004) and
interestingly also non-drug addictions such as internet addiction (Ho et al., 2014) or
pathological gambling (Lorains et al., 2011).
Our model of the depression and addiction comorbidity combining the olfactory
bulbectomy (OBX) and operant drug self-administration seems to mimic the clinical
situation quite well showing increased rate of drug taking similarly as in clinical reports
(Kushnir et al., 2013, Pettinati et al., 2013). Specifically, in this model the depressive-like
rats showed higher intake of amphetamine (Holmes et al., 2002), methamphetamine
(Kucerova et al., 2012), synthetic CB1 receptor agonist WIN55,212-2 (Amchova et al.,
2014), ketamine (Babinska and Ruda-Kucerova, 2016) and 10% alcohol (Grecksch and
Becker, 2015). This indicates an analogous reaction of animals in the OBX model towards
drugs of abuse with substantially different mechanisms of action. Therefore, we expect a
general distortion of the reward mechanisms in the OBX model. We tried to shed some
light on the matter by evaluating extracellular levels of dopamine before and after a drug
151
challenge and assessed a comprehensive profile of neurotransmitters in the nucleus
accumbens shell in the OBX model. We observed lower basal levels of extracellular
monoamines, i.e. dopamine, serotonin and their metabolites, and increased levels of
glutamate and GABA. However, after a challenge dose of methamphetamine we detected a
higher release of monoamines, lower levels of glutamate and no effect on GABA (RudaKucerova
et al., 2015b). Interestingly, in the study on WIN55,212-2 self-administration we
observed an apparently opposite effect of the challenge dose of the drug on dopamine
release in the nucleus accumbens shell (Amchova et al., 2014). Following Figure 6 and
Figure 7 show data on the dopamine release reported in the corresponding studies.
(Source: Ruda-Kucerova et al., 2015b)
Figure 6: relative dopamine release in the nucleus accumbens shell after methamphetamine
challenge dose
152
(Source: Amchova et al., 2014)
Both figures depict the same variable – a percent of basal dopamine level after the drug
dose in intervals of 20 minutes. First, there is a high difference between the overall effects
of the challenge dose. There is only moderate - 40% - release after WIN55,212-2 dose
while methamphetamine dose has induced a 400% to 500% increase of the release. This
can be explained by different mechanism of action of psychostimulant and cannabimimetic
drugs. Psychostimulants act as indirect sympathomimetic agents producing a strong release
of monoamines (Chiu and Schenk, 2012, Yu et al., 2015). Similar results of WIN55,212-2
(Lecca et al., 2006, Scherma et al., 2016) and methamphetamine (Izawa et al., 2006, Kai et
al., 2015), effects on dopamine release in the nucleus accumbens shell were already
reported.
There is a clearly different response of dopamine release in OBX animals to a challenge
dose in both studies. We observed no effect in the OBX animals after the WIN55,212-2
treatment while there was significantly increased dopamine release after acute
methamphetamine dose. The explanation is likely to be related to the drug doses used in
the experiments. In case of WIN55,212-2 a dose of 0.3 mg/kg was chosen as this is the
usual amount which is self-administered by control animals and was also found in the selfadministration
part of this study. However, the other set of experiments aimed to evaluate
methamphetamine effects on many neurotransmitters and a dose known to exert a very
strong effect was selected - 5 mg/kg. The usual operant intake of methamphetamine by
Figure 7: relative dopamine release in the nucleus accumbens shell after WIN55,212-2
challenge dose
t
153
control animals is around 2 mg/kg and it rises up to 3 mg/kg in OBX rats. Similarly, we
reported that OBX rats doubled their intake of WIN55,212-2 compared to control group.
Taken together it seems that OBX rats need a higher dose to experience the same
dopamine release in the nucleus accumbens shell and consequent rewarding effects which
explains increased drug taking in the operant paradigms. Furthermore, the higher relative
release of dopamine shown in the OBX rats after 5 mg/kg of methamphetamine may be
baseline dependent. Figure 8 shows an unpublished comparison of the data presented in
the paper as relative values (%). The dopamine release is depicted in absolute values
(pg/ml) and the different basal level can be appreciated. In this comparison the peak levels
are not statistically different.
(Source: unpublished comparison of data from Ruda-Kucerova et al., 2015b)
Figure 8: absolute values (pg/ml) of dopamine in the nucleus accumbens shell after
methamphetamine challenge dose
154
Findings from both studies point to a differential dose-response to drugs of abuse in the
OBX model. We hypothesize a shift of the theoretical dose-response curve to right (Figure
9) in the OBX model. This assumption needs to be tested in order to conclude the matter.
Figure 9: theoretical dose-response curve
(Source: author's own figure)
This hypothesis can also be disputed by the fact that our in vivo microdialysis
measurements evaluated only acute effects of the drugs. Besides the dose-response
assessment of acute drug dose also a chronic study should be performed in order to
estimate the effects of long-term exposure to the drug. Interestingly, the increased intake in
the OBX animals in operant self-administration studies usually develops over time, usually
after two or three weeks.
Relapse-like behaviour was not found to be consistently increased in the OBX group.
While this effect was seen in studies using psychostimulants, i.e. methamphetamine
(Babinska et al., 2016) and cocaine (Frankowska et al., 2014), we found an opposite effect
in a ketamine self-administration study (Babinska and Ruda-Kucerova, 2016). However,
the relapse after a period of abstinence induces quite different neurochemical adaptations
than chronic exposure to the drug (See, 2005, Robinson, 1993, Self, 1998). Therefore,
extracellular dopamine levels should be again assessed in this model of relapse to explain
the reinstatement behaviours of the OBX animals.
155
5.2. Schizophrenia-addiction comorbidity
We have only recently extended our research of psychiatric dual disorders to an animal
model of schizophrenia and drug abuse comorbidity. Also, we introduced a paradigm of
alcohol drinking to our laboratory. This method is based on the drinking in the dark
approach, where animals have a time limited access to alcohol during the dark phase of the
light cycle (Samson et al., 1988, Czachowski, 2005). We have chosen this paradigm
because it resembles the operant self-administration studies. To model schizophrenia-like
phenotype in rats we employed a neurodevelopmental model, because human
epidemiological data supports the fact that pre-perinatal environmental factors such as
malnutrition, infection and obstetric complications increase the risk of developing
schizophrenia (Brown et al., 2012). This knowledge has stimulated the development of
models based on direct pre-perinatal damage of the central nervous system, which replicate
several behavioural and neurochemical changes linked to the disease. In line with this
approach, rats prenatally exposed to methylazoxymethanol (MAM), an antimitotic agent
that methylates DNA, show behavioural (hyperactivity, cognitive and social deficits,
disruption of pre-pulse inhibition) and histopathological (hyperdopaminergia) patterns
similar to those observed in schizophrenia (Lodge et al., 2009, Lodge and Grace, 2009).
The advantage of neurodevelopmental over pharmacological (acute) models of
schizophrenia is the ability to perform behavioural and neurochemical investigations in the
absence of confounding drugs and identification of new classes of antipsychotics by the
use of agents operating on multiple pharmacological mechanisms (Micale et al., 2013).
So far we have published only one study with this dual disorder animal model which
includes methamphetamine self-administration and alcohol drinking in a
neurodevelopmental model of schizophrenia (Ruda-Kucerova et al., 2016). The most
interesting finding of this study was that the female sex and schizophrenia-like phenotype
induced by the prenatal MAM exposure may work synergistically to enhance alcohol
consumption. There are other reports evaluating alcohol intake in other animal models of
schizophrenia. Most of the papers presented findings from neonatal ventral hippocampal
lesion (NVHL) model (Tseng et al., 2009, O'Donnell, 2012). Interestingly, in this model a
key role of adolescence period was proven when a moderate exposure to alcohol was
shown to sensitize the animals towards the same drug in adult age. The authors did not find
differences in alcohol intake during adolescence or adult age without previous exposure
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(Jeanblanc et al., 2014). A similar phenomenon was reported in the MAM model in the
vulnerability to diazepam at adult age with adolescent exposure (Du and Grace, 2013).
Therefore, adult animals in the MAM model might exhibit a different drug-taking
behaviour after their early exposure to the drug. The part of our study focused on
methamphetamine self-administration did not reveal any important significant differences
between MAM and control animals. Negative results were also reported earlier in case of
cocaine IV self-administration in the MAM model (Featherstone et al., 2009).
The usefulness of the MAM model in the study of dual disorders can be questioned.
However, increased reactivity to amphetamine psychostimulants is routinely used as a test
of positive-like symptoms of schizophrenia in the model (Gill et al., 2011, Lodge and
Grace, 2009). Therefore, a hypothesis of distorted reward mechanism could be valid.
However, so far the paradigm which would reveal these changes was not found.
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5.3. Sex-differences in drug abuse
Sex differences in drug abuse were repeatedly assessed in both clinical studies (Becker and
Hu, 2008) and animal models (Roth et al., 2004). Our team has contributed to the research
by evaluation of oestrogen levels on methamphetamine self-administration. Furthermore,
we included a variable of potential development of behavioural sensitization showing a
decreased drug intake in previously sensitized rats of both genders (Kucerova et al., 2009).
Later, we aimed at evaluation of relapse-like behaviour accepting physiological oestrous
cycle of female rats as a confounding factor. Despite this limitation we observed higher
drug-seeking behaviour after a period of abstinence, which reflects higher motivation of
females to find methamphetamine (Ruda-Kucerova et al., 2015a). Together, our data
indicate that despite a strong influence of the sex hormones, intact females do exhibit
different relapse-like behaviour. This is in accordance with previous pre-clinical (Cox et
al., 2013) and clinical studies (Grella and Lovinger, 2011).
There are already published many studies exploring all phases of drug abuse (i.e.
acquisition, maintenance, withdrawal symptoms, abstinence, relapse) in both women and
female animal subjects. However, gender specific issues in the dual disorders are still
rarely assessed in the pre-clinical setting. So far, we attempted to assess differential
reactivity of female MAM rats towards drugs of abuse and we actually found increased
vulnerability in females in the MAM model towards alcohol drinking (Ruda-Kucerova et
al., 2016).
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6. Conclusion and future perspectives
Drug addiction is a serious condition harmful to all aspects of the afflicted person’s life.
Substance abuse is often comorbid with other psychiatric disorders where it may be either
a primary cause or a secondary consequence of the disease. In order to develop innovative
treatment strategies for these psychiatric dual disorders a downward translation to animal
models is essential.
On the field of depression and addiction dual disorder modelling we propose a well
validated combination of olfactory bulbectomy (OBX) and operant self-administration of
drugs for testing of new anti-addiction treatments specific for depressive individuals. The
ultimate aim of our research is to identify a pharmacological mechanism responsible for
the OBX induced distortions in the reward pathways and test drugs which may reverse
these neuroplastic changes. Such candidate drug might be low dose ketamine. There is
currently an extensive research on its antidepressant effects in both preclinical studies
(Scheuing et al., 2015) and clinical trials (Newport et al., 2015, Xu et al., 2015). We
performed a pilot experiment evaluating the effect of acute pre-treatment (5 mg/kg of
ketamine 30 minutes before the session) on IV self-administration of methamphetamine
and observed a strong suppression of the drug-taking behaviour (unpublished data). This
line of research deserves further investigation in order to explore this phenomenon in depth
and publish a concise study.
There are only few preclinical studies aiming for evaluation of addictive behaviours in
schizophrenia-like phenotype. In future we will design studies for identification of
experimental approach sensitive enough to reflect the hypothesized distortions in the
reward processes of the neurodevelopmental model of schizophrenia induced by prenatal
administration of methylazoxymetanol acetate (MAM). Exposing adolescent rats to
addictive substances and testing their abuse liability later in life will be the first because
adolescence seems to be a vulnerable period as reported in an alcohol drinking study in
another neurodevelopmental model of schizophrenia (Jeanblanc et al., 2014) and in
vulnerability to benzodiazepines in the MAM model (Du and Grace, 2013).
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The research on dual disorders should be extended to evaluation of sex differences in the
animal models. So far, we have identified higher sensitivity of female MAM rats towards
alcohol drinking. Female OBX rats are rarely used but the available evidence suggests they
also exhibit a depressive-like phenotype (Stepanichev et al., 2016, Stock et al., 2000,
Pudell et al., 2014) and may provide a useful tool to study sex differences in drug taking
behaviours in context of a depression and addiction dual disorder.
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7. List of papers related to the habilitation thesis
7.1. Publications in extenso in journals with IF
Ruda-Kucerova J, Babinska Z, Amchova P, Stark T, Drago F, Sulcova A, Micale V.
Reactivity to addictive drugs in the methylazoxymethanol (MAM) model of schizophrenia
in male and female rats. World J Biol Psychiatry. 2016, doi:
10.1080/15622975.2016.1190032, in press.
IF (2015) 4.159
Citations (WOS): 0
Babinska Z, Ruda-Kucerova J, Amchova P, Merhautova J, Dusek L, Sulcova A.
Olfactory bulbectomy increases reinstatement of methamphetamine seeking after a forced
abstinence in rats. Behav Brain Res. 2016, 297: 20-7, doi: 10.1016/j.bbr.2015.09.035.
IF (2015) 3.002
Citations (WOS): 0
Ruda-Kucerova J, Amchova P, Havlickova T, Jerabek P, Babinska Z, Kacer P, Syslova
K, Sulcová A, Sustkova-Fiserova M. Reward related neurotransmitter changes in a model
of depression: An in vivo microdialysis study. World J Biol Psychiatry. 2015, 16(7): 521-
35. doi: 10.3109/15622975.2015.1077991.
IF 4.159
Citations (WOS): 0
Kucerova J, Babinska Z, Horska K, Kotolova H. The common pathophysiology
underlying the metabolic syndrome, schizophrenia and depression. A review. Biomed Pap
Med Fac Univ Palacky Olomouc Czech Repub. 2015, 159(2): 208-14. doi:
10.5507/bp.2014.060.
IF 0.924
Citations (WOS): 2
This paper is included in this thesis as Appendix 1.
161
Amchova P, Kucerova J, Giugliano V, Babinska Z, Zanda MT, Scherma M, Dusek L,
Fadda P, Micale V, Sulcova A, Fratta W, Fattore L. Enhanced self-administration of the
CB1 receptor agonist WIN55,212-2 in olfactory bulbectomized rats: evaluation of possible
serotonergic and dopaminergic underlying mechanisms. Front Pharmacol. 2014, 5: 44. doi:
10.3389/fphar.2014.00044.
IF 3.802
Citations (WOS): 5
Micale V, Kucerova J, Sulcova A. Leading compounds for the validation of animal
models of psychopathology. Cell Tissue Res. 2013 Oct;354(1):309-30. doi:
10.1007/s00441-013-1692-9.
IF 3.333
Citations (WOS): 8
This paper is included in this thesis as Appendix 2.
Kucerova J, Pistovcakova J, Vrskova D, Dusek L, Sulcova A. The effects of
methamphetamine self-administration on behavioural sensitization in the olfactory
bulbectomy rat model of depression. Int J Neuropsychopharmacol. 2012, 15(10): 1503-11.
doi: 10.1017/S1461145711001684.
IF 5.641
Citations (WOS): 8
Kucerova J, Vrskova D, Sulcova A. Impact of repeated methamphetamine pretreatment
on intravenous self-administration of the drug in males and estrogenized or nonestrogenized
ovariectomized female rats. Neuro Endocrinol Lett. 2009, 30(5): 663-70.
IF 1.047
Citations (WOS): 13
162
7.2. Publications in extenso in journals without IF
Ruda-Kucerova J, Amchova P, Babinska Z, Dusek L, Micale V, Sulcova A. Sex
differences in the reinstatement of methamphetamine seeking after forced abstinence in
Sprague-Dawley rats. Front Psychiatry. 2015, 6: 91. doi: 10.3389/fpsyt.2015.00091.
Citations (WOS): 1
Amchová P, Kučerová J. Pohlaví a drogová závislost: od animálních modelů ke klinické
praxi. Česká a Slovenská Psychiatrie, Praha: Česká lékařská společnost J.E.Purkyně, 2015,
111(2): 72 -78.
This paper is included in this thesis as Appendix 3.
Babinská Z, Kučerová J. Spoločné neurobiologické mechanizmy depresie a
metamfetamínovej závislosti. Alkoholizmus a drogové závislosti, Bratislava: Obzor, 2014,
49(3): 127-152.
This paper is included in this thesis as Appendix 4.
Kucerova J, Tabiova K, Drago F, Micale V. Therapeutic potential of cannabinoids in
schizophrenia. Recent Pat CNS Drug Discov. 2014, 9(1): 13-25. Review.
This paper is included in this thesis as Appendix 5.
Kucerova J, Pistovcakova J, Vrskova D, Dusek L, Sulcova A. Aripiprazole does not
influence methamphetamine I.V. self-administration in rats. Activitas nervosa superior
rediviva, 2010. 52(4): 261-266.
This paper is included in this thesis as Appendix 6.
Kucerova J, Sulcova. Comparison of behavioural sensitization to ecstasy in mouse males
and ovariectomized females with and without oestrogen substitution. Activitas Nervosa
Superior, 2008. 50(1-2): 18-19.
163
Kučerová J. Vliv behaviorální senzitizace a pohlaví na metamfetaminovou závislost u
člověka a ve zvířecím modelu. Alkoholismus a drogové závislosti, 2008. 43(5): 295-309.
Kucerova J, Novakova J, Landa L, Sulcova A. Gender differences in cannabinoid and
ecstasy interacting effects in mice. Homeostasis in Health and Disease, 2006. 44(1-2): 95-
96.
7.3. Book chapters
Micale, V., Tabiová, K., Kučerová, J. and Drago, F., Role of the endocannabinoid system
in depression: From preclinical to clinical evidence. 2014, in Cannabinoids,
Endocannabinoids, and Modulation of Emotion, Memory, and Motivation, Fattore &
Campolongo, editors, Springer Science+Business Media New York 2015, DOI
10.1007/978-1-4939-2294-9_5, pp. 97-129 (Scopus)
Kučerová, J., Babinská, Z., Fattore, L.: Behavioral rodent models of eating disorders
Appetite, Nova Science Publishers, Inc., ISBN: 978-1-63117-241-0, pp. 71-96. (Scopus)
164
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Low-Dose and Very Low-Dose Ketamine among Patients with Major Depression:
a Systematic Review and Meta-Analysis. Int J Neuropsychopharmacol, in press,
doi: 10.1093/ijnp/pyv124.
181
187. Yahyavi-Firouz-Abadi, N. & See, R. E. 2009. Anti-relapse medications: preclinical
models for drug addiction treatment. Pharmacol Ther, 124, 235-47.
188. Yu, S., Zhu, L., Shen, Q., Bai, X. & Di, X. 2015. Recent advances in
methamphetamine neurotoxicity mechanisms and its molecular pathophysiology.
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189. Yui, K., Ikemoto, S., Ishiguro, T. & Goto, K. 2000. Studies of amphetamine or
methamphetamine psychosis in Japan: relation of methamphetamine psychosis to
schizophrenia. Ann N Y Acad Sci, 914, 1-12.
190. Zellner, M. R., Watt, D. F., Solms, M. & Panksepp, J. 2011. Affective
neuroscientific and neuropsychoanalytic approaches to two intractable psychiatric
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Biobehav Rev, 35, 2000-8.
182
9. List of figures
Figure 1: normal rat brain and OBX rat brain...................................................................11
Figure 2: location of burr holes drilled for OBX procedure..............................................12
Figure 3: IV self-administration session...........................................................................13
Figure 4: in vivo microdialysis principle..........................................................................15
Figure 5: forced swim test procedure ...............................................................................16
Figure 6: relative dopamine release in the nucleus accumbens shell after methamphetamine
challenge dose...............................................................................................................151
Figure 7: relative dopamine release in the nucleus accumbens shell after WIN55,212-2
challenge dose...............................................................................................................152
Figure 8: absolute values (pg/ml) of dopamine in the nucleus accumbens shell after
methamphetamine challenge dose..................................................................................153
Figure 9: theoretical dose-response curve ......................................................................154
183
10. Appendices
Appendix 1 ………………………………………………………………………………184
Kucerova J, Babinska Z, Horska K, Kotolova H. The common pathophysiology
underlying the metabolic syndrome, schizophrenia and depression. A review. Biomed Pap
Med Fac Univ Palacky Olomouc Czech Repub. 2015, 159(2): 208-14. doi:
10.5507/bp.2014.060.
Appendix 2………………………………………………………………………….……191
Micale V, Kucerova J, Sulcova A. Leading compounds for the validation of animal
models of psychopathology. Cell Tissue Res. 2013 Oct;354(1):309-30. doi:
10.1007/s00441-013-1692-9.
Appendix 3 ………………………………………………………………………………213
Amchová P, Kučerová J. Pohlaví a drogová závislost: od animálních modelů ke klinické
praxi. Česká a Slovenská Psychiatrie, Praha: Česká lékařská společnost J.E.Purkyně, 2015,
111(2): 72 -78.
Appendix 4……………………………………………………………………………….220
Babinská Z, Kučerová J. Spoločné neurobiologické mechanizmy depresie a
metamfetamínovej závislosti. Alkoholizmus a drogové závislosti, Bratislava: Obzor, 2014,
49(3): 127-152.
Appendix 5………………………………………………………………………….……246
Kucerova J, Tabiova K, Drago F, Micale V. Therapeutic potential of cannabinoids in
schizophrenia. Recent Pat CNS Drug Discov. 2014, 9(1): 13-25. Review.
Appendix 6…………………………………………………………………………….…259
Kucerova J, Pistovcakova J, Vrskova D, Dusek L, Sulcova A. Aripiprazole does not
influence methamphetamine I.V. self-administration in rats. Activitas nervosa superior
rediviva, 2010. 52(4): 261-266.
Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015 Jun; 159(2):208-214.
208
The common pathophysiology underlying the metabolic syndrome,
schizophrenia and depression. A review
Jana Kucerovaa,b
, Zuzana Babinskaa,b
, Katerina Horskac
, Hana Kotolovac
Background. There is a growing interest in metabolic alterations in patients with psychiatric disorders due to their
increased risk for metabolic syndrome (MetS) development. Inflammation is known to underlie the pathophysiology
of schizophrenia and depression as well as MetS. Vulnerability factors for schizophrenia/depression and MetS hence
appear to be shared.
Methods and Results. Based on aWeb of Science search, this review examines current evidence for MetS pathophysiology
involving dysregulation of adipose tissue signaling – adipokines and pro-inflammatory cytokine, both also known
to be aberrant in schizophrenia/depression. Further, gender differences in the incidence and course of schizophrenia/
depression were reported.The disturbances linked to the MetS are also described.Therefore, this review further maps
the gender differences in the psychiatric-metabolic comorbidities.
Conclusion. There is evidence supporting a pathological predisposition to MetS in both schizophrenia and depression
in both humans and animal models. This predisposition is dramatically enhanced by antipsychotic medication.
Further, there are gender differences from clinical findings suggesting women with schizophrenia/depression are more
vulnerable to MetS development. This has not yet been assessed in animal studies. We suggest further validation of
existing schizophrenia and depression animal models for the assessment of metabolic disturbances to provide tools
for developing new antipsychotics and antidepressants with“metabolically inert”profile or improving the metabolic
status in schizophrenic/depressed patients.
Key words: metabolic syndrome, schizophrenia, depression, sex/gender differences, adipokines, leptin, adiponectin,
resistin, AFABP
Received: August 13, 2014; Accepted: November 12, 2014; Available online: December 5, 2014
http://dx.doi.org/10.5507/bp.2014.060
a
Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic
b
Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno
c
Department of Human Pharmacology and Toxicology, Faculty of Pharmacy, Veterinary and Pharmaceutical University Brno
Corresponding author: Jana Kucerova, e-mail: jkucer@med.muni.cz
INTRODUCTION
Psychiatric disorders are commonly associated with
increased morbidity and mortality largely due to other
medical conditions such as cardiovascular diseases, diabetes,
respiratory and infectious diseases. There is a rapidly
growing interest in the assessment of metabolic disturbances
in patients diagnosed with psychiatric disorders
(especially schizophrenia and depression) due to their
higher risk of metabolic syndrome (MetS) than the general
population. In addition, growing evidence indicates
a role of inflammation in the pathophysiology of these
diseases.
In the US and European populations, the prevalence
of MetS in psychiatric patients ranges from 25-56% (depending
on definition) and it constitutes major health and
financial burdens1-3
. It is estimated to be up to 50% higher
than age-matched healthy control populations4
with expectation
to rise to 59% by 2020 (ref.5
). Moreover, not
only western populations are affected but also eastern
regions report a high prevalence. More particularly, Asian
populations report a mean prevalence of 20-40% (ref.6
),
South American regions with 14-30% and Australia with
20-30% (ref.1
). Further, younger age groups also show a
growing rate of MetS incidence7
. A number of reports
confirm the psychiatric populations have a substantially
higher incidence of MetS than the general population,
especially in the case of schizophrenia8
and depression9
.
Obesity and MetS development are also known to
differ according to gender as a result of differences in
the amount and distribution of body fat and differences
in adipose tissue metabolism and function between the
sexes10
. The incidence of MetS was reported to be higher
in women than in men in Arabic populations11
, a finding
consistent with European populations12
. Therefore,
a gender-specific approach may be more effective for the
treatment and prevention of MetS development.
The need for reviewing current knowledge on the
shared pathophysiology linking MetS and psychiatric
disorders is supported by increasing numbers of publications
on the topic of comorbid MetS and schizophrenia/
depression. Web of Science search (performed in August
2014) for “metabolic syndrome” and “schizophrenia*” in
publication titles currently provides 186 records, with the
oldest from the year 2002 including 11 reviews. However,
most papers (and reviews) focus on the side-effects of anti-
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209
psychotics and not the pathophysiological causes according
to the disorders per se resulting in MetS. Analogous
search for depression (“depress*”) returns 161 publications
with the oldest from the 2003 including 5 review
papers.
The aim of this review was to evaluate the available
findings on the link between MetS and schizophrenia/
depression with a focus on gender differences.
METABOLIC SYNDROME AND INFLAMMATION
The epidemic spread of MetS has resulted in over 2
billion overweight and obese adults worldwide13
. This syndrome
is defined as a complex of risk factors closely related
to the development of atherosclerosis and subsequent
cardiovascular morbidity together with type-2 diabetes.
In particular, these factors comprise mainly abdominal
distribution of adipose tissue (abdominal obesity), dyslipidemia,
hypertension and distortion of glycemic homeo-
stasis14
. The seriousness of this pathology lies in increased
mortality due to cardiovascular conditions and diabetes
type 2. Compared to normal populations, the incidence
of myocardial infarction and stroke are 3-fold higher in
MetS patients; the risk of type 2 diabetes development is
5-fold higher15
. Individuals with MetS also often manifest
pro-thrombotic and pro-inflammatory states reflected by
higher blood levels of pro-inflammatory cytokines such
as interleukin-6 (IL-6), interleukin-12 (IL-12), tumor
necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and
interferon-γ (ref.16
).
Dysregulation of adipokines as biomarkers of adipose
tissue metabolism plays an essential part in all obesityrelated
diseases. Most relevant adipokines known to be
dysregulated in MetS are leptin, adiponectin (both supporting
insulin signaling functions), resistin and adipocyte
fatty acid binding protein (AFABP). Also pro-inflammatory
cytokines are considered adipokines such as interleukins,
TNF-α and C-reactive protein (CRP), all generally
suppressing insulin signaling functions.
Leptin is a peripheral signaling protein that regulates
the hypothalamic satiety center and adipose reserves in
the body17,18
. It is key mediator of energy homeostasis involving
regulation of appetite, lipid catabolism and inhibition
neurotransmitter neuropeptide Y, a known appetite
stimulator19
. Plasma levels of leptin are generally higher in
obese patients, which is considered to be a leptin-tolerant
state20,21
.
Adiponectin is a protein that inhibits inflammatory reactions
and protects against metabolic disease, by a wide
range of mechanisms, including anti-diabetic, anti-inflammatory
and anti-sclerotic22
. It is involved in the regulation
of carbohydrate and lipid metabolism, inhibiting gluconeogenesis
in liver and increasing the transport and utilization
of free fatty acids in the periphery. Furthermore,
it significantly affects the function of insulin and plays an
important role in energy homeostasis, causing a decrease
in body weight without affecting food intake. However, it
is believed that it also directly influences the regulation
of appetite and weight control21,23
.
Resistin is a peptide hormone produced by mature
adipocytes and regulates insulin sensitivity24
. Its inhibitory
effect on the differentiation of adipocytes probably underlies
its role in the feedback between nutritional status
and adipogenesis. Its plasma levels increase in correlation
with inflammatory markers including CRP, soluble TNF-α
receptor-2, IL-6 and lipoproteins in combination with
phospholipase A2 under pathophysiological conditions
related to inflammation25
. Based on preclinical evidence,
resistin may represent a key link between inflammation
and its metabolic consequences21,26-28
.
AFABP is a newly discovered adipokine found at higher
plasma levels in patients who have the MetS. Patients
with higher levels of AFABP have worse prognosis and
increased cardio-metabolic risk factors, reversible by
atorvastatin treatment29
. Further, TNF-α is constitutively
expressed in adipose tissue and this condition leads to
insulin resistance in animal models of obesity which supports
the face validity of the models. Plasma levels of
AFABP in humans closely correlates with degree of obesity
and the development of insulin resistance and positively
correlates with waist circumference, blood pressure
values, and parameters of lipid metabolism, serum fasting
insulin and insulin resistance index21,30
.
The growing evidence has resulted in the formulation
of the inflammatory hypothesis of insulin resistance and
MetS (ref.31-33
) and MetS is also associated with other inflammatory
diseases such as rheumatoid arthritis34
showing
secondary development of MetS on an inflammatory
basis. The hypothesis assumes obesity is a consequence
of excessive caloric intake representing a sub-clinical inflammatory
process, which induces insulin resistance
and following clinical and biochemical manifestations of
MetS as demonstrated in numerous studies. For review
see Alemany et al.31
Moreover, the inflammation is mediated
by pro-inflammatory cytokines produced by macrophages
which tend to populate the growing adipose tissue
in obesity at higher rates35
. In mouse obesity models, an
up-regulation of specific genes for macrophages - macrophage
inflammatory protein 1α (MIP-1α), monocyte
chemoattractant protein-1 (MCP-1), macrophage-1 antigen
(MAC-1), macrophage surface glycoproteins F4/80
and CD68; and genes promoting inflammatory processes
in white adipose tissue are found. Molecular mechanisms
leading to macrophage activation in obesity/ MetS are
not fully understood. However, participation of adipokines
(adiponectin, leptin, complement factor C3, MCP-
1, cytokines, free fatty acids) is assumed36
. Activated
macrophages release several cytokines and chemokines
such as TNF-α (ref.37
), IL-1, IL-6 and MCP-1, distorting
adipocyte sensitivity to insulin, which then in turn promote
further activation and infiltration of macrophages.
Therefore, impaired insulin signaling in adipocytes may
lead to massive lipolysis, necrosis and development of
insulin resistance33,38
.
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210
PSYCHIATRIC DISORDERS
AND INFLAMMATION
There is accumulating evidence suggesting that schizophrenia
is associated with increased serum levels of proinflammatory
cytokines, namely IL-1, IL-6, TNF-α and
high-sensitivity CRP, even in patients with minimal or no
exposure to antipsychotics39
.
Prenatal infections are also hypothesized to have serious
impact on the brain, which is supported by validation
of several neurodevelopmental animal models of schizophrenia
based on immune or toxic prenatal insult namely:
Immune: polyIC (polyriboinosinic-polyribocytidilic acid)
model40
, toxic: MAM (methylazoxymethanol acetate)
model41
or Δ9-THC (Δ9-tetrahydrocannabinol) model42
.
Together with certain genetic factors, these findings provide
convincing evidence that inflammation is a major
factor in the pathology of this disorder43
.
There is a well-established concept of depression
strongly associated with inflammation. An abundance of
both clinical and preclinical data reported increases in
pro-inflammatory cytokines such as IL-1, IL-6, TNF-α and
CRP in depressed patients44-47
. Similar findings were also
discovered in preclinical studies48
including the olfactory
bulbectomized rodent model of depression49
.
PSYCHIATRIC DISORDERS AND METABOLIC
SYNDROME
Schizophrenia and metabolic syndrome
Obesity or MetS are common in schizophrenic patients.
MetS has an incidence of 3-4% in the general
population, but up to 10% in schizophrenic patients even
before initiation of the treatment with antipsychotics8
which often results in typical changes in lipid metabo-
lism50
. It appears that not only antipsychotic treatment
but the pathophysiology of schizophrenia itself is linked
to MetS development suggesting a common underlying
pathway- chronic inflammatory abnormality of cyto-
kines43
. Therefore, vulnerability factors for development
of schizophrenia, diabetes, and MetS seem to be shared
and interconnected. In patients with schizophrenia the
risk is further greatly increased by the use of antipsychotic
medication as reviewed repeatedly elsewhere8,51-55
.
More specifically, elevated blood levels of adiponectin
have been reported in schizophrenia56
and there is a correlation
between serum leptin levels and body weight51
.
In addition, leptin concentration was shown to play an
important part in the negative feedback against dopamine
activity connected to positive symptoms of schizophre-
nia57
. Furthermore, it is well known that antipsychotic
treatment induces clinically relevant weight gain and rise
in fasting plasma glucose levels58
.
Despite the high clinical relevance, relatively little
research has been done in preclinical models of schizophrenia,
which could then contribute to targeted drug
development for MetS treatment in psychotic patients.
Studies conducted in animals were mostly related to the
evaluation of metabolic effects in all classes of antipsychotics
rather than the assessment of the relation between
MetS and schizophrenic phenotype per se. These drugs
were shown to notably disturb lipid metabolism in drug
naïve Sprague-Dawley rats as well as in kainic acid treated
Fisher rats used as a model of schizophrenia59,60
. This indicates
that schizophrenic-like phenotype in rodent models
and antipsychotic medication does lead to increase in
vulnerability to metabolic disturbances further confirming
their validity and translational potential evaluating
gender differences, a key importance for developing new
therapeutic strategies as described in the corresponding
section of this text.
DEPRESSION AND METABOLIC SYNDROME
Multiple lines of evidence confirm higher incidence of
MetS in depressed patients. A 50% higher prevalence of
depression has been reported in individuals with MetS in
an Australian population61
and a 4-fold increased risk for
MetS in patients with lifetime major depression episode in
a Lithuanian population62
. Similar outcomes in a German
population were found63
. Also, different ethnic groups
were compared with consistent finding of higher MetS
prevalence in depressed African-American, Caucasian64
,
and Asian women65
. Furthermore, not only major depression
but also bipolar disorder has been shown to have
association with MetS (ref.66
). However, MetS seems to
be specifically linked to depression as it has been repeatedly
shown that anxiety is not associated with metabolic
disturbances62,67
.
There are also clinical studies showing no68
or only
partial association of MetS symptoms (lipid profile) with
depression69
. However, recently, Pan et al. published an
extensive meta-analysis (the first of its kind) reporting a
strong link between depressive disorders and development
of MetS in both genders. The results indicate a convincing
bidirectional association between depression and MetS
(ref.9
). Further support (although sporadic) for the association
between MetS and depression could provide a case
study of a (Caucasian) woman treated with pioglitazone
and showing strong antidepressant effect70
.
Suggested mechanisms underlying both disorders
include HPA axis dysregulation following the inflammatory
reaction. Moreover, two subtypes of depression
(melancholic and atypical depression) were identified
to be associated with the inflammatory and metabolic
dysregulation. This highlights the possibility that not all
forms of major depression possess this association with
MetS (ref.71
). Changes in important adipokine levels in
depressed patients were reported compared to a healthy
population suggesting predisposition to MetS development
in depressed patients. In agreement, a “leptin hypothesis
of depression” was formulated as low levels of
leptin have been found in association with depression in
humans as well as depressive behaviors in rodents. It was
suggested that both leptin insufficiency and leptin resistance
may contribute depressive status72
. These findings
are translated and further supported by several animal
studies73
including pharmacological experiments where
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211
leptin induced antidepressive-like behaviour in a forced
swimming test, a commonly used test for evaluation of
depressive-like phenotype in rodents74
.
Blood levels of another adipokine – adiponectin
– were found to be reduced in Brazilian patients with
major depression before antidepressant treatment. The
authors of the study (Leo et al. 2006) conclude that the
reduced availability of circulating adiponectin is likely to
have an impact on mood state75
. Similar findings were
reported in an Italian population76
and later in a US popu-
lation77
. However, contradictory findings were reported in
a Korean study showing higher levels of adiponectin in
depressed individuals78
. The variability could be due to
antidepressant treatment which seems to increase adiponectin
levels79
. Yet, studies reporting no difference before
and after treatment have also been published80
.
Regarding resistin, a positive association between
blood levels and free cortisol concentrations were found
in depressed patients. Resistin levels were normalized
when patients remitted after pharmacological treatment
but not in non-remitters80
.
ROLE OF GENDER IN THE METABOLIC
SYNDROME AND PSYCHIATRIC DISORDERS
Schizophrenia and MetS: implications of gender
Sex-specific differences in the epidemiology, onset and
course of schizophrenia are repeatedly reported. More
specifically, men have approximately 4-times higher tendency
to develop schizophrenia and the first symptoms
usually appear at a younger age81
. On the other hand,
women tend to suffer more from comorbid depression
while men it is drug addiction. In addition, gender differences
in response to antipsychotic treatment are reported.
However, a clear explanation is yet to be provided.
Influences of sex hormones, sexual dimorphism of the
brain, metabolic differences and social factors were so
far only proposed as partial explanations82,83
. Gender differences
in MetS comorbidity with schizophrenia were
found repeatedly before and after the initiation of the antipsychotic
treatment with women being approximately
3-times more prone to develop MetS (ref.8
).
Furthermore, preclinical studies have recorded gender
differences suggesting a greater vulnerability (increase
in body weight and metabolic changes) in female rats,
specifically, the most suitable model for antipsychoticinduced
weight gain appears to be the female SpragueDawley
rat84
. In contrast, male rats showed no significant
changes in body mass yet still exhibited metabolic disturbances
such as increased visceral fat mass and hormonal
changes59
. Nevertheless, increased adiposity was reported
in both genders and seems to be adequately modeled in
rodents with a schizophrenic phenotype. It has not yet
been established which experimental paradigms most accurately
reflect weight gain and metabolic abnormalities
in schizophrenia, known to be increased by antipsychotic
treatment in humans85
.
Depression and MetS: implications of gender
In the case of depression, reports of gender differences
are only recently emerging. A similar strength of the
overall association has been reported between metabolic
risk factors in men and women, but in males, several factors
were associated with depressive symptoms, while in
females the association was confined to waist circumference
only86
. Earlier, a stronger association of MetS with
depression was found in the female US population compared
to male group87
similarly in the Israeli population88
.
However, negative findings have been published as well89
.
Furthermore, depressed women showed significantly
higher leptin levels than a control group both before and
after the response to antidepressant treatment, whereas no
difference was found between the male patients and their
controls. The improvement of depression with antidepressant
treatment was shown to cause a further elevation of
leptin levels, in both female and male patients. Therefore,
clinical response to antidepressant treatment seems to be
linked to leptin metabolism90
. In addition, adiponectin levels
were found to be dysregulated in men with depression
while no differences were observed in that of women78
.
CONCLUSION
In summary, there is likely a metabolic predisposition
to MetS in both schizophrenia and depression patients
which is evidence of underlying pathophysiology in both
humans and animal models. This predisposition is enhanced
by antipsychotic medication in psychotic patients.
In agreement, numerous authors in clinical fields have already
suggested screening for MetS in psychiatric patients
to combat increased rates of morbidity and mortality from
non-psychiatric reasons in these patients with early lifestyle
and pharmacotherapeutic interventions. Specifically,
in schizophrenic patients, routine consultation with a diabetologist
has been suggested52
.
Moreover, development of new anti-inflammatory
treatments for the dual pathology of schizophrenia and
MetS has been proposed91
, which also could be a useful
approach alleviating the cognitive symptoms of schizo-
phrenia92
. This evidence concerns newly diagnosed patients
as well, with chronic treatment with antipsychotics
being a well-known risk factor for MetS development.
Thus, a follow-up monitoring of metabolic abnormalities
in patients on second generation antipsychotics is
strongly recommended by the consensus of the American
Diabetes Association, American Psychiatric Association,
American Association of Clinical Endocrinologists, and
North American Association for the Study of Obesity93
.
In depression, a closer monitoring for MetS (ref.61,63,94
)
together with development of disease-modifying thera-
pies95
and special regards to sex-differences96
have been
repeatedly suggested. Further, the current scientific literature
has suggested the validation of existing animal
models and the development of newer models to better
reflect psychiatric diseases97,98
.
Furthermore, the possible gender differences in clinical
findings (suggesting women with schizophrenia/de-
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212
pression are more vulnerable to MetS development) are
not yet assessed in animal studies. In the light of evidence
on gender differences in MetS development in psychiatric
disorders, sex differences should be taken into account in
future preclinical and clinical studies. However, the lack
of validated animal models for assessment of metabolic
disorders comorbid in psychiatric diseases is problematic.
Such validation of existing animal models of schizophrenia
and depression could provide a useful tool for
developing innovative pharmacotherapeutic solutions
with “metabolically inert” profile or even improving the
metabolic status of psychiatric patients.
The endocannabinoid system targeting drugs are an
important source of candidates and have already been proposed
for the treatment of schizophrenia99,100
and mood
disorders101
. Endocannabinoid targeting drugs effective
in reducing abdominal obesity have been identified102
.
More specifically, these drugs act through CB1 receptor
inverse agonism. Unfortunately, marketing of the first
drug, rimonabant, was discontinued for psychiatric sideeffects,
namely inducing depressive states and suicidal
ideas103,104
. However, newer cannabinoid compounds are
emerging and a strong influence on appetite, metabolism
and energy homeostasis is consistently reported105-107
.
Most importantly, preclinical studies are constantly widening
the range of new candidate molecules108-110
.
ACKNOWLEDGEMENT
This work was supported by the project of specific research
at the Masaryk University (MUNI/A/0886/2013),
Project of the Internal Grant Agency (IGA) VFU
Brno (48/2014/FaF) and the project “CEITEC
- Central European Institute of Technology”
(CZ.1.05/1.1.00/02.0068) from European Regional
Development Fund.
Author contributions: All authors contributed to manuscript
writing and final approval.
Conflict of interest statement: None declared.
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REVIEW
Leading compounds for the validation of animal
models of psychopathology
Vincenzo Micale & Jana Kucerova & Alexandra Sulcova
Received: 15 March 2013 /Accepted: 1 July 2013
# Springer-Verlag Berlin Heidelberg 2013
Abstract Modelling of complex psychiatric disorders, e.g.,
depression and schizophrenia, in animals is a major challenge,
since they are characterized by certain disturbances in
functions that are absolutely unique to humans. Furthermore,
we still have not identified the genetic and neurobiological
mechanisms, nor do we know precisely the circuits in the
brain that function abnormally in mood and psychotic disorders.
Consequently, the pharmacological treatments used are
mostly variations on a theme that was started more than
50 years ago. Thus, progress in novel drug development with
improved therapeutic efficacy would benefit greatly from
improved animal models. Here, we review the available
animal models of depression and schizophrenia and focus
on the way that they respond to various types of potential
candidate molecules, such as novel antidepressant or antipsychotic
drugs, as an index of predictive validity. We conclude
that the generation of convincing and useful animal
models of mental illnesses could be a bridge to success in
drug discovery.
Keywords Depression . Schizophrenia . Animal models .
Antipsychotics . Antidepressants
Introduction
Animal models in neuroscientific research are of irreplaceable
value. They are important tools for the assessment of
pathological mechanisms, for the testing of hypotheses that
cannot be addressed in clinical studies and for the development
of novel pharmacological treatment (Nestler et al.
2002). Psychiatric disorders such as depression and schizophrenia
(SCZ) are difficult to replicate in a laboratory animal.
At the same time, no animal model is able to fully mimic
any mental illness, as these are characterized by specific disturbances
in functions that are absolutely unique to humans, such
as markedly diminished interest, thought disorders and hallucinations
(American Psychiatric Association 2000). However, a
general approach is to reproduce particular symptoms of psychiatric
diseases (i.e., attention/cognitive deficits) in laboratory
animals or to develop models (i.e., the forced swim test) to
identify novel compounds as potential treatments (Cryan et al.
2002; Meyer et al. 2009). Ideally, an animal model should reflect
the human psychiatric disease in terms of face validity (i.e.,
reproduce the symptoms of the human mental disease), construct
validity (i.e., replicate the neurobiological abnormalities)
and predictive validity (i.e., response to the pharmacological
treatment in a way that predicts the effects of that treatment in
humans). Nevertheless, none of the available animal models are
able to mimic all the aspects of neuropsychiatric disorders, in
terms of neurobiological mechanisms and disease symptoms
and most likely never will. Therefore, the lack of knowledge
regarding the mechanisms that underlie diseases such as depression
and SCZ, their comorbidity and symptomatic overlap
between them (i.e., patients with psychotic depression) is associated
with the partial efficacy of the present pharmacological
armoury. This raises the central question to be addressed in this
review: are current animal models reliable tools with a predictive
validity for the development of novel therapeutic compounds?
This work was supported by the project “CEITEC—Central European
Institute of Technology” (CZ.1.05/1.1.00/02.0068) from the European
Regional Development Fund.
The authors declare no conflicts of interest.
V. Micale (*) :J. Kucerova :A. Sulcova
CEITEC—Central European Institute of Technology, Masaryk
University, Kamenice 5, 625 00 Brno, Czech Republic
e-mail: vincenzomicale@inwind.it
J. Kucerova
Department of Pharmacology, Faculty of Medicine, Masaryk
University, Brno, Czech Republic
Cell Tissue Res
DOI 10.1007/s00441-013-1692-9
191
Table 1 Behavioural effects of clinically prescribed antidepressants in validated animal models of depression (SSRI selective serotonin reuptake
inhibitor, SNRI serotonin and noradreniline reuptake inhibitors)
Rodent model of depression Behavioural antidepressant-like
response
Drug treatment
Olfactory bulbectomy ↓ Hyperactivity in the open field test
(after chronic drug administration)
Tianeptine, sertraline (Kelly and Leonard 1994), desipramine
(Kelly and Leonard 1996), amitryptiline (Stockert et al. 1988),
imipramine (Roche et al. 2008), citalopram (Hasegawa et al.
2005; Nguyen et al. 2009), fluvoxamine (Saitoh et al. 2007),
fluoxetine (Freitas et al. 2013; Machado et al. 2012; Roche et al.
2007), buspirone (Sato et al. 2010), agomelatine (Norman et al.
2012), tiagabine (Pistovcakova et al. 2008)
Learned helplessness ↓ Number of failures to escape shock Imipramine (Besson et al. 1999; Demontis et al. 1993; Gambarana
et al. 1995; Geoffroy et al. 1991; Ishida et al. 2005; Iwamoto
et al. 2005; Iwata et al. 2006; Joca et al. 2003; Martin and Puech
1991; Martin et al. 1987; Meloni et al. 1993; Takamori et al.
2001), desipramine (Beck and Fibiger 1995; Besson et al. 1999;
Centeno and Volosin 1997; Duman et al. 2007; Joca et al. 2006;
Martin et al. 1987; Rojas-Corrales et al. 2004; Rusakov and
Valdman 1983), chlorimipramine (Rusakov and Valdman
1983), clomipramine (Martin and Puech 1991; Martin et al.
1987; Millan et al. 2001), amitriptyline (Besson et al. 1999;
Caldarone et al. 2003; Rusakov and Valdman 1983), trazodone
(Rusakov and Valdman 1983), tranylcypromine, mianserine
(Takamori et al. 2001), venlafaxine (Millan et al. 2001; RojasCorrales
et al. 2004), fluvoxamine (Iwamoto et al. 2005; Martin
and Puech 1991; Rojas-Corrales et al. 2004; Takamori et al.
2001), mirtazapine (Slattery et al. 2005), fluoxetine (Iwamoto
et al. 2005; Marco and Laviola 2012; Marcussen et al. 2008;
Page and Abercrombie 1997; Reines et al. 2008; Shumake et al.
2010; Zazpe et al. 2007), paroxetine (Zazpe et al. 2007),
sertraline (Duman et al. 2007), St. John’s wort extract
(Chatterjee et al. 1998), buspirone (Lucki 1991; Martin and
Puech 1991); citalopram (Martin and Puech 1991; Millan et al.
2001), escitalopram (Reed et al. 2008), zimelidine (Dabrowska
et al. 2008; Joca et al. 2006), lamotrigine (Consoni et al. 2006),
agomelatine (Bertaina-Anglade et al. 2006; Dagyte et al. 2011;
Popoli 2009; Tardito et al. 2010)
Forced swim test ↓ Time of immobility (↑ swimming or
climbing activities) (after acute drug
administration)
Amitryptiline (Caldarone et al. 2003), tianeptine (Della et al. 2012;
Kelly and Leonard 1994; Solich et al. 2008), imipramine
(Bourin et al. 2004; Della et al. 2012; Kulkarni and Dhir 2007;
Paulke et al. 2008; Schulte-Herbrueggen et al. 2012; Zanelati
et al. 2010), desipramine (Robles-Molina et al. 2012; Simpson
and Kelly 2012; Will et al. 2003), venlafaxine (Kulkarni and
Dhir 2007), sertraline (Kelly and Leonard 1994; Leggio et al.
2008; Rogoz and Skuza 2006), paroxetine (Akagawa et al.
1999; Leggio et al. 2008), reboxetine (Cryan et al. 2005b; Wong
et al. 2000), phenelzine (Bourin et al. 2002; Will et al. 2003),
tranylcypromine, agomelatine (Bourin et al. 2002, 2004),
fluoxetine (Bourin et al. 2004; Cryan et al. 2005b; Kulkarni and
Dhir 2007; Reed et al. 2008; Rogoz and Skuza 2006),
paroxetine (Karanges et al. 2011), moclobemide (Cryan et al.
2005b), pramipexol (Rogoz and Skuza 2006; SchulteHerbrueggen
et al. 2012), mirtazapine (Muguruza et al. 2013),
St. John’s wort extract (Paulke et al. 2008), citalopram (Leggio
et al. 2008; Nguyen et al. 2009; Tamburella et al. 2009, 2013),
escitalopram (Nguyen et al. 2013; Reed et al. 2008),
clomipramine (Consoli et al. 2005, 2007; Leggio et al. 2008;
Micale et al. 2006, 2008a, 2008b; Tamburella et al. 2009, 2010,
2013)
False positive results Amphetamines (Cryan et al. 2002), caffeine (Slattery and Cryan
2012)
Tail suspension test ↓ Time of immobility (after acute drug
administration)
Mianserine, nomifensine, viloxazine (Steru et al. 1985),
amitryptiline (Caldarone et al. 2003; Steru et al. 1985),
desimipramine (Berrocoso et al. 2013; O’Leary et al. 2007;
Steru et al. 1985), imipramine (Berrocoso et al. 2013; Kulkarni
and Dhir 2007; Liu and Gershenfeld 2001), reboxetine (O’Leary
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Status of current animal models of depression
and their pharmacological validation
Unfortunately, an animal model that perfectly includes the
aetiology, pathophysiology and symptoms of depression while
allowing an evaluation of the responses to treatments remains
difficult to envisage. Although the generation of genetically
modified mice could result in animal models mimicking genetic,
biochemical or behavioural characteristics of human
depression, we have to keep in mind the role of major confounding
factors such as background strain, neurodevelopment
or interactions between genetic and environmental factors
during the interpretation of any findings (Urani et al. 2005).
However, various models, each with specific limitations, are
able to reproduce most of the aetiological factors and symptoms
of the disease or possess a satisfactory predictive value
for identifying new compounds. On this basis, we review the
validation of rodent models of depression, such as bilateral
olfactory bulbectomy (OBX), learned helplessness, the forced
swim test (FST) or the tail suspension test (TST) and the
chronic mild stress (CMS) or chronic social stress paradigm,
according to the effects of pharmacological interventions that
have successfully achieved antidepressive-like activities in
animals and treatment efficacy in depressive patients.
Olfactory bulbectomy
OBX results in behavioural (i.e., hyperactive response in the
open field paradigm) and neurochemical (i.e., changes in the
endocrine, immune and neurotransmitter systems) alterations
in rats (Cairncross et al. 1975; Jesberger and Richardson 1985;
Kelly et al. 1997) and mice (Hellweg et al. 2007; Zanelati et al.
2010; Zueger et al. 2005); the alterations resemble some of
those seen in depressed patients and are reversed by chronic
treatment with clinically approved or potential antidepressants
(Tables 1 and 2). Since the olfactory system in rodents is part of
the limbic region in which the amygdala and hippocampus
contribute to emotional behaviour, OBX affects the corticalhippocampal-amygdala
circuit, which also seems to be dysfunctional
in depressed patients (Song and Leonard 2005).
Interestingly, a dysregulation of the functionality of the central
reward pathway in bulbectomized rats has also been reported,
suggesting that it may have an impact on the development of
depression/addiction comorbidity. Thus, OBX could be a useful
animal model of these dual diagnosis disorders (Kucerova
et al. 2012).
Learned helplessness
Learned helplessness might model in animals a human situation
of unpredictable and uncontrollable events leading to
consequences: “stress-coping depression”. Thus, the animal
model is considered to provide specificity towards antidepressant
pharmacotherapy (Chourbaji et al. 2005; Christensen
1993; Maier 1984; Miller and Seligman 1976; Seligman and
Beagley 1975; Sherman et al. 1982; Vollmayr and Henn
2001). Animals exposed to inescapable and unavoidable electric
shocks in one situation later fail to escape shock in a
Table 1 (continued)
Rodent model of depression Behavioural antidepressant-like
response
Drug treatment
et al. 2007; Wong et al. 2000), tianeptine (Berrocoso et al. 2013),
fluoxetine (Berrocoso et al. 2013; Kulkarni and Dhir 2007;
Muguruza et al. 2013; O’Leary et al. 2007), mirtazapine
(Muguruza et al. 2013), venlafaxine, duloxetine (Berrocoso
et al. 2013; Kulkarni and Dhir 2007), citalopram (Berrocoso
et al. 2013)
Chronic mild stress ↑ Responsiveness to rewards (after
chronic drug administration)
Fluoxetine (Jindal et al. 2013; Muscat et al. 1992; Mutlu et al.
2012), maprotiline (Muscat et al. 1992), minaserin (Cheeta et al.
1994), imipramine (Marston et al. 2011; Norman et al. 2012;
Papp et al. 1996; Przegalinski et al. 1995), buspirone (Papp et al.
1996; Przegalinski et al. 1995), ipsapirone (Przegalinski et al.
1995), agomelatine (Bourin et al. 2004; Dagyte et al. 2011),
risperidon (Marston et al. 2011), citalopram (Herrera-Perez et al.
2010; Przegalinski et al. 1995), escitalopram (Christensen et al.
2012), tianeptine (Mutlu et al. 2012)
Social stress—
repeated defeat
Resident-intruder ↓ Agressivity, ↑ flight Acute: SSRIs, SNRIs, tricyclics (Mitchell and Neumaier 2005)
↑ Aggressivity Chronic: SSRIs, SNRIs, tricyclics (Mitchell and Neumaier 2005)
↑ Ambulation in open field test Fluoxetine, reboxetine (Rygula et al. 2006, 2008)
Group-housed vs.
singly-housed
aggressive partner
↑ Ambulation in open field test Chronic: citalopram, valproate, felbamate (Pistovcakova et al.
2005; Sulcova 1999)
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193
different situation in which escape is possible. A drug is
considered to be effective as an antidepressant if the learned
helplessness is reduced (the number of failures to escape is
decreased). However, we need to assess a depressive-like
phenotype in experimental animals and exclude some subjects
from the study. In mice, approximately 30 % of individuals
reportedly become helpless after shock exposure. However,
the remaining animals show helpless behaviour with high
escape latency and thus a low number of failures to escape
might be attributable to variable pain sensitivity (Chourbaji
et al. 2005). Parameters for inescapable shock and the testing
of learned helplessness to minimize artifacts have been stated
in a study published elsewhere (Chourbaji et al. 2005). Two
rat lines have also been established by selective breeding,
namely helpless and non-helpless, which differ in neurochemical
and behavioural parameters that are known to be related to
depression (Henn and Vollmayr 2005).
Forced swim test and tail suspension test
These two tests are widely used paradigms specifically developed
to test new antidepressants. In the FST (also known
as Porsolt’s test; Porsolt et al. 1977), rodents are forced to
swim in an inescapable cylinder and will eventually adopt a
characteristic immobile posture that is interpreted as a passive
stress-coping strategy or depression-like behaviour (behavioural
despair). The FST has shown its ability to detect a
broad spectrum of substances that are therapeutically effective
in human depression, as these drugs shift passive-stress
coping towards active coping, which is detected as reduced
immobility (Table 1). Furthermore, the quantity of the different
movements, such as climbing or swimming behaviour,
has a predictive value for differentiating between noradrenergic
(NAergic) and serotonergic (5-HTergic) activity (Cryan
et al. 2002). However, care must be taken with regard to the
strain (variations have been shown between inbred and outbred
mice and rats) used for the test because of differential
spontaneous locomotor activity possibly reducing the duration
of immobility (Crawley et al. 2007; Petit-Demouliere
et al. 2005). False positive results can be obtained when testing
drugs with psychostimulant activity, e.g., amphetamines, caffeine
(Cryan et al. 2002; Slattery and Cryan 2012).
Similar assumptions and interpretations to those for the
FS, can be drawn from the TST (Steru et al. 1985). In this
test, mice are suspended by their tails for a defined period of
time during which their immobility is decreased by several
antidepressants. The percentage of animals showing passive
behaviour should be counted and then compared with that
after vehicle or active drug treatment, as several mouse
strains have been shown to be essentially resistant to tailsuspension-induced
immobility (Cryan et al. 2005a). The
test however is sensitive to acute treatment only and its
validity for non-monoamine antidepressants is uncertain
(Berrocoso et al. 2013; Cryan et al. 2005b).
Chronic mild stress
Chronic mild stress procedures (food or water deprivation,
45° cage tilt, intermittent illumination, soiled cage, paired
housing or low-intensity stroboscopic illumination), applied
for a period of several consecutive weeks decrease the responsiveness
to rewards (consumption of a 1 % sucrose
solution) in rats or mice; this is reversed by chronic administration
of antidepressant drugs. This “chronic mild stress
model” is considered to represent anhedonia in depression
(Papp et al. 1996; Willner 1984, 1997; Willner et al. 1992).
In comparison with other animal models of depression, it has
been evaluated as a high perspective research approach,
despite its procedural complexity and difficult reproducibility
(Porsolt 2000). Chronic treatment with clinically used
antidepressants normalizes sucrose drinking (Table 1).
Drug-withdrawal-induced anhedonia
A withdrawal from abuse of psychoactive compounds (e.g.,
cocaine, amphetamines) is known to be associated with
states of depression in humans and depressive-like states in
animals (Barr and Phillips 1999; Jang et al. 2013; Renoir
et al. 2012). The animal model “drug-withdrawal-induced anhedonia”
is based on experimental experience with laboratory
rodents; upon their withdrawal from long-term treatment with
psychostimulatory agents, they show mild food and water
avoidance as depressive-like symptoms (anhedonia) in response
to rewards in various paradigms, e.g., place preference, i.v. drug
self-administration, electric intracranial self-stimulation or sucrose
solution preference (Barr and Phillips 1999; Cryan and
Mombereau 2004). Rates of reward responding is increased by
subsequent treatment with antidepressants, e.g., imipramine and
amitriptyline (Kokkinidis et al. 1980).
Chronic social stress
Repeated social stress was suggested as an aethologically
relevant animal model of depression in mice (Keeney and
Hogg 1999), rats (Rygula et al. 2005) and tree shrews (Fuchs
2005). Any behaviour indicative of social conflict such as
threat, attack, fight or escape, avoidance or subordination is
called agonistic behaviour and encompasses the actions of
both the instigator and the victim (Scott 1966). Compared
with control individuals, the animals that are subjected to
repeated agonistic encounters exhibit significantly reduced
locomotor activity in the open field test, which, in turn is
normalized by previously clinically proven or potential antidepressants,
e.g., citalopram or valproate and by potential
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194
antidepressants, e.g., felbamate (Pistovcakova et al. 2005;
Sulcova and Pistovcakova 2008; Table 1).
Alterations of hypothalamic-pituitary-adrenal functions
have been established in states of depression and stress,
including social stress conditions, in both humans and animals
(Blanchard et al. 2001; Kubera et al. 2011; Mathews
et al. 2006; Morris et al. 2012). In rodents, social defeat and
subordination are stressful, especially in males (Blanchard
et al. 2001; Martinez et al. 1998). Animals that are subjected
to repeated agonistic encounters are used for testing potential
antidepressant treatment effects (Mitchell 1994, 2005;
Sulcova 1999). The same stress procedure results in increased
release of corticosterone and dopamine (DA).
Felbamate decreases NA concentrations and inhibits the
stress-induced rise in corticosterone and DA. Modulation
of stress hormone release has been suggested to be induced
by the action of felbamate on glutamate neurotransmission
and neuroendocrine changes might contribute to behavioural
effects of the drug (Pistovcakova et al. 2005). The moodstabilizing
action of felbamate and other anti-epileptic drugs
has been proposed by clinicians for further verification
(Cavanna et al. 2010).
Current leading compounds for development of new
antidepressants
Pharmacological analyses of action of clinically approved
antidepressants support the predictive validity of the animal
models presented. However, consideration of the behavioural
and molecular phenotypes corresponding to the human
disorders suggests that these models are also useful for the
improvement of our knowledge of the neuronal mechanisms
of the disease, the biomarkers of its specific symptoms and
the integration of basic and clinical methodologies (translational
medicine) for the development of new antidepressants
(Borsini 2012; Cryan et al. 2002; Dzirasa and Covington
2012; Kluge et al. 2011; Neumann et al. 2011; Rupniak
2003). Taking into account that the 5-HT hypothesis of depression
has not been abandoned (Albert and Benkelfat 2013),
the targets of potential relevance as treatments for mood disorders
are also those involved in the regulation of several other
neuronal systems in the brain, including the opioid system
(Pradhan et al. 2011), the cholinergic system (Drevets et al.
2013), the endocannabinoid system (Marco and Laviola 2012;
Micale et al. 2013), the neuropeptidergic signalling system
(Griebel and Holsboer 2012), the melatoninergic system
(Lanfumey et al. 2013) and the glutamatergic system
(Connolly and Thase 2012; Hashimoto 2011; Javitt 2012;
Machado-Vieira et al. 2012; Mathews et al. 2012; Serafini
et al. 2013; Tokita et al. 2012). Thus, attention should be given
to compounds influencing these systems, which have been
shown to produce antidepressant-like effects in animal models.
Currently, the compounds that modulate glutamatergic neurotransmission
are reported to hold the greatest promise for the
development of new antidepressants (Serafini et al. 2013).
Suggested mechanisms are based on the antagonistic influence
on ionotropic N-methyl-D-aspartate (NMDA) receptors, the
modulation of metabotropic glutamate receptors, especially
the negative modulation of mGlu2/3 and mGlu5 receptors
(Chaki et al. 2013) and the positive modulation of mGlu2
and mGlu7 receptors (Sanacora et al. 2012). The animal model
studies with leading glutamatergic compounds are cited in
Table 2.
Status of current animal models of SCZ
and their pharmacological validation
SCZ, described by Kraepelin in 1896 as a dementia praecox,
is a unique human disorder for which modelling in animals
might prove problematic because of the lack of a uniform set
of symptoms in patients and indictions of the heterogeneity
of the disorder. Thus, a greater understanding of the disorder
might arise from modelling specific signs and symptoms, as
opposed to the entire syndrome. In line with this strategy,
several efforts have been directed at developing animal
models that allow the translation of the symptomatology in
SCZ and prediction of antipsychotic activity. Although positive
symptoms such as hallucinations and delusions cannot be
measured in animals, the most reliable behavioural indices of
positive symptoms in animal models are hyperlocomotor activity
and behavioural stereotypes that mimic the psychomotor
agitation and presence of stereotyped behaviour in acutely
psychotic patients (Young et al. 2010). The rationale for the
use of these indices is based upon the principle that the
hyperfunction of the mesolimbic DAergic system, which
seems to be involved in the enhanced locomotor activity and
stereotyped behaviours, is consistent with the clinical conditions
in which enhanced subcortical DAergic activity plays a
pivotal role in precipitating positive symptoms (Murray et al.
2008). The loss of selective associative learning in the form of
the disruption of latent inhibition, which is also induced by
hyperdopaminergic activity at the subcortical level, seems to
be another cross-species translational index relevant to positive
symptoms of SCZ (Weiner 2003). Indeed, some behavioural
aspects of SCZ can be modelled and objectively
assessed in rodents. More specifically, anhedonia and social
behaviour as hallmarks of negative symptoms in humans can
be assessed in rodents, together with prepulse inhibition,
which reflects disrupted sensory gating abilities both in
schizophrenic patients and in experimental animal models
(Young et al. 2010). Finally, the various cognitive aspects
affected in the disease, as identified by the NIH Measurement
and Treatment Research to Improve Cognition in Schizophrenia
(MATRICS) initiative (Marder and Fenton 2004), can be
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experimentally addressed in animal models by the use of specific
test batteries (Peleg-Raibstein et al. 2012). Among the
several approaches used to create experimental animal models
of SCZ, which also include the lesion model (Jones et al. 2011)
and genetic-based preparations (Inta et al. 2010), we will examine,
in the following discussion, (1) pharmacological models
and (2) neurodevelopmental models that are the most used in
drug discovery studies (Tables 3, 4)
Pharmacological models
Hyperfunction of the DAergic system in the mesolimbic
pathway was the original tenet for the occurrence of SCZ;
thus, the first animal models were developed on the basis of
the pharmacological manipulation of the DAergic system in
an attempt to mimic this dysregulation (Carlsson et al. 2001).
In rodents, repeated treatment with the DA-releasing agent
amphetamine induced a persistent sensitization exaggerating
the hyperactivity caused by an acute amphetamine challenge,
which was prevented by antipsychotic pre-treatment. This model
is supported by the observation that chronic psychostimulant
abuse can lead to psychotic episodes, whereas low doses of
amphetamine worsen the symptoms (Featherstone et al. 2007).
Amphetamine sensitization is also characterized by deficits in
prepulse inhibition or latent inhibition and in prefrontal-cortexdependent
cognitive tasks, whereas hippocampal function is
unaltered (Peleg-Raibstein et al. 2012; Russig et al. 2002,
2005; Tenn et al. 2005). Furthermore, it is accompanied by
neurochemical (i.e., increase in DA, NA and 5-HT efflux in
nucleus accumbens, striatum or prefrontal cortex) and structural
changes (i.e., reduction of parvalbumin and brain-derived
neurotrophic factor expression in the medial prefrontal cortex
and hippocampus, respectively) (Doucet et al. 2013; Morshedi
and Meredith 2007; Motawaj and Arrang 2011; Salomon et al.
2006). However, it fails to induce any deficits in social activity
as an index of negative symptoms and therefore limits the
conformity to available human data (Srisurapanont et al.
2003, 2011). Similarly, the preferential DA receptor agonist
apomorphine has induced a SCZ-like phenotype in rodents
(Peleg-Raibstein et al. 2012). Overall, behavioural changes
induced by DA-stimulating drugs have been employed as
models of psychosis or cognitive-related abnormalities but they
fail to capture cardinal aspects of negative symptoms.
The glutamate hypothesis of SCZ has been developed
from the observation that NMDA receptor antagonists induce,
in normal humans, a psychosis-like state (plus negative
and cognitive symptoms) that closely resembles SCZ, leading
to the establishment of glutamatergic models of SCZ
(Javitt 2012). In animals, acute phencyclidine (PCP) treatment
induces hyperactivity and disruption of prepulse inhibition;
this is reversed by atypical but not typical antipsychotics
(Mouri et al. 2007). However, both classes of antipsychotic
agents are able to counteract the ketamine-induced
deficits, suggesting a different involvement of D2 receptors
in the PCP or ketamine effects (Neill et al. 2010). Acute PCP
treatment affects social activity and sucrose consumption, as
indices of negative symptoms and various different cognitive
domains (Mouri et al. 2012; Turgeon and Hulick 2007).
More conflicting results have been obtained from repeated
PCP treatment, which elicits reduced (Snigdha and Neill
2008) or no effects (Sams-Dodd 2004) on social behaviour
and an improvement in negative-like symptoms (Brigman
et al. 2009). PCP-induced deficits have also been found in
various cognitive domains, which are counteracted by atypical
antipsychotics (Amitai et al. 2007; Kunitachi et al.
2009). However, in clinical practice, antipsychotics do not
Table 2 Antidepressant-like effects of glutamatergic leading compounds in animal models of depression (NMDA N-methyl-D-aspartate, AMPA αamino-3-hydroxy-5-methylisoxazole-2-proprionic
acid)
Pharmacological mechanism Compound: animal models (references)
NMDA receptor antagonist Ketamine: learned helplessness, tail suspension test (Koike et al. 2011), forced swim test (Engin et al. 2009;
Lindholm et al. 2012), chronic mild stress (Garcia et al. 2009)
mGlu2/3 receptor antagonist MGS0039: tail suspension test (Koike et al. 2011), learned helplessness (Yoshimizu et al. 2006), olfactory
bulbectomy (Palucha-Poniewiera et al. 2010), forced swim test (Kawasaki et al. 2011); LY341495: tail
suspension test (Chaki et al. 2004; Koike et al. 2013)
mGlu2/3 receptor allosteric
negative modulator
RO4491533: tail suspension test (Campo et al. 2011)
mGlu2 receptor allosteric
potentiator
THIIC: forced swim test (Fell et al. 2011)
mGlu5 receptor uncompetitive
antagonist
MTEP: tail suspension test, forced swim test (Belozertseva et al. 2007; Li et al. 2006)
mGlu5 receptor negative allosteric
modulator
GRN-529: tail suspension test, forced swim test (Hughes et al. 2013)
mGlu7 receptor allosteric agonist AMN082: tail suspension test, forced swim test (Bradley et al. 2012)
NMDA receptor glycine-site partial
agonist
GLYX 13: learned helplessness, forced swim test (Ashton and Moore 2011; Burgdorf et al. 2013)
AMPA receptor potentiator LY 451646: forced swim test (Lindholm et al. 2012)
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Table3Pharmacologicalmodelsofschizophrenia(5-HTserotonin,AMYamygdala,ARIaripiprazole,BDNFbrain-derivedneurotrophicfactor,BLAbasolateralamygdala,CBcerebellum,CLZ
clozapine,DAdopamine,DRD2dopamineD2receptor,FCfrontalcortex,GLUglutamate,HPChippocampus,HPhaloperidol,mPFCmedialprefrontalcortex,NDnotdetermined,NEnoradrenalin,
NOR-1nuclearorphanreceptor1,PFCprefrontalcortex,PPIprepulseinhibition,PVparvalbumin,OLAolanzapine,RISrisperidone)
DrugPositive-
like
symptoms
Negative-
like
symptoms
Spatial/
working
memory
Latent
inhibition
Prepulse
inhibition
Neurochemical
changes
AntipsychoticresponseReferences
Dopamineagonists
AmphetamineYesNoDeficitDeficitDeficit↑MesolimbicDA
↑NEand5-HTinthe
PFC
↓PVinthemPFC
↓BDNFintheHPC
DeficitsreversedbyCLZ
andHP
Doucetetal.2013,Featherstoneetal.2007,
MorshediandMeredith2007,Motawajand
Arrang2011,Peleg-Raibsteinetal.2012,
Russigetal.2002,2005,Salomonetal.2006,
Srisurapanontetal.2003,2011,Tennetal.2005
ApomorphineYesNDDeficitDeficitDeficit↓mGluR5inthePFCPPIdeficitreversedbyCLZGeyerandEllenbroek2003,Gourgiotisetal.2012,
Lengetal.2003,Meloetal.2009,Posch
etal.2012,Shaoetal.2010
NMDAreceptorantagonists
PhencyclidineYesYesDeficitDeficitDeficit↓DAandGLUinthe
PFC
↓PVintheFC,HPCand
CB
↓CaMKIIinthePFC
↓ERKintheHPCand
AMY
↑5-HTinthePFC
DeficitsreversedbyHP,
CLZ,
ARI,RISandOLA
Amitaietal.2007,Bullocketal.2009,Kunitachi
etal.2009,Lietal.2011,Mourietal.2007,
2012,Nodaetal.2000,Pollardetal.2012,
TurgeonandHulick2007
KetamineYesYesDeficitDeficitDeficit↓PVintheHPCDeficitsreversedbyHP,CLZ
andRIS
EnomotoandFloresco2009,Gamaetal.2012,
Gaoetal.2009,Maeharaetal.2011,
Neilletal.2010,Pitsikasetal.2008,
Razouxetal.2007,Rujescuetal.2006
Dizocilpine(MK-
801)
YesYesDeficitDeficitDeficit↑GLUand5-HTinthe
mPFC
↓PVinthemPFC,HPC
andBLA
DeficitsreversedbyHP,
CLZ,
OLAandRIS
FeinsteinandKritzer2013,Gaisler-Salomon
etal.2008,Gururajanetal.2012,Lopez-Gil
etal.2007,2012,Mutluetal.2012,Ozdemir
etal.2012,Romonetal.2011,Ueharaetal.
2012,WiescholleckandManahan-Vaughan2013
5-HTagonist
Lysergicacid
diethylamide
(LSD)
YesYesNDNDDeficit↑DRD2,5-HT2cand
NOR1inthemPFC
CLZreversedpositive
symptoms.HPhasno
effectsonPPIdisruption
Marona-Lewickaetal.2011,Morenoetal.2011,
2013,Ouagazzaletal.2001,Paleniceketal.2010
Muscarinicacetylcholinereceptorantagonist
ScopolamineYesYesYesDeficitDeficitNDPPIdeficitreversedbyCLZ
andHP
BarakandWeiner2010,2011b,Depoortereetal.
2007,Guanetal.2010,Haradaetal.2012,
Johnsonetal.2005,ShannonandPeters1990,
SingerandYee2012
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197
improve cognition in patients; thus, further studies are necessary
to assess the mechanisms underlying the PCP effect
on cognition. Interestingly, the recent use of genetically modified
mice has revealed that various components of the
glutamatergic systems, such as specific glutamate receptor subtypes
or various components of their intracellular transduction
mechanism, might be involved in the pathophysiology of SCZ
(Inta et al. 2010). Hallucinogens such as lysergic acid
diethylamide (LSD) or cholinergic receptor antagonists, e.g.,
scopolamine, have induced, in humans and animals, psychoticlike
effects, thus supporting the 5-HTergic or cholinergic hypothesis
of SCZ, respectively. Therefore, the full potential of 5-HTor
cholinergic manipulations in preclinical research of SCZ needs to
be further validated (Barak 2009; Vollenweider et al. 1998).
Neurodevelopmental models
In the last few decades, human epidemiological data have supported
the finding that pre-perinatal environmental factors such
as malnutrition, infection and obstetric complications increase
the risk of the development of SCZ (Brown et al. 2013). This
knowledge has stimulated the development of models based on
direct pre-perinatal damage of the central nervous system (CNS);
such models replicate several behavioural and neurochemical
Table 4 Neurodevelopmental models of schizophrenia (5-HT serotonin,
AMY amygdala, CLZ clozapine, DA dopamine, dHPC dorsal hippocampus,
GLU glutamate, HPC hippocampus, HP haloperidol, MAM
methylazoxymethanol, mPFC medial prefrontal cortex, NAc nucleus
accumbens, PFC prefrontal cortex, PV parvalbumin, OLA olanzapine,
RIS risperidone, SER sertindole, vHPC ventral hippocampus, VTA
ventral tegmental area)
Experimental method Positive-
like
symptoms
Negative-
like
symptoms
Spatial/
working
memory
Latent
inhibition
Prepulse
inhibition
Neurochemical
changes
Antipsychotic
response
References
Prenatal manipulation
Prenatal MAM
exposure
Yes Yes Deficit Deficit Deficit ↑ DA activity
at the VTA
↓ PV and
mGlu5 in
the mPFC
↔ Reelin in
the HPC
Hyperactivity
of DA
neurons in
the VTA
reduced by
HP and
SER
Gastambide et al. 2012,
Lodge et al. 2009, Lodge
and Grace 2009, Matricon
et al. 2010, Moore et al.
2006, Snyder et al. 2012,
Valenti et al. 2011,
Zimmerman et al. 2013
Prenatal polyinosinic:
polycytidylic acid
exposure
Yes Yes Deficit Deficit Deficit ↓ PV in the
HIP
↓ DA in the
mPFC and
vHPC
↑ 5-HT in the
AMY and
NAc
↓ Reelin in the
dHPC
↑ GAD67 in
the vHPC
Deficits are
reversed by
RIS and
CLZ
Bitanihirwe et al. 2010,
Cardon et al. 2010,
Harvey and Boksa 2012,
Meyer et al. 2009, 2010,
Piontkewitz et al. 2009,
2011, 2012, Vuillermot
et al. 2012, Wolff and
Bilkey 2010
Postnatal manipulation
Postweaning isolation
rearing
Yes Yes Deficit Deficit Deficit ↑ Mesolimbic
DA
↑ GAD67 in
the AMY
↓ PV and reelin
in the vHPC
↓ CB1 and
GluR1in the
PFC
↑ Plasma
tryptophan
metabolites
Deficits
reversed by
HP, OLA,
RIS and
CLZ
Cassidy et al. 2010, GilabertJuan
et al. 2012, Harte
et al. 2007, Hermes et al.
2011, Marsden et al. 2011,
Moller et al. 2011, 2013,
Zamberletti et al. 2012a,
2012b
Neonatal ventral
hippocampal lesion
Yes Yes Deficit Deficit Deficit ↑ DA in the
PFC
↓ PV in the
HPC
↓ GAD67 in
the mPFC
Deficits
reversed by
HP, CLZ
and RIS
Bringas et al. 2012, Lee et al.
2012, Macedo et al. 2012,
Naert et al. 2013,
O’Donnell 2012,
Richtand et al. 2006,
Swerdlow et al. 2012
Cell Tissue Res
198
changes linked to the disease. In agreement with this
approach, rats exposed in utero on gestional day 17 to
methylazoxymethanol (MAM), an antimitotic agent that methylates
DNA, show behavioural (hyperactivity, cognitive
and social deficits or prepulse inhibition disruption) and
histopathological (decreased parvalbunin expression,
hyperdopaminergia) patterns similar to those observed in
SCZ (Lodge et al. 2009; Lodge and Grace 2009). Although
the MAM model seems to have face validity for SCZ symptoms
and construct validity in terms of the structural and
DAergic changes observed, only a few recent studies have
been performed to detect the antipsychotic activity of current
agents (Belujon et al. 2012; Valenti et al. 2011) or novel
compounds (Brown et al. 2013; Gastambide et al. 2012,
2013; Gill et al. 2011) and thus the predictive validity of this
model is not extensively established. Similarly, maternal administration
of the viral mimetic polyinosinic:polycytidylic
acid induces, in the offspring, a spectrum of neurochemical
and behavioural SCZ-related changes that were partially reversed
by antipsychotics (Bitanihirwe et al. 2010; Ozawa et al.
2006). An alternative approach makes use of environmental
manipulations during postnatal brain development and maturation,
such as maternal separation, isolation rearing, early
handling or brain lesions. These procedures are based on the
hypothesis that they can deflect the physiological development,
within the CNS, of an aberrant maturation process prone
to the emergence of psychotic-like behaviour and of social,
cognitive or attention/gating deficits that are sensitive to the
existing antipsychotics.
The advantage of neurodevelopmental over pharmacological
models of SCZ is the ability to perform behavioural and
neurochemical investigations in the absence of confounding
drugs and to identify new classes of antipsychotics by the use
of agents operating on multiple pharmacological mechanisms.
New potential pharmacological targets in the treatment
of SCZ: lessons from animal models
Current pharmacological treatment for SCZ is primarily focused
on modulating DA and 5-HT signalling, which is generally
effective in treating positive symptoms. However, it is
less effective in treating the negative and cognitive symptoms
and can induce several side effects, such as the extrapyramidal
side effect, weight gain and diabetes mellitus. Furthermore, a
significant proportion of patients are refractory to all current
treatments; thus, the development of new approaches for
treating SCZ is urgently needed (Keefe 2007). At the same
time, we are becoming increasingly aware that the pathophysiology
underlying SCZ cannot merely be explained by simple
changes in monoamine signalling but involves more complex
alterations in activity through key brain circuits that are critical
for sensory, cognitive and emotional processing (Lisman et al.
2008; Marek et al. 2010). These brain circuits are modulated
by DA and 5-HT, by the major excitatory and inhibitory
neurotransmitters glutamate and GABA, which are critical
for signalling through these circuits and by acetylcholine.
Thus, all these factors represent potential targets for pharmacological
intervention (Table 5). Based on the hypothesis that
impaired NMDA function in important cellular compartments
of the limbic forebrains might represent a critical feature
underlying the pathophysiology of SCZ, the mGlu2/3 receptor
agonists (Cartmell et al. 1999; Fabricius et al. 2011; Hackler
et al. 2010; Harich et al. 2007; Hikichi et al. 2013; Johnson
et al. 2005, 2011; Moghaddam and Adams 1998; Nakazato
et al. 2000; Patil et al. 2007; Profaci et al. 2011; Schlumberger
et al. 2009; Takamori et al. 2003), the mGlu2- (Galici et al.
2005; Harich et al. 2007; Nikiforuk et al. 2010) and mGlu5positive
allosteric modulators (PAMs; Clifton et al. 2013;
Darrah et al. 2008; Gastambide et al. 2013; Gilmour et al.
2013; Horio et al. 2012; Kinney et al. 2005; Kjaerby et al.
2013; Schlumberger et al. 2009, 2010; Stefani and
Moghaddam 2010; Vales et al. 2010) and the mGlu group
III orthosteric agonists (Palucha-Poniewiera et al. 2008;
Wieronska et al. 2012, 2013) have all shown preclinical
efficacy in reversing SCZ-like symptoms in several experimental
models. Although the positive results have not been
fully confirmed by clinical trials, the mGlu receptor ligands
seem to represent the first non-dopamine D2 receptor-based
antipsychotics (Hashimoto et al. 2013). To obtain a more
efficient NMDA receptor activation through an increased
synaptic glycine concentration, selective glycine transporter-
1 (GlyT-1) inhibitors have been shown to be effective in
specific preclinical models of SCZ (Alberati et al. 2012;
Hagiwara et al. 2013; Chen et al. 2010; Karasawa et al.
2008; Nagai et al. 2012; Shimazaki et al. 2010; Yang et al.
2010). Although definitive trials remain ongoing, encouraging
results to date have been reported (Javitt 2012). Several lines
of evidences suggest that alterations in central muscarinic or
nicotinic cholinergic neurotransmission are involved in the
pathophysiology of SCZ (Jones et al. 2012). Thus, based on
the above premise, the M1/M4 muscarinic acetylcholine receptor
(mAChR) agonist xanomeline (Barak and Weiner
2011b; Jones et al. 2005; Thomsen et al. 2010; Woolley
et al. 2009), the M1 or M4 PAMs (Brady et al. 2008; Chan
et al. 2008; Jones et al. 2005; Thomsen et al. 2010; Vanover
et al. 2008) and the α7 nAChr agonist/activators (Barak 2009;
Feuerbach et al. 2009; Hauser et al. 2009; Rezvani et al. 2010;
Roncarati et al. 2009; Wallace and Porter 2011; Wishka et al.
2006) have been shown to be effective in animal studies.
Despite the promising preclinical data, additional studies are
needed to develop more selective mAChRs subtype compounds
(i.e., molecules without agonistic activity at M2 and
M3 mAChRs) to avoid undesirable cholinergic side effects
(Langmead et al. 2008). Among the phosphodiesterases
(PDEs), which are a class of enzymes within the intracellular
Cell Tissue Res
199
Table 5 Leading compounds in experimental models of schizophrenia
(5-HT serotonin, mAChR muscarinic acetylcholine receptor, MAM
methylazoxymethanol, nAChR nicotinic acetylcholine receptor, ND
not determined, NMDA N-methyl-D-aspartate, PAMs positive allosteric
modulators, PDE phosphodiesterase, PPI prepulse inhibition)
Drugs Animal models Positive-like
symptoms
Negative-
like
symptoms
Cognitive
dysfunctions
Sensorimotor
gating
deficits in
PPI
References
mGlu2/3 agonists
LY354740,
LY404039,
LY379268,
MGS0008
MGS0028
BINA
CBiPES
Amphetamine, NMDA
antagonist, Neonatal ventral
hippocampal lesion
Improvement ND Improvement Improvement Cartmell et al. 1999,
Fabricius et al. 2011,
Hackler et al. 2010,
Harich et al. 2007,
Hikichi et al. 2013,
Johnson et al. 2005,
2011, Moghaddam
and Adams 1998,
Nakazato et al. 2000,
Patil et al. 2007,
Profaci et al. 2011,
Schlumberger et al.
2009, Takamori
et al. 2003
mGlu2 PAM
LY487379 Amphetamine, NMDA
antagonist
Improvement ND Improvement Improvement Galici et al. 2005,
Harich et al. 2007,
Nikiforuk et al. 2010
mGlu5 PAM
CDPPB
ADX47273
CPPZ
LSN2463359
LSN2814617
Amphetamine, NMDA
antagonist, MAM
Improvement Improvement Improvement Improvement Clifton et al. 2013,
Darrah et al. 2008,
Gastambide et al. 2012,
Horio et al. 2012,
Kinney et al. 2005,
Kjaerby et al. 2013,
Schlumberger et al.
2009, 2010, Stefani
and Moghaddam 2010,
Vales et al. 2010,
Vardigan et al. 2010
mGlu group III orthosteric agonists
LSP1-2111
ACPT-I
Amphetamine, NMDA
antagonist,
Improvement Improvement Improvement ND Palucha-Poniewiera et al.
2008, Wieronska et al.
2012, 2013
Glycine transporter 1 inhibitors
RG1678
Sarcosine
d-Serine
Amphetamine
NMDA antagonist
Polyinosinic:
polycytidylic acid
Improvement Improvement Improvement Improvement Alberati et al. 2012,
Hagiwara et al. 2013,
Chen et al. 2010,
Karasawa et al. 2008,
Nagai et al. 2012,
Shimazaki et al.
2010, Yang et al. 2010
M1/M4 mAChR agonists
Xanomeline Amphetamine
NMDA antagonist
Scopolamine
Improvement Improvement Improvement Improvement Barak and Weiner 2011a,
Thomsen et al. 2010,
Woolley et al. 2009
M1/M4 mAChR PAMs
TBPB
LY2033298
BQCA
AC-260584
VU0152100
Amphetamine
Apomorphine
Scopolamine
Improvement ND Improvement Improvement Bradley et al. 2010,
Brady et al. 2008,
Chan et al. 2008,
Jones et al. 2008,
Vanover et al. 2008
Cell Tissue Res
200
signal transduction cascade associated with brain abnormalities
in SCZ, PDE4 and PDE10A seem to be novel therapeutic
targets (Andreasen et al. 2011). Interestingly, specific PDE4 or
PDE10A inhibitors ameliorate positive symptoms and
cognitive/attention deficits (Davis and Gould 2005; Grauer
et al. 2009; Kanes et al. 2007; Schmidt et al. 2008; Siuciak
et al. 2008; Smith et al. 2013; Weber et al. 2009). Several
compounds are currently undergoing clinical testing, mostly
in clinical phase I trials in which SCZ is the leading indication
(Kehler 2013). Studies on histamine function in the CNS have
focused largely on the effects mediated via H3 receptor signalling.
Hence, H3 receptors antagonists or inverse agonists
have advanced into clinical assessment based on their effectiveness
as cognition enhancers in experimental models of
human diseases such as attention deficit hyperactivity disorder,
SCZ and Alzheimer’s disease (Brown et al. 2013; Fox
et al. 2005; Ligneau et al. 2007; Mahmood et al. 2012;
Medhurst et al. 2007; Raddatz et al. 2012; Southam et al.
2009; Vohora and Bhowmik 2012). In addition, the serotonin
5-HT6 receptors have been identified as a potential target for
the treatment of cognitive deficits in various disorders (Mitchell
and Neumaier 2005). The 5-HT6 receptor is almost exclusively
expressed in brain areas associated with learning and memory
and a large number of studies have shown that 5-HT6 antagonists
(de Bruin et al. 2013; Mitchell et al. 2006; Mohler et al.
2012) and 5-HT6 agonists (Burnham et al. 2010; Kendall et al.
2011; Nikiforuk et al. 2013) have beneficial effects in several
domains of cognition. Although the explanation for their similar
pro-cognitive effect is unavailable, they might act on various
neuronal subpopulations (Kendall et al. 2011; Schechter
et al. 2008) and trigger diverse signalling pathways (Yun et al.
2007).
Conclusive remarks and future prospectives
In conclusion, the development of reliable and predictive
animal models for neuropsychiatric disorders is a major
challenge for assuring successful drug development. The
field desperately needs better animal models of depression
Table 5 (continued)
Drugs Animal models Positive-like
symptoms
Negative-
like
symptoms
Cognitive
dysfunctions
Sensorimotor
gating
deficits in
PPI
References
α7 nAChR agonist/activator
SSR180711
RG3487
SEN12333
TC-5619
MEM3454
JN403
Amphetamine, apomorphine
NMDA antagonist
Improvement Improvement Improvement Improvement Barak 2009, Feuerbach
et al. 2009, Hauser et al.
2009, Rezvani et al. 2010,
Roncarati et al. 2009,
Wallace and Porter 2011,
Wishka et al. 2006
PDE4/PDE10A inhibitors
Rolipram
Papaverine
TP-10
MP-10
Vp1-15
THPP-1
Amphetamine
NMDA antagonist
Improvement Improvement Improvement Improvement Davis and Gould 2005,
Grauer et al. 2009,
Kanes et al. 2007,
Schmidt et al. 2008,
Siuciak et al. 2008,
Smith et al. 2013,
Weber et al. 2009
H3 antagonists/inverse agonists
ABT-239
Pitolisant
GSK-189254
GSK207040
Irdabisant
A-431404
Amphetamine
NMDA antagonist
MAM
Improvement ND Improvement Improvement Brown et al. 2013,
Fox et al. 2005,
Ligneau et al. 2007,
Mahmood et al. 2012,
Medhurst et al. 2007,
Raddatz et al. 2012,
Southam et al. 2009
5-HT6 agonists/antagonists
EMD386088
E-6801
PRX-07034
GSK-742457
NMDA antagonist
Scopolamine
ND ND Improvement No effect Burnham et al. 2010,
de Bruin et al. 2013,
Kendall et al. 2011,
Mohler et al. 2012,
Nikiforuk et al. 2013
Cell Tissue Res
201
and SCZ because of the partial efficacy of present pharmacological
treatment. Without improved models of human
disease, we cannot know whether particular molecular and
cellular findings in animals are relevant to the clinical situations.
Improved animal models of depression could come
from various sources, such as mutant mice exhibiting particular
depressive symptoms or human genetic studies identifying
the genetic abnormalities that increase an individual’s
risk. Given the complexity of the neurobiological mechanisms
involved in the SCZ, the recreation of the diversity of
the disease in a single animal model might not be possible.
Thus, the development and use of symptom-focused tests is
important, whereby the goal is to replicate specific symptoms
such as anhedonia or the seven cognitive domains as
identified by the NIH-MATRICS consensus committee,
which are impacted in SCZ, rather than the entire syndrome.
Therefore, novel potential pharmacological targets (see
Tables 2, 5) and positive control compounds will probably
be needed for each of these domains. Nevertheless, all the
findings reviewed above suggest that the identification of
candidate compounds and the validation of efficacious treatments
that can be used as positive controls in the development
of new preclinical paradigms remain to be of paramount
importance.
Acknowledgments The authors are grateful to Mr. Hynek Anthony
Spicka (London, UK), Tony Fong and Vanessa Raileanu (Toronto,
Canada) for their kind help with manuscript preparation and proofreading.
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Čes a slov Psychiat 2015; 111(2): 72–78
Souhrn
Amchová P, Kučerová J. Pohlaví a drogová
závislost: od animálních modelů
ke klinické praxi
Drogová závislost je závažný zdravotní
i psychosociální problém, který kromě
organického poškození organismu může
vést až k neschopnosti plnit normální
sociální funkce a role ve společnosti
a rodině. Z klinických zkušeností je patrné,
že závislost má u žen a u mužů odlišné
charakteristiky, a přestože absolutní
počet žen užívajících návykové látky je
nižší než počet mužů, což je dáno především
sociálními faktory, míra eskalace,
potíže s ukončením a frekvence relapsů
po abstinenci jsou u žen výrazně vyšší
než u mužů. Tyto odlišnosti mezi pohlavími
budou v budoucnu bezpochyby
vyžadovat odlišné strategie léčby drogové
závislosti u žen a mužů. Preklinické
studie látkových závislostí byly dlouho
vedeny výhradně na samcích laboratorních
zvířat, neboť je známo, že estrální
cykly samic výrazně ovlivňují chování
i laboratorní výsledky. Ale i zde je v poslední
době kladen stále větší důraz právě
na identifikaci behaviorálních a neurochemických
rozdílů mezi pohlavími
za účelem vývoje personalizovaných léčebných
řešení. Plazmatické koncentrace
ženských a mužských pohlavních hormonů
ovlivňují chování jedince, včetně
adiktivního, velmi významně, ale byly již
zaznamenány i rozdíly v mozkové struktuře
závislé na pohlaví u člověka i experimentálních
zvířat. Mezi další možné
příčiny těchto rozdílů lze zařadit odlišPetra
Amchová1,2
Jana Kučerová1,2
1
Farmakologický ústav,
Lékařská fakulta,
Masarykova Univerzita, Brno
2
Výzkumná skupina
experimentální a aplikované
neuropsychofarmakologie,
CEITEC – Středoevropský
technologický institut,
Masarykova Univerzita, Brno
Kontaktní adresa:
PharmDr. Jana Kučerová, Ph.D.
Farmakologický ústav LF MU
Kamenice 5
625 00 Brno
e-mail: jkucer@med.muni.cz
souborný článek
Pohlaví a drogová závislost:
od animálních modelů
ke klinické praxi
Tato práce vznikla díky podpoře
interního projektu Lékařské
fakulty Masarykovy Univerzity
(MUNI/11/InGA09/2012),
projektu Specifického výzkumu
na Masarykově univerzitě
(MUNI/A/1116/2014) a projektu
„CEITEC – Středoevropský
technologický institut“
(CZ.1.05/1.1.00/02.0068)
z Evropského fondu regionálního
rozvoje.
Summary
Amchová P, Kučerová J. Gender and
drug addiction: from the animal models
to human medicine
Drug addiction is a serious medical and
psychosocial problem which leads to organic
harm of the body as well as distortion
of the normal functioning of affected
persons within the society and family.
There is a large body of clinical evidence
suggesting differential characteristics of
the disorder in men and women. Despite
the absolute number of female drug
abusers is lower than the male ones,
women usually show higher escalation
rate, more frequent relapses and more
difficulties when discontinuing the drug
use. These gender specific differences
will require specific treatment strategies
for men and women in the near future.
Preclinical studies of drug addiction
were carried out with male subject only
for a long time because significant influence
of the estrous cycle is well known in
terms of behavioural and neurochemical
effects but recently this approach has
been abandoned on order to identify
the gender differences and develop new
more specific treatments. Levels of male
and female gonadal hormones strongly
affect the behaviour of people which
concerns the addictive one as well. However,
there have been reported also gender
dependent differences in the brain
structure in both humans and animals.
Another source of the gender differences
comprise metabolic adjustments
leading to pharmacokinetic changes of
213
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Čes a slov Psychiat 2015; 111(2): 72–78
Úvod
Drogová závislost je zdrojem závažných zdravotních
a psychosociálních problémů, které kromě organického
poškození organismu mohou vést až k selhání normálního
sociálního zařazení jedince. Pohlavní rozdíly v těchto
fenoménech vyplývají již z epidemiologických dat. Česká
republika dlouhodobě patří mezi země s největší konzumací
alkoholu na světě, kde 15 % dospělých pije alkohol
pravidelně a velmi často (denně nebo obden). U mužů je
výskyt 23 %, u žen 8 %. Pití alkoholických nápojů alespoň
jednou týdně uvedla více než polovina mužů a více než
čtvrtina žen. Současných kuřáků tabákových výrobků je
u nás 31 %, mužů 36,5 %, žen 26,3 %.1
Monitoring prevalence ostatních návykových látek se
rutinně provádí pouze na základě evidence všech uživatelů
drog, kteří za  daný rok alespoň jedenkrát navštíví
některé ze zařízení, která poskytují péči osobám užívajícím
drogy. Tab. 1 uvádí prevalenci těchto osob v závislosti
na pohlaví za rok 2013.
Drogová závislost má u žen a mužů odlišné charakteristiky:
počínaje volbou návykové látky, její dávky, přes
zahájení a průběh, až po tendenci k relapsu v období abstinence.
Tyto odlišnosti mezi pohlavími budou v budoucnu
bezpochyby vyžadovat odlišné strategie léčby závislosti
u žen a mužů. Cílem tohoto článku je identifikovat možné
zdroje těchto rozdílů a poskytnout přehled dostupných
humánních i animálních dat.
K možným zdrojům lze řadit:
1) 	genetické odlišnosti související s geny nesenými pohlavními
chromosomy,
2) 	odlišnou farmakokinetiku a farmakodynamiku,
3) 	odlišné hladiny pohlavních hormonů a
4) 	socio-kulturní rozdíly.
nosti v metabolismu, které způsobují
rozdíly ve farmakokinetice návykových
látek včetně rozdílů v množství tuku,
vody a svalové hmoty, a to i na úrovni
mozku. Dalšími faktory jsou rozdíly
farmakodynamické, jejichž podkladem
může být odlišná konektivita neuronálních
drah a neuromediátorových systémů,
které jsou již prenatálně modifikovány
pohlavními hormony a chromosomy.
Především farmakodynamické rozdíly
vedou k subjektivním rozdílům v účinku
návykových látek a následně i variabilitě
v tendenci k rozvoji drogové závislosti,
toleranci či senzitizaci k látce. Cílem této
práce je poskytnout přehled současných
znalostí z klinických i preklinických studií
a mechanismů, které jsou podkladem
pro mezipohlavní rozdíly v průběhu drogové
závislosti.
Klíčová slova: animální modely, drogová
závislost, farmakodynamika, farmakokinetika,
geny, mezipohlavní rozdíly, pohlavní
hormony, socio-kulturní rozdíly.
the drugs including different fat deposition,
amount of water in the body or
proportion of skeletal muscles. Pharmacodynamic
changes could be under
lied by changes in connectivity of the
neuronal tracts and neurotransmitter
systems which are modified prenatally
by gonadal hormones or chromosomes.
These pharmacodynamic specificities
are the major source of subjective differences
in the effects of the abused drugs
and to the variable tendency to develop
the addiction, tolerance or sensitization
to the drug. The aim of this review article
is to provide a survey of the current
knowledge on the gender differences in
drug addiction based on both clinical
and preclinical studies preklinických together
with the mechanisms responsible
for such differences in the course of the
drug addiction.
Key words: animal models, drug addiction,
gender differences, pharmacodynamics,
pharmacokinetics, sex hormones,
genes, socio-cultural differences.
Tab. 1. Prevalence uživatelů drog léčených v roce 2013 v ČR (zdroj:
Hygienická stanice hl. m. Prahy)
Návyková látka Muži
(počet)
Muži
(%)
Ženy
(počet)
Ženy
(%)
Opiáty 1232 73,3 443 26,4
Kokain 13 68,4 6 31,6
Stimulancia 4587 66,7 2260 32,9
Hypnotika, sedativa 27 40,9 39 59,1
Halucinogeny 1 33,3 2 66,7
Kanabinoidy 822 76,3 251 23,3
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Animální modely drogové závislosti
Tato práce čerpá z klinických i preklinických dat, proto je
nutné alespoň stručně popsat základní animální modely,
které se ve výzkumu používají. V těchto modelech lze
jasně izolovat jednotlivé fáze rozvoje a průběhu drogové
závislosti. Preklinický výzkum tak umožňuje lépe pochopit
neurobiologii drogové závislosti a poskytuje možnost
studovat jednotlivé proměnné, které mají vliv na adiktivní
chování. Takové studie umožňují analýzu změn v chování
zvířat po expozici návykovým látkám i sledování hladin
neurotransmiterů v příslušných mozkových drahách. Zvířecí
modely mají samozřejmě své limity vycházející z rozdílnosti
člověka a zvířete na všech úrovních. Rozvoj závislosti
na návykové látce je komplexní proces ovlivněný
mnoha faktory a animální model nemůže obsáhnout stav
člověka ve všech aspektech. Většina animálních modelů
vychází z předpokladu, že droga je pozitivní posilovač
a stimuluje tak subjekt k vyhledávání další dávky (positive
reinforcement). Motivace k opakovanému užití drogy
může být na druhou stranu i z důvodu jejího averzivního
účinku (negative reinforcement) za účelem odstranění nepříjemných
pocitů (tzv. abstinenční syndrom) po přerušení
opakované aplikace látky.
K  modelům založeným na  pozitivním posilovacím
účinku drogy patří operantní intravenózní autoaplikace
látek (IV drug self-administration; IVSA). Ten slouží
ke sledování příjmu drogy, jeho eskalace a motivace zvířete
pracovat pro další dávku. Dále sem lze zařadit model
intrakraniální autostimulace (intracranial self-stimulation,
ICSS), který spočívá v implantaci elektrody do mozku
zvířete tak, aby elektricky stimulovala oblasti motivace
a odměny po příslušném chování.
Stimulem pro vyvolání cravingu může být, kromě
samotné drogy, i  prostředí (conditioned place preference,
CPP) nebo podnět (např. injekční stříkačka), který
drogu připomíná. Na stejném principu jako CPP, kde má
zvíře spojeno prostředí s pozitivním zážitkem (po aplikaci
drogy), je založen i test averze k místu (place aversion)
s očekávaným opačným efektem (jako negativní zážitek se
užívá např. mírný elektrický výboj).2
Genetické odlišnosti
související s geny nesenými
pohlavními chromosomy
Existují klinické i preklinické studie dokazující vliv samotných
pohlavních chromosomů (XX, XY), anebo kombinace
chromosomů a hormonů na pohlavní rozdíly. Psychická
závislost (tzv. habit formation) na kokainu může
být ovlivněna nikoliv pouze pohlavními hormony, jak se
předpokládá, že tomu je u fyzické závislosti po opakovaném
podání látky, či při rozvoji behaviorální senzitizace,3
ale vlivem pohlavních chromosomů.
Pro tento výzkum se v preklinických studiích stala ideálním
modelem transgenní myš, které byl z chromosomu
Y odstraněn gen Sry určující vznik varlat. U  zvířete se
pak vyvinuly vaječníky, přestože jeho gonozomy zůstaly
XY.4
Další studie využívající tohoto modelu zkoumala vliv
amfetaminu na  odměnu vyvolanou umělou elektrickou
mozkovou stimulací (ICSS model) a  došla k  závěru, že
amfetamin zesiluje systém odměny u genotypu XY, zatímco
u XX nikoliv. Tento výsledek autoři odůvodňují rozdílnou
citlivostí dopaminergního systému podmíněného
pohlavním dimorfismem mozku, který vzniká v důsledku
odlišného vývoje nezávisle na hormonálních hladinách.5
Rozdíly dané odlišnou
farmakodynamikou
a farmakokinetikou
Klinické údaje
V závislosti na návykové látce dochází k ovlivnění jednotlivých
neuromediátorových systémů (dopaminergní,
serotonergní, noradrenergní, opioidní, cholinergní,
GABA-ergní, glutamátergní, endokanabinoidní a další)
a ke změnám v jednotlivých částech mozku zodpovědných
za systém odměny (kortikolimbický systém). Hladiny
neurotransmiterů a mediátorů po podání látky jsou
jiné u žen než u mužů, mezipohlavní rozdíl se také vyskytuje
v objemu šedé a bílé hmoty míšní.
Existuje hypotéza, že k  predispozici závislosti na  návykových
látkách může vést hypertrofie striata, která byla
prokázána u  lidí užívajících amfetamin.6
Avšak vysoký
objem striata může být také kompenzací toxicity vyvolané
vysokými hladinami dopaminu v bazálních gangliích.
Dalším společným znakem chronických uživatelů amfetaminu
je snížený objem šedé hmoty mozkové.6
Také užívání kokainu je spojeno s poškozením neuronů
a gliových buněk v šedé i bílé hmotě mozkové předního laloku.
U abstinujících jedinců dochází k jejich reparaci, a to
v mnohem vyšší míře u žen v porovnání s muži.7
Dlouhodobé
užívání alkoholu taktéž vede k úbytku objemu šedé
i bílé hmoty u obou pohlaví, ale u žen v porovnání s muži
markantněji.8
Denier et al. ve své studii se závislými na heroinu
dávají do  souvislosti úbytek objemu šedé hmoty
ve frontální oblasti mozku a snížený průtok krve v téže
oblasti. U abstinujících závislých na metamfetaminu byl
měřen průtok krve v týlním laloku a ve střední čáře, kdy
u obou struktur byl naměřen nižší průtok u mužů ve srovnání
se ženami.9
Důvodem odlišného vlivu návykové látky v  ženském
a mužském těle může být specifická farmakokinetika drogy,
jak bylo např. popsáno u alkoholu: ženy mají nižší procento
celkové tělesné vody, menší vliv prvního průchodu játry
(„first-pass“ efekt), a pomalejší metabolismus alkoholu
v důsledku nižších hladin alkohol-dehydrogenázy v žaludeční
sliznici, nebo farmakodynamické rozdíly, podmíněné
např. rozdílným počtem, lokalizací či expresí receptorů
v centrálním nervovém systému. V porovnání s muži jsou
ženy více ohroženy intoxikací alkoholu10
a  mají i  horší
a rychleji nastupující chronické následky, jako atrofie mozku,
onemocnění srdce, kosterních svalů a jater.11
Je již dlouho známo, že chronické užívání metamfetaminu
působí neurotoxicky na dopaminergní neurony, kdy
nejvíce postiženy jsou frontální a subkortikální oblast,12
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a zdá se, že u mužů se projevují neurodegenerativní změny
ve větší míře než u žen. Kromě již zmíněného sníženého
průtoku krve a zmenšeného objemu šedé hmoty dochází
k  poklesu cerebrálního glukózového metabolismu
v bílé hmotě ve frontální oblasti, což koreluje se sníženými
funkcemi v této oblasti, a to pouze u mužů. U žen k této
metabolické změně nedochází, což může být vysvětleno
tím, že funkční glukózový metabolismus umožňuje reparaci
gliových buněk.13
Dalším vysvětlením je pak možné
neuroprotektivní působení estrogenu.14
Preklinické údaje
Mezipohlavní rozdíly mohou být kromě hormonálních
vlivů vysvětleny především na základě odlišností
v mozkové organizaci. Bylo prokázáno, že samice potkanů
po ovariektomii, tzn. nezávisle na hormonech, si aplikují
kokain rychleji a ve větším množství než samci (model
IVSA).15
To znamená, že existuje rozdíl v nervových systémech
zprostředkujících lokomočně pátrací chování v závislosti
na pohlavní diferenciaci mozku v časných stadiích
vývoje.16
Studie zabývající se touto problematikou však ne
vždy skutečně používají gonadektomizovaná zvířata, což
je hlavním limitem pro interpretaci výsledků ve smyslu
nezávislosti na hormonálních hladinách.
Rozdíly v hladinách
pohlavních hormonů
Pohlavní hormony působí na systém odměny a stresu,
čímž ovlivňují dva nejčastější důvody příjmu drogy:
pro potěšení a za účelem uvolnění stresu. To se potvrdilo
jak v preklinických, tak v klinických studiích, přestože
hormonální změny u zvířat přesně nekopírují změny hladin
hormonů u lidí.
Klinické údaje
Hormonální odlišnosti se nabízejí jako první vysvětlení
mezipohlavních rozdílů v oblasti zneužívání návykových
látek. Pohlavní hormony neslouží pouze k regulaci reprodukčních
procesů, ale mají také vliv na kognitivní a afektivní
funkce. Interpretaci klinických údajů nicméně komplikuje
praktická obtížnost sledování jejich aktuálních
hladin v průběhu menstruačního cyklu a jejich přímá korelace
s adiktivním chováním, což značně limituje i počet
publikovaných klinických studií.
Když se pokusíme zjednodušit průběh menstruačního
cyklu podle převládajících hladin hormonů, charakterizuje
folikulární fázi vysoká hladina estrogenů, zatímco
v luteální fázi dominuje progesteron. Byla provedena klinická
studie, ve které byly nalezeny rozdíly v subjektivních
účincích amfetaminu na ženský organismus v těchto dvou
fázích cyklu. Objektivní účinek drogy, hodnocený pomocí
krevního tlaku a srdeční frekvence, byl v obou fázích
cyklu prakticky stejný. Naproti tomu subjektivní efekt amfetaminu,
hodnocený několika dotazníky (míra euforie,
bažení, přátelských pocitů, energie apod.), se významně
odlišoval – ve folikulární fázi byly tyto účinky signifikantně
vyšší než v luteální fázi.17
Carpenter et al. později publikovali
metaanalýzu 13 studií sledujících ženy kuřačky a jejich
pokusy přestat kouřit v závislosti na menstruačním
cyklu. Ženy inklinovaly ke zvýšenému cravingu (dychtění
po droze) a dysforiím v pozdní luteální fázi oproti folikulární
fázi.18
Jedním z vysvětlení je schopnost estradiolu
potlačovat úzkostné chování, a tudíž ženy, které přestanou
kouřit ve folikulární fázi, mají větší naději na úspěch. Tyto
odlišnosti byly zaznamenány i pomocí měření funkčního
MRI vyšetření – BOLD fMRI (blood-oxygen-level dependent
functional magnetic resonance imaging), kde bylo
prokázáno, že v průběhu folikulární fáze jsou u žen více
aktivovány oblasti mozku spojené s  odměnou (střední
mozek, striatum, levá frontopolární prefrontální kůra)
ve srovnání s luteální fází.19
Vliv mají i exogenně podané hormony, např. estradiol
zvyšuje pozitivní subjektivní účinek amfetaminu během
folikulární fáze.20
Analogická data byla zaznamenána
u kokainu, kde navíc exogenně podaný progesteron snižoval
vliv drogy, a to pouze u žen, zatímco u mužů se jeho
efekt po akutním podání neprojevil.21
Progesteron byl použit
již v několika klinických studiích jako podpůrná farmakoterapie
při odvykání závislosti na kokainu a nikotinu
u žen se slibnými výsledky.22
Méně pozornosti v  souvislosti s  drogovou závislostí
má testosteron, a to u žen i u mužů. Fluktuace jeho hladin
u mužů patrně souvisí především se sklonem k relapsu drogové
závislosti, který může být odstartován v souvislosti se
sexuální aktivitou, vzhledem k tomu, že muži často subjektivně
uvádějí relaps do souvislosti s příjemnými pocity
(např. výhra, obecněji jakýkoli druh odměny). To je zvlášť
patrné u psychostimulancií.23
Hladiny testosteronu u žen
se mění v závislosti na menstruačním cyklu, ale také v různých
sociálních situacích (sport, výhry, prohry, pláč dítěte).
Zdá se, že když žena začne užívat psychostimulancia s cílem
ulevit depresi či úzkosti, riziko následného rozvoje závislosti
se výrazně liší podle fáze menstruačního cyklu.24
Muži a ženy se liší i v klinických projevech závislosti.
Míra eskalace, potíže s  ukončením a  frekvence relapsů
po abstinenci je u žen výrazně vyšší než u mužů.25
Tyto
výsledky jsou patrné u alkoholu, tabáku, marihuany, kokainu
i opioidů.16,26
Nejprozkoumanější je z tohoto pohledu
fumátorství. Ženy kouří v kratších intervalech, tzn. více
cigaret za jednotku času a přestat kouřit je pro ně obtížnější
než pro muže.16
Stejně jako pohlavní hormony ovlivňují adiktivní chování
a s tím spojené mezipohlavní rozdíly, tak i naopak, návykové
látky mají vliv na uvolňování a metabolismus hormonů.
Příkladem je alkohol, kokain a marihuana, jejichž
užívání je spojováno se sníženými hladinami luteinizačního
hormonu a testosteronu a zvýšenými hladinami progesteronu,
adrenokortikotropního hormonu (ACTH) a  kortikotropinu,
což vede k ohrožením plodnosti žen27
i mužů.28
S těmito změnami charakteristickými pro jednotlivé návykové
látky bude rovněž nutné počítat při vývoji případných
hormonálních terapií drogových závislostí.
Preklinické údaje
U pokusných zvířat (nejčastěji hlodavců) se projevil vliv
pohlavních hormonů ve všech fázích experimentálně
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navozené drogové závislosti. V souladu s klinickými důkazy
samice potkanů v době zvýšených hladin estradiolu
a snížených hladin progesteronu (tj. estrus) vyhledávají
drogy nejintenzivněji. Tento modulační vliv estradiolu se
vysvětluje prostřednictvím zvýšeného vyplavování dopaminu
v mezokortikolimbických strukturách, což je dopaminergní
dráha spojená s procesy odměny. V případě
podání opioidů přistupuje k vyplavení dopaminu další
mechanismus, a to aktivace mí a kappa opioidních receptorů
v okruhu odměny.
S hladinou estrogenu souvisí i relaps, který je obecně
častější u  žen, a  samice potkanů vykazují v  příslušných
modelech rovněž vyšší vulnerabilitu. Touha po droze se
v animálních modelech měří v testu autoaplikace drogy
po určité době abstinence. Potkaní samice v estru vykazují
trend zvýšeného bažení po droze.29
Analogicky jako u žen, i u zvířat potlačuje progesteron
posilující vliv estrogenu. Progesteron jako potenciální léčba
závislostí je již klinicky zkoumán.22
Dalším důkazem
vlivu progesteronu na adiktivní chování jsou jeho nízké
hladiny (samice potkana v estru) odpovídající vyšší motivaci
k autoaplikaci kokainu. U samic s přirozeným estrálním
cyklem byly naměřeny nízké hladiny progesteronu
ve fázi vyhledávání drogy i při relapsu.30
Limitujícím faktorem je v této oblasti léčba závislosti
na alkoholu, u kterého se regulující vliv pohlavních hormonů
nepotvrdil. Ani ovariektomie, ani exogenně podaný
progesteron neovlivnil míru užívání alkoholu u zvířat. Navíc
u lidí se projevují mezipohlavní rozdíly v alkoholismu
obráceně než u hlodavců (muži pijí více než ženy, samci
méně než samice). Alkoholovou závislost však u člověka
ovlivňuje mnoho komplexních faktorů (sociální, genetické,
hormonální a neurobiologické), které tento fenomén
vyvolávají a které je nemožné modelovat u zvířat v jejich
úplnosti.31
Socio-kulturní rozdíly
Společnost od každého pohlaví očekává jiné chování, tzv.
genderové stereotypy, které se projevují také u lidí se syndromem
závislosti. Tato genderová specifika se promítají
do prevalence, preference užívané látky, motivace k užití,
rodinné anamnézy, poskytování sexuálních služeb aj.
Obecně lze tvrdit, že muži užívají drogy častěji než ženy.
Tento celosvětový fenomén se vysvětluje jak příležitostí
a přístupem k droze, což platí zejména v asijských státech
(např. v Indii nebo Íránu je ze všech drogově závislých
cca 7 % žen), tak vnímáním role ženy v socio-kulturním
kontextu (nejen ve východních zemích, ale i v Evropě
a USA). Podstatou genderové specifičnosti je tzv. fenomén
dvojí stigmatizace či dvojí deviace, kdy žena je stigmatizována
primárně za samotné užívání návykové látky,
a navíc za to, že zklamala ve své roli matky/pečovatelky.24
Ženy začínají s užíváním drog v dřívějším věku než muži
a také míra eskalace a kvantita je u většiny návykových
látek markantnější u žen v porovnání s muži. Muži mají
většinou jinou motivaci k experimentování s návykovými
látkami než ženy, obecně muži sáhnou primárně po droze
z důvodu jejího posilujícího účinku (pro pocit vzrušení
nebo posílení jejich sociální pozice), naopak u žen je
více pravděpodobné, že využijí drogy k léčbě psychického
stavu (většinou úzkost, deprese, sociální izolace). Tato
genderová rozdílnost v iniciativě odpovídá i tomu, že více
mužů užívá ilegální drogy (cannabis, opiáty, kokain), ženy
naopak ve větší míře zneužívají léky na předpis (opiáty, sedativa).
Ženy mají také v anamnéze častěji než muži různé
formy fyzického zneužívání, jemuž jsou v dětství a/nebo
v dospělosti vystavovány, a díky tomu vstupují do procesu
vzniku závislosti již s existujícím psychickým zatížením,
což může být další důvod, proč se u nich rozvine závislost
na látce rychleji než u mužů.32
Zahraniční výzkumy potvrzují, že ženy vyhledávají
odbornou pomoc v  souvislosti s  užíváním návykových
látek méně často než muži, ale pokud do léčby nastoupí,
jejich výsledky a míra úspěšnosti léčby jsou podobné jako
u mužů. Důvodů, proč ženy váhají při vyhledávání odborné
pomoci, se naskytuje více. V první řadě je to již zmiňované
společenské stigma a dále také strach z odebrání
dětí, proto mají ženy větší tendenci svou závislost skrývat,
jejich okolí reaguje opožděně. Rovněž si častěji obstarávají
peníze na  nákup drog prostřednictvím sexuálního průmyslu,
a pokud zažádají o institucionální pomoc, musejí
vyjít alespoň částečně z anonymity. Navíc většina zařízení
je zaměřena na řešení problémů spojených s užíváním ilegálních
drog, jež častěji užívají muži.24
Závěr
K přiblížení neurobiologických rozdílů mezi pohlavími
v rozvoji drogové závislosti nám mohou posloužit následující
3 koncepty:
1) 	závislost na návykových látkách postihuje u žen jiné
mozkové oblasti než u mužů,
2) 	závislost má mnoho klinických projevů, které vedou
k maladaptivnímu chování a
3) 	závislost může předcházet přítomnost psychopatologie
před začátkem užívání drogy.
Společnost do jisté míry určuje mezipohlavní rozdíly
v užívání návykových látek, což se odráží na vyšší prevalenci
drogově závislých mužů. Naopak u žen hrozí vyšší
riziko vzniku závislosti již po akutní aplikaci drogy v souvislosti
s  fluktuujícími hladinami hormonů (podle fáze
menstruačního cyklu), které ovlivní subjektivní náladu
a účinky drogy.24
V mozku žen se více stimuluje dopaminergní
systém na počátku expozice drogy, a to ve smyslu
většího uvolňování dopaminu i zvýšené inhibice zpětného
vychytávání, proto u žen dochází k rychlejší eskalaci
příjmu návykové látky a  rozvoji závislosti.24
Chronické
užívání drogy vede k adaptačnímu hypodopaminergnímu
stavu ve striatu u obou pohlaví, avšak u žen je tento efekt
díky vyšší reaktivitě dopaminergního systému výraznější.
Snížené hladiny dopaminu v periodách abstinence se projevují
silnou dysforií a anhedonií, ke zvýšení hladin dopaminu
již nestačí přirozené druhy odměny (jídlo apod.)
a dochází k většímu sklonu k relapsu.5
Droga navíc může
být formou samoléčby depresivních stavů charakterizovaných
sníženými hladinami monoaminů,33
jak ukazují
i preklinické studie s návykovými látkami z různých sku-
pin.34,35
Chronické užívání téměř všech návykových látek
je spojeno také se zvýšeným vyplavováním noradrenalinu,
217
strana 77
Čes a slov Psychiat 2015; 111(2): 72–78
který přispívá k negativnímu afektivnímu stavu: úzkost,
podrážděnost. U žen je stav horší díky zvýšené noradrenergní
a kortikotropní aktivitě.24
Léčba závislostí zatím nemá jasný koncept a  existuje
jen málo účinných postupů. Vzhledem k tomu, že existují
mezipohlavní rozdíly ve vzniku, průběhu i následcích
závislosti na drogách, měla by se i léčba závislostí zaměřit
jednotlivě na obě pohlaví, aby byla co nejúčinnější. Např.
naltrexon a  disulfiram snižují užívání kokainu u  mužů,
avšak ne u  žen.36,37
Léčba je často zacílená na  zmírnění
negativních abstinenčních příznaků, jako dysforie, úzkost,
podrážděnost, čímž se sníží frekvence užívání drog
a recidivy. K potlačení subjektivních negativních pocitů se
osvědčily inhibitory acetylcholinesterázy, avšak účastníci
studií byli pouze muži.24
Animální experimenty zásadním způsobem obohacují
dostupné údaje o neurobiologických podkladech mezipohlavních
rozdílů ve všech fázích látkových závislostí. Při
interpretaci dat získaných z experimentů na zvířatech je
třeba počítat s jejich limity při predikci reaktivity lidského
organismu, která se může zásadně lišit v mnoha ohledech.
Lidský organismus může odbourávat dané léčivo či návykovou
látku odlišně (farmakokinetické rozdíly), může
disponovat jinou receptorovou a enzymatickou výbavou
a dalšími zvláštnostmi. Přes svá omezení však mohou animální
behaviorální modely na tomto poli, obdobně jak je
jednoznačně prokazováno i v jiných oblastech medicíny,
přinášet validní informace o  reaktivitě celého organismu,
možných interakcích jednotlivých látek a především
o slibných možnostech ovlivnění drogové závislosti.
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Robert Rusina, Radoslav Matěj a kolektiv
NEURODEGENERATIVNÍ ONEMOCNĚNÍ
nemoc, Huntingtonova nemoc, progresivní supranukleární
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a parkinsonismu (farmakologické i nefarmakologické intervence,
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neuropsychiatrické projevy demencí a možnosti
jejich ovlivnění. Součástí knihy je i problematika právní
a etická, zahrnující sdělování diagnózy, otázku řidičských
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Prakticky zaměřená publikace je
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Přehledně a prakticky jsou
diskutovány etiopatologické, klinické,
neuropsychologické a neuroradiologické
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219
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Send Orders for Reprints to reprints@benthamscience.net
Recent Patents on CNS Drug Discovery, 2014, 9, 13-25 13
Therapeutic Potential of Cannabinoids in Schizophrenia
Jana Kucerova1,2
, Katarina Tabiova1,2
, Filippo Drago3
and Vincenzo Micale1,3
*
1
CEITEC (Central European Institute of Technology) Masaryk University, Brno, Czech Republic; 2
Department of
Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czech Republic; 3
Department of Clinical and
Molecular Biomedicine, Section of Pharmacology and Biochemistry, University of Catania, Catania, Italy
Received: February 04, 2014; Revised: March 03, 2014; Accepted: March 06, 2014
Abstract: Increasing evidence suggests a close relationship between the endocannabinoid system and schizophrenia. The
endocannabinoid system comprises of two G protein-coupled receptors (the cannabinoid receptors 1 and 2 [CB1 and CB2]
for marijuana’s psychoactive principle 9
-tetrahydrocannabinol), their endogenous small lipid ligands (namely anandamide
[AEA] and 2-arachidonoylglycerol [2-AG], also known as endocannabinoids), and proteins for endocannabinoid
biosynthesis and degradation. It has been suggested to be a pro-homeostatic and pleiotropic signalling system activated in
a time- and tissue-specific manner during pathophysiological conditions. In the brain, activation of this system impacts the
release of numerous neurotransmitters in various systems and cytokines from glial cells. Hence, the endocannabinoid system
is strongly involved in neuropsychiatric disorders, such as schizophrenia. Therefore, adolescence use of Cannabis
may alter the endocannabinoid signalling and pose a potential environmental risk to develop psychosis. Consistently,
preclinical and clinical studies have found a dysregulation in the endocannabinoid system such as changed expression of
CB1 and CB2 receptors or altered levels of AEA and 2-AG . Thus, due to the partial efficacy of actual antipsychotics,
compounds which modulate this system may provide a novel therapeutic target for the treatment of schizophrenia. The
present article reviews current available knowledge on herbal, synthetic and endogenous cannabinoids with respect to the
modulation of schizophrenic symptomatology. Furthermore, this review will be highlighting the therapeutic potential of
cannabinoid-related compounds and presenting some promising patents targeting potential treatment options for schizo-
phrenia.
Keywords: 9
-tetrahydrocannabinol, animal models, antipsychotics, cannabidiol, cannabis, CB receptors, endocannabinoid
system, schizophrenia.
1. INTRODUCTION
1.1. Current Pharmacological Approach for the Treatment
of Schizophrenia
Schizophrenia (SCZ) is a chronic mental disorder affecting
about 1 % of the population worldwide. It is characterized
by three broad clusters of symptoms which result in
enormous personal suffering, as well as social and economic
burden. These symptom domains include positive symptoms
such as delusions, hallucinations, disorganized speech and
behaviour; negative symptoms including anhedonia and social
withdrawal; and cognitive impairments in sensory information
processing, attention, working memory and executive
functions [1]. They occur in different combinations, differing
degrees of severity and in a changing pattern over
time in each patient. Thus, SCZ is regarded as a complex and
highly heterogeneous disorder. Hyperfunction of dopaminergic
(DAergic) system in the mesolimbic pathway was the
original tenet of the theory underlying the basis of SCZ
because antipsychotic drugs blocked dopamine D2
receptors (D2Rs) and amphetamine which indirectly
*Address correspondence to this author at the CEITEC (Central European
Institute of Technology) Masaryk University, Kamenice 5, 625 00 Brno,
Czech Republic; Tel: +420549494624; Fax: +420549492364; E-mail:
vincenzomicale@inwind.it
increases the release of dopamine (DA) exacerbated positive
symptoms and thus led to the dopamine hypothesis of
schizophrenia [2]. The treatment of SCZ was revolutionized
more than 50 years ago with the discovery - by serendipity
rather than design - that chlorpromazine and haloperidol
(called today typical neuroleptics or the first generation antipsychotics)
alleviate the psychotic manifestations such as
hallucinations and delusions by blocking the D2Rs. From the
1970’s the second generation or atypical antipsychotics (including
clozapine, olanzapine, risperidone and aripiprazole)
were developed. These drugs still act mainly by DA antagonism
in the central nervous system (CNS) but their effects
are mediated by serotonin receptor subtypes (5-HT2A/5HT2c),
D3R and/or D4R in addition to D2Rs. This class is
also known as Multi-acting Receptor Targeted Antipsychotics
(MARTA) and has less tendency to produce unwanted
extrapyramidal side effects and hyperprolactinemia [3]. Although
current pharmacological armamentarium is generally
effective treating positive symptoms, it is less effective in
treating the negative and cognitive symptoms. In addition, it
can induce several side effects resembling Parkinson’s disease
(known as extrapyramidal side effects) and metabolic
syndrome. Furthermore, a significant proportion of patients
are refractory to the available drugs. Thus, there is a need to
develop new approaches for treating SCZ and appropriate
animal models for preclinical testing [4, 5]. It is well accepted
that the pathophysiological mechanisms underlying
2212-3954/14 $100 00+ 00 © 2014 Bentham Science Publishers
246
14 Recent Patents on CNS Drug Discovery, 2014, Vol. 9, No. 1 Kucerova et al.
SCZ cannot be explained by simple changes in monoamine
signalling such as DA and 5-HT but involves more complex
alterations in brain circuits including glutamate, GABA and
acetylcholine [6]. Thus, all these neurotransmitters could
represent potential targets for pharmacological intervention
[4]. In accordance, a driven focus on rational discovery of
highly selective drugs with new mechanisms such as the
glutamatergic, cholinergic neurotransmission or neuropeptidergic
signalling affecting intracellular signal transduction
pathways appeared in the past decade. Unfortunately, none
of these drugs have reached the market yet [7]. Therefore,
the partial efficacy of current pharmacological armamentarium,
since approximately one third of psychotic patients
are non-responders raises the central question to be addressed
in this review: Should the pharmacological exploitation
of the endocannabinoid system (ECS) be a promising
therapeutic approach for treatment of the behavioural dimensions
which are dysregulated in SCZ?
1.2. Cannabis and Schizophrenia: Clinical Evidence
Cannabis (or marijuana) is the most frequently abused
illicit “recreational” substance in the Western society. Its
popularity is due to its capacity to alter sensory perception,
to induce euphoria and to increase sociability. Although the
association between Cannabis sativa and psychopathologic
conditions had been known for thousands of years, only in
the last 50 years the identification of the chemical structure
of marijuana components, cloning of specific cannabinoid
receptors and discovery of the ECS in the brain has triggered
an exponential growth of studies to explore its real effects on
mental health [8, 9]. The Cannabis plant contains over 100
terpenophenolic pharmacologically active compounds,
known as cannabinoids. Of these, 9
-tetrahydrocannabinol
(THC), characterized in 1964 [10], was identified as the
main psychoactive component of Cannabis and later shown
to act as a direct agonist on cannabinoid CB1 and CB2 receptors.
Other cannabinoids include cannabidiol (CBD),
cannabichromene and cannabigerol which do not induce any
THC-like psychoactivity. They act via several mechanisms,
including modulation of endocannabinoid system tone [11-
13], interaction with transient receptor potential vanilloid 1
(TRPV1) channels [11] and serotonin 5-HT1A receptors [14],
and enhancement of adenosine signalling [15, 16]. As recently
reviewed, the above mentioned mechanisms could
underlie the positive effects induced by CBD treatment in
preclinical and clinical studies of several disorders [17, 18].
In addition, accumulating evidence suggests that the recreational
use of Cannabis during adolescence increases the
relative risk for psychotic disorders. However, it is still unknown
whether Cannabis use is an independent risk factor
for SCZ or simply that the high prevalence of Cannabis use
in SCZ patients as an attempt of self-medication due to Cannabis’s
euphoric effects and increased sociability to relieve
negative symptoms [19, 20]. Furthermore its use may instead
contribute as an environmental risk factor in vulnerable individuals
with genetic mutation of COMT (Catechol-Omethyltransferase)
enzymes [21] given that the majority of
Cannabis users do not develop SCZ. Multiple lines of evidence
have shown that frequent Cannabis consumption
could down regulate anandamide (AEA) signalling in
schizophrenic but not in healthy individuals. Also, it is associated
to brain abnormalities in regions which are known to
be rich in CB1 receptors such as the anterior and posterior
cingulated cortex, as suggested by magnetic resonance imaging
studies [22-25]. Although the exact relationship between
Cannabis and SCZ is not fully elucidated, alterations of ECS
elements as receptors and their endogenous activators seem
to be involved in pathophysiology of SCZ. More specifically,
previous studies have reported an increase in CB1 receptor
binding in prefrontal area of brains from schizophrenic
patients [26-31]. However, other studies failed to
demonstrate any alteration [32] or reduction of CB1 density
on the neuronal surface [33] and CB1 mRNA expression
[34]. This contradiction might result from other neuroplastic
alterations which further complicate the situation as another
study detected lower CB1 receptor density but no differences
on the level of CB1 mRNA expression [35]. Although several
confounding factors such as Cannabis consumption,
treatment with antipsychotics or different biochemical techniques
used for the determination of CB1 receptors density
and proteosynthesis might explain the apparent opposite results;
in general, the presence of a dysfunction in CB1 receptors
in selected brain regions of patients is supported. Furthermore,
polymorphisms in the CB1 receptor gene CNR1,
which could be correlated with an increased probability to
develop psychosis, have also been described. Yet, the data
are still controversial [23, 36-39].
Recently, the potential involvement of CB2 receptors in
the pathogenesis of SCZ has been also supported by clinical
findings. Patients with first-episode psychosis have a decreased
expression of peripheral CB2 receptors in comparison
to healthy controls [40, 41], which is in accordance with
preclinical studies [42]. Thus, the altered expression of both
receptors in SCZ patients confirms that they possess a certain
homeostatic role.
Besides CB receptor dysfunctions, alteration in endocannabinoid
levels seems to be implicated in the pathophysiology
of SCZ as well. AEA levels have been found elevated in
cerebrospinal fluid which were negatively correlated with
psychotic symptoms and normalized by treatments with
typical antipsychotics [43, 44]. In contrast, Muguruza et al.
showed in cerebellum, hippocampus and prefrontal cortex
of schizophrenic subjects lower AEA and higher 2arachidonylglycerol
(2-AG) levels [45]. Considering the
glutamate hypothesis of SCZ and the role of 2-AG in the
modulation of glutamatergic neurotransmission, this could
represent an adaptive response to reduce glutamatergic hyperactivity
in schizophrenics. Yet, it must be taken in to account
that these alterations in opposite directions may be due
to the different regulation of 2-AG and AEA levels under
both physiological and pathological conditions [46]. Moreover,
the difference of endocannabinoid levels in cerebrospinal
fluid may be related to alterations in peripheral amounts
of endocannabinoids, so the neuronal origin of the AEA and
2-AG in the cerebrospinal fluid remain conjectural [45].
Evidence of potential endocannabinoid signalling dysregulation
in SCZ is also supported by the decreased expression of
endocannabinoid synthesizing enzymes NAPE (N-acylphosphatidylethanolamine
phospholipase) and DAGL (diacylglycerol
lipase) in the peripheral blood mononuclear cells
of patients with first episode of psychosis [40].
247
Endocannabinoid System and Schizophrenia Recent Patents on CNS Drug Discovery, 2014, Vol. 9, No. 1 15
Based on the evidence presented above, functional abnormalities
in the endocannabinoid system could be involved
in the pathophysiology of SCZ; thus there is increasing interest
to explore potential antipsychotic properties of compounds
modulating the endocannabinoid signalling.
2. THE ENDOCANNABINOID SYSTEM (ECS)
The endogenous cannabinoid system (ECS) is a neuromodulatory
system which is involved in a variety of physiological
processes both in the brain and in the periphery.
Within the CNS, it acts at the level of inhibitory and excitatory
synapses in brain regions involved in emotional or nonemotional
processes, and mediates the effects of THC, the
main psychoactive constituent of Cannabis [47]. Increasing
evidence suggest that altered EC signalling could play a role
in the pathophysiology of several diseases such as pain and
inflammation [48]; immunological disorders [49, 50]; neurodegenerative
[9] and stress-related conditions [51]; obesity,
metabolic [52, 53] and cardiovascular [54] diseases;
cancer [55], gastrointestinal [53, 56] and hepatic [57] disorders.
However, the exact pathophysiological mechanisms
through which the ECS plays are not clearly understood at
present.
The ECS consists of: (1) the cannabinoid receptors CB1
and CB2 [58-60], (2) the endogenous cannabinoid CB receptor
agonists, AEA and 2-AG [61, 62], (3) a specific and not
yet identified cellular uptake mechanism and [63, 64],
(4) the enzymes for endocannabinoid biosynthesis: N acylphosphatidylethanolamine-selective
phosphodiesterase or
glycerophosphodiesterase E1 and diacylglycerol lipase or
[65, 66]; or degradation: fatty acid amide hydrolase (FAAH)
and monoacylglycerol lipase (MAGL) [67, 68], respectively
for AEA and 2-AG. Despite it is well accepted that AEA is
an endogenous agonist for cannabinoid CB1 receptors in the
brain, some of the typical cannabimimetic effects of AEA
are still present in transgenic mice lacking CB1 receptors.
These effects may be due to AEA capability to act as a full
agonist for the TRPV1 channels [69] resulting in mechanisms
distinct from CB1 and CB2 receptors activation.
However, additional “players” which target TRPV1 and/or
CB1 receptors, including putative CB1 antagonist peptides
like hemopressins, peroxisome proliferator-activated receptor-
(PPAR- ) and (PPAR- ) ligands, such as oleoylethanolamide
(OEA) or palmitoylethanolamide (PEA), and Narachidonoyl-dopamine
(NADA) are described as potential
members of this signalling system. Although the existence of
a third cannabinoid receptor subtype has been also suggested
[70], to date only CB1 and CB2 receptors are recognized as
G protein coupled receptors for endocannabinoids [71].
The cannabinoid CB1 and CB2 receptors which are encoded
by two different genes on human chromosomes: 6q14q15
(CNR1) and 1p36.11 (CNR2), are 7 transmembrane Gi/o
coupled receptors that share 44 % protein identity but they
display different pharmacological profiles and patterns of
expression, a dichotomy that provides a unique opportunity
to develop pharmaceutical approaches. The cannabinoid CB1
receptors are highly expressed in the basal ganglia, frontal
cortex, hippocampus and cerebellum. They are expressed
with a moderate/low density in the amygdala, nucleus accumbens,
medulla, periaqueductal gray and thalamus [72];
as well as they are also described in non-neuronal cells of the
brain such as microglia, oligodendrocytes and astrocytes
[73]. Within these cortical areas, they are expressed at the
GABAergic interneurons and glutamatergic neurons, which
are the two major neuronal subpopulations expressing the
CB1 receptors [74]. These neurotransmitter systems represent
the two major opposing players regulating the excitation
state of the brain; GABAergic interneurons being inhibitory
and glutamatergic neurons being excitatory. Recent studies
have demonstrated that CB1 receptors are also located in
neurons of the dorsal raphe nucleus and in the nucleus coeruleus
which are the major source of serotonin and noradrenalin
in the brain [75, 76]. Thus, the direct or indirect modulation
by monoamine activity on GABA and glutamate neurons
could underlie the psychotropic and non-psychotropic
effects of CB1 activation, respectively.
The cannabinoid CB2 receptors, also activated by AEA
and 2-AG, are mostly peripherally located on immunological
tissues. CB2 receptors are also detected in glia cells and in
neurons of several brain regions such as cerebral cortex,
amygdala, hippocampus, hypothalamus and cerebellum but
in a much lesser extent [77, 78]. They play an important part
in regulation of pain and inflammation even though recent
data also suggest their involvement in emotional and nonemotional
processes [79]. The observation that the elements
of such neuromodulator system are prevalent throughout the
neuroanatomical structures and circuits implicated in emotionality
provides a rationale for the preclinical development
of agents targeting the ECS to treat multiple psychiatric disorders
including SCZ.
3. EFFECTS OF PHARMACOLOGICAL MANIPULATION
OF THE ENDOCANNABINOID SIGNALLING
IN PRECLINICAL AND CLINICAL STUDIES OF
SCHIZOPHRENIA
Schizophrenia is a unique human disorder characterized
by specific clinical manifestations such as delusions, thought
disorders and hallucinations, which was described in 1896
by Kraepelin as dementia praecox. Due to the nature of the
disease it is impossible to develop an animal model which
would fully mimic its symptoms [1]. Thus, a greater understanding
of the disorder might arise from modelling specific
signs and symptoms, rather than mimicking the entire syndrome.
In accordance with this strategy, the most reliable
behavioural indices of positive symptoms in experimental
models are hyperlocomotor activity and behavioural stereotypes
which mimic the psychomotor agitation and presence
of stereotyped behaviours in acutely psychotic patients since
positive symptoms such as hallucinations and delusions cannot
be measured in animals [80]. These are based on the rationale
that the hyperfunctioning of the mesolimbic DAergic
system, which seems to underlie the enhanced locomotor
activity and stereotyped behaviour, is consistent with the
human conditions where an enhanced subcortical DAergic
activity plays a pivotal role to precipitate positive symptoms
[81]. However, some behavioural aspects of SCZ seem to be
modelled and objectively assessed in rodents. More specifically,
hallmarks of negative symptoms, deficits in social behaviour
and anhedonia, can be assessed both in humans and
rodents with the pre-pulse inhibition (PPI) as an index of
disrupted sensory gating abilities both in schizophrenic pa-
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16 Recent Patents on CNS Drug Discovery, 2014, Vol. 9, No. 1 Kucerova et al.
tients and in experimental animal models [82]. Interestingly,
the various cognitive deficits in SCZ, as identified by the
NIH (National Institute of Health) Measurement and Treatment
Research to Improve Cognition in Schizophrenia
(MATRICS) initiative, could be experimentally assessed by
the use of specific rodent behavioural tasks [83]. As recently
estimated, more than 20 different rodent models of SCZ have
been developed, which fit into four different categories depending
on the type of manipulation, namely 1) pharmacological,
2) genetic, 3) lesion based and 4) neurodevelopmental
models. First experimental models were developed on the
basis of the theory that SCZ is a disorder related to an excessive
DAergic activity; accordingly, the DAergic agents such
as amphetamine and apomorphine attempt to mimic this feature.
However, due to the increased understanding of the
genetic basis and potential involvement of glutamate and
adverse environmental insults, different experimental manipulations
for animal models of SCZ have also been developed
[84].
Since the identification of cannabinoid CB1 and CB2
receptors and their endogenous ligands (AEA and 2-AG), a
key aspect to assess the function and therapeutic potential of
the ECS for SCZ treatment has been the availability of selective
pharmacological tools. They vary from directly acting
compounds, such as agonists and inverse agonists, to agents
that enhance indirectly endocannabinoid signalling by affecting
the cellular reuptake of endocannabinoids (experimental
agents: AM404 or VDM11) or by inhibiting the hydrolytic
enzymes FAAH and MAGL (experimental agents: URB597,
AA-5-HT or JZL184). However, several additional elements
which can be described as potential members of the
ECS such as ligands (i.e. noladin, virodhamide), receptors
(GPR55, PPAR , TRPV1) and synthetic or degradative
pathways, could participate in the mechanism of action of
the compounds described above [85].
3.1. Studies on Positive- and Negative-like Symptoms
Different substances modulating the endocannabinoid
signalling have been evaluated in several animal (mostly
pharmacological) models for affecting positive- and/or negative-like
symptoms of SCZ, as summarized in Table 1. In a
recent study, Spano et al. have shown that chronic exposure
to the CB1 agonist WIN55,212-2 reduces phencyclidine
(PCP)-induced hyperlocomotion [86], in agreement with
previous studies, showing a reduction of cocaine- or quinpirole-
induced hyperactive behaviour by direct CB1 activation
[87, 88]. Interestingly, stereotyped and hyperlocomotor behaviours,
an index of positive-like symptoms, were also reduced
by the non-psychoactive component of Cannabis sativa
cannabidiol (CBD) [89-92]. Although in a recent study
CBD failed to reverse the amphetamine-induced hyperactivity,
it elicited certain neuroprotective effects [93]. In additional,
CBD prevented human experimental psychosis. More
specifically, it was effective in open case reports and clinical
trials in psychotics with a remarkable safety profile, [94-97]
as well as it and others phytocannabinoids such as cannabidiolic
acid, tetrahydrocannabivarin, tetrahydrocannabivarin
acid, cannabichromene, cannabichromenic acid, cannabigerol,
and cannabigerolic acid were patented for their
use in combination with one or more anti-psychotic medicaments
to prevent or treat psychosis and psychotic disorders
[98]. Yet, it is still unknown the exact mechanism(s) of action
underlying its antipsychotic effects, but it is clear that
CBD does not only act through ECS (as weak partial antagonist
at CB1/CB2 receptors or inhibitor of AEA hydrolysis
and reuptake), but also activates serotonin 5-HT1A or adenosine
receptors or targets nuclear receptors of the PPAR family
as well as modulates ion channels including TRPV1 [18].
Regardless the exact mechanism of action, attention has been
focused on the potential therapeutic use of CBD in further
mental diseases such as mood (i.e. anxiety and depression)
and neurodegenerative (Alzheimer’s or Parkinson’s disease)
disorders [17].
In the last years, selective antagonist/inverse agonists of
CB1 receptors were some of the most promising molecules
in pharmacological research for the treatment of obesity and
addictive disorders. The first such compound was rimonabant
(SR141716) [99] introduced into clinical practice as
antiobesity agent in several countries. However, due to the
higher incidence of psychiatric side effects such as anxiety,
depression and suicidal tendencies, rimonabant was very
soon withdrawn from the market [100]. In contrast to CBD,
the ability of CB1 antagonists on positive-like symptoms is
still under debate due to the contradictory results. In 1999,
Poncelet et al. reported that rimonabant as well as clozapine
or haloperidol antagonized the hyperlocomotor activity induced
by d-amphetamine, cocaine and morphine in gerbils
[101]. Potential therapeutic effects on positive symptoms
were then also confirmed by Tzavara and colleagues in the
PCP animal model [102]. However, in other studies, it failed
to ameliorate the hyperlocomotor activity [103-105] or instead
increased stereotype behaviour [106]. Although the
discrepancies among these studies could be due to interspecies
differences, or physiochemical differences between
drugs or experimental models, the preclinical data described
above suggest that the CB1 blockade might have a limited
potential to treat positive symptoms. In line with this concept,
AVE1625 (drinabant, so far reported as the CB1 antagonist)
has partially reversed the positive-like symptoms in
experimental models with an improved side effects profile
[107].
Recently, attention has been drawn to the expression of
CB2 receptor in the CNS [77, 78]. Further supporting that
cannabinoid CB2 receptors may play a role in psychiatric
disorder, it has been seen that pharmacological or genetic
CB2 receptor blockade increased susceptibility to develop
positive-like symptoms [41]. As a result, the CB2 agonist
beta-caryophyllene has been recently patented for potential
efficacy for SCZ treatment [108]. The ECS seems to play a
role in the social behaviour of rodents and the resistance of
negative symptoms to pharmacological interventions; therefore,
the effects of pharmacological modulation of endocannabinoid
signalling on the social deficits of experimental
models of schizophrenia have been recently examined. Direct
activation of CB1 receptors through the use of CB1 agonists
WIN55,212-2 or CP55,940 reversed the PCP-induced
social deficits [86, 109]. Interestingly, the pharmacological
enhancement of endocannabinoid levels via systemic treatment
with the FAAH inhibitor URB597 also reversed the
social deficits in the PCP model, but at the same time elicited,
as well as the cannabinoid CB1 blockade, harmful effects
in the social behaviour of control animals, maybe by
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Endocannabinoid System and Schizophrenia Recent Patents on CNS Drug Discovery, 2014, Vol. 9, No. 1 17
Table 1. Effects of pharmacological modulation of the endocannabinoid system on schizophrenia-like symptoms.
a) Positive-like symptoms
Mechanism Drug: Effective Dose
(Range tested)
Animals Models Behavioral Response Positive Control Ref.
WIN: 6 (3-6) mg/kg, i.p. Wistar rats cocaine (10 mg/kg, i.p.) hyperlocomotion not determined [88]
CP: (0.0025-
0.01 mg/kg, s.c.)
Cebus
monkeys
d-amphetamine (0.25 mg/kg, s.c.) no effect on arousal
and stereotypy
not determined [103]
CP: 0.1/0.25 (0.01-
0.25) mg/kg, i.p.
Wistar rats quinpirole (0.5 mg/kg, i.p.) hyperlocomotion not determined [87]
CB1/CB2
receptor
agonists:
WIN: 0.3 mg/kg/day,
i.v. for 14 days
Lister
Hooded rats
PCP (2.5 mg/kg, i.p.) hyperlocomotion not determined [86]
RIM: 1/3 (0.3-3) mg/kg,
i.p.
Gerbils cocaine (5-15 mg/kg, i.p.)
d-amphetamine (2.5 mg/kg, i.p.)
morphine (4 mg/kg, i.p.)
WIN-55,212-2 (1 mg/kg, i.p.)
hyperlocomotion in
habituated gerbils
clozapine
(3 mg/kg, i.p.)
haloperidol
(0.1 mg/kg, i.p.)
[101]
RIM: 3-10 mg/kg, i.p. Bl6 mice PCP (4 mg/kg, i.p.)
d-amphetamine (2.5 mg/kg, i.p.)
hyperlocomotion not determined [102]
RIM: (0.0005-5) mg/kg,
i.p.
Wistar rats d-amphetamine (3 mg/kg, i.p.) no effect on hyperlocomotion
and stereo-
typy
haloperidol
(0.25 mg/kg, i.p.)
[104]
RIM: 0.1-0.5 (0.1-
0.75) mg/kg, s.c.
Cebus
monkeys
d-amphetamine (0.25 mg/kg, s.c.) arousal
no effect on stereotypy
not determined [103]
RIM: 1 (0.1-1) mg/kg,
i.p.
Wistar rats SKF38393 (0.05-1 mg/kg, s.c.)
quinpirole (0.25 mg/kg, s.c.)
stereotypy not determined [106]
RIM: 1 mg/kg, i.p. SpragueDawley
rats
amphetamine (1 mg/kg, i.p.) no effect on
hyperlocomotion
not determined [105]
CB1 an-
tagonists:
AVE: 1- 3-10 mg/kg,
i.p.
Wistar rats MK-801 (0.05 mg/kg, i.p.) disrupted LI risperidone
(0.01-1 mg/kg, i.p.)
[107]
CB2
antagonist:
AM630: 3-30 mg/kg, i.p. Bl6/JJ mice MK-801 (0.5 mg/kg, i.p.)
methamphetamine (2 mg/kg, i.p.)
hyperlocomotion not determined [41]
AEA reuptake
inhibi-
tor:
AM404: 10 μg/rat, i.c.v. Wistar rats quinpirole (0.25 mg/kg, i.p.) hyperlocomotion not determined [156]
CBD: 30/60 (15-
60) mg/kg, i.p.
Swiss mice d-amphetamine (5 mg/kg, i.p.)
ketamine (60 mg/kg, i.p.)
hyperlocomotion haloperidol
(0.15-0.6 mg/kg,
i.p.)
clozapine
(1.25-5 mg/kg, s.c.)
[91]
CBD: 20 (5-20) mg/kg,
i.p.
SpragueDawley
rats
THC (1 mg/kg, i.p.) hyperlocomotion not determined [90]
CBD: 50 (1-
50) mg/kg/day, i.p. for 3
weeks
Bl6/Jarc
mice
d-amphetamine (5 mg/kg, i.p.) hyperlocomotion not determined [89]
Non-
psychotropic
cannabinoid:
CBD: (15-60) mg/kg,
i.p. for 7 days
Wistar rats d-amphetamine (2 mg/kg, i.p.) no effect on
hyperlocomotion
not determined [93]
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18 Recent Patents on CNS Drug Discovery, 2014, Vol. 9, No. 1 Kucerova et al.
(Table 1) contd…..
b) Negative-like Symptoms
Mechanism Drug: Effective Dose
(Range tested)
Animals Models Behavioral Response Positive Control Ref.
WIN: 0.3 mg/kg/day,
i.v. for 14 days
Lister
Hooded rats
intermittent PCP
(2.5 mg/kg/day, i.p.)
social deficit in PCP
social deficit in con-
trol
not determined [145]
CP: 0.01 mg/kg, i.p. Wistar rats PCP (5 mg/kg/day, i.p.) twice a
day for 7 days
social deficit in PCP not determined [109]
CB1/CB2
agonists:
THC: 2.5 mg/kg/day,
i.p. (PND37-39);
5 mg/kg/day, i.p.
(PND40-43);
10 mg/kg/day, i.p.
(PND44-47)
SpragueDawley
rats
maternal deprivation aggressive behavior
of female in the SI
no effect in the FST
not determined [157]
AM251: 0.5 mg/kg/day,
i.p. for 3 weeks
Lister
Hooded rats
PCP (2.58 mg/kg/day, i.p.) for 4
weeks
immobility time in
the FST
clozapine
(5 mg/kg/day, i.p.)
for 3 weeks
[115]
AM251: 0.5 mg/kg/day,
i.p. for 3 weeks
Lister
Hooded rats
social isolation aggressive behavior
in the SI
not determined [129]
CB1 antago-
nists/invesre
agonists:
AM251: 3 (0.3-
3) mg/kg, i.p.
RIM: 0.3/1 (0.1-
1) mg/kg, i.p.
Wistar rats PCP (5 mg/kg/day, i.p.) twice a
day for 7 days
social withdrawal in
control rats
not determined [109]
TRPV1 an-
tagonist:
capsazepine: 1 (1-
10) mg/kg, i.p.
Wistar rats PCP (5 mg/kg/day, i.p.) twice a
day for 7 days
social withdrawal in
control rats
not determined [109]
FAAH in-
hibitor:
URB597:
0.1/0.3/1 mg/kg, i.p.
Wistar rats PCP (5 mg/kg/day, i.p.) twice a
day for 7 days
social withdrawal in
PCP rats
social withdrawal in
control rats
not determined [109]
Table 1. The table summarizes the effects of direct pharmacological manipulation of the endocannabinoid signalling on positive and negative-like symptoms in rodent models of
schizophrenia. Acronyms: THC: 9
-tetrahydrocannabinol, AEA: anandamide, CBD: cannabidiol, CP: CP55940, FAAH: fatty acid amide hydrolase, FST: forced swim test, i.c.v.:
intracerebroventricular, i.p.: intraperitoneal, i.v.: intravenous, LI: latent inhibition, PCP: phencyclidine, PND: postnatal day, RIM: rimonabant (SR141716), s.c.: subcutaneous, SI:
social interaction, TRPV1: transient receptor potential vanilloid 1 channels, WIN: WIN55,212-2.
disturbing the ECS tone through the activation of TRPV1
channels [109, 110]. In accordance, it has been seen that
chronic Cannabis consumption improves negative symptoms
in schizophrenic subjects [111, 112], as well as it also induces
an amotivational syndrome, which mimics negative
symptoms in non schizophrenics [113]. This suggests different
effects of cannabinoids on healthy or schizophrenic subjects.
Chronic treatment with the CB1 receptor antagonist
AM251 counteracted the aggressive behaviour and reversed
the PCP-induced immobility in the forced swim test which
was accompanied by the rescue of CB1 receptor functionality
in a neurodevelopmental animal model based on a social
isolation procedure [114, 115]. Although the genetic CB1
disruption in mice was also able to counteract the PCPinduced
social deficit [116] further supporting the potential
antipsychotic properties of the CB1 blockade, human experimental
studies have so far shown controversial results.
More specifically, Meltzer et al. have not seen a clinical improvement
in schizophrenic patients after rimonabant treatment.
In contrast, Kelly et al. found a significant reduction
of psychotic symptomatology in obese patients with SCZ
[117, 118]. Thus, further clinical studies are necessary to
elucidate the therapeutic potential of CB1 antagonists. To
date, several compounds of this pharmacodynamic profile
have been patented for potential efficacy for treating SCZ
symptoms [119-123].
3.2. Studies on Cognitive/Attention Deficits
It has become clear that SCZ cannot be reduced to its
psychotic symptoms and the cognitive deficits of these patients
are the most debilitating and remain resistant to
treatment. Thus, the development of new drugs has been
hampered by the lack of existing drugs for treating the cognitive
impairment in schizophrenic patients, since there is
not gold standard positive control drug that can be used in
cognitive assays. Thus, in light of the high density of cannabinoid
CB1 receptors in cortical regions involved in cognition
and memory processes, the cognitive effects of the
modulation of the endocannabinoid signalling could be one
of the potential pharmacological targets for the SCZ treatment.
The existing evidence of involvement of ESC in the
cognitive/attention processes in animal models of SCZ is
presented in the Table 2.
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Endocannabinoid System and Schizophrenia Recent Patents on CNS Drug Discovery, 2014, Vol. 9, No. 1 19
Table 2. Effects of pharmacological modulation of the endocannabinoid system on cognitive/attention deficits of a schizophrenialike
phenotype.
Mechanism Drug: Effective
Dose (Range tested)
Animals Models Behavioral Response Positive Control Ref.
THC: 0.5 mg/kg/day,
i.p. for 3 weeks
Lister
Hooded rats
PCP (2.58 mg/kg/day, i.p.) for
4 weeks
cognitive deficit clozapine
(5 mg/kg/day, i.p.) for
3 weeks
[126]
THC: 0.3/1/3 mg/kg,
i.v.
SpragueDawley
rats
social isolation disruption of PPI not determined [144]
WIN: 3 mg/kg, i.p. Bl6/J psychosocial stress disruption of PPI not determined [143]
THC: 2.5 mg/kg/day,
i.p. (PND35-37);
5 mg/kg/day, i.p.
(PND38-41);
10 mg/kg/day, i.p.
(PND42-45)
SpragueDawley
rats
maternal deprivation cognitive deficit in control
female
not determined [157]
CB1/CB2
receptor
agonists:
WIN: 0.3 mg/kg/day,
i.v. for 14 days
Lister
Hooded rats
chronic PCP
(2.5 mg/kg/day, i.p.)
disruption of PPI
cognitive deficit
not determined [145]
RIM: (0.3-5) mg/kg,
i.p.
SpragueDawley
rats
apomorphine (0.5 mg/kg, s.c.)
MK-801 (0.1 mg/kg, s.c.)
d-amphetamine (5 mg/kg, s.c.)
no effect on PPI clozapine (10 mg/kg,
i.p.); olanzapine
(10 mg/kg, i.p.);
haloperidol
(0.3 mg/kg,1 i.p.)
[104]
RIM: 3 (0.3-
3) mg/kg, i.p.
Swiss mice apomorphine (3 mg/kg, i.p.) disruption of PPI not determined [150]
AM251:
0.5 mg/kg/day, i.p.
for 3 weeks
Lister
Hooded rats
PCP (2.58 mg/kg/day, i.p.) for
4 weeks
cognitive deficit clozapine
(5 mg/kg/day, i.p.) for
3 weeks
[130]
AM251:
0.5 mg/kg/day, i.p.
for 3 weeks
Lister
Hooded rats
social isolation cognitive deficit not determined [129]
AM251:
0.5 mg/kg/day, i.p.
for 3 weeks
Lister
Hooded rats
social isolation disruption of PPI not determined [114]
AM251: 1 mg/kg,
i.p.
Wistar rats PCP (5 mg/kg/day, i.p.) twice
a day for 7 days
cognitive deficit in PCP
cognitive deficit in
control
not determined [110]
CB1 antago-
nists/inverse
agonists:
RIM:
0.75/1/3 mg/kg, s.c.
AM251:
1.4/1.8 mg/kg, s.c.
SpragueDawley
rats
PCP (1.25 mg/kg, s.c.) disruption of PPI clozapine (7.5 mg/kg,
i.p.)
[149]
CB2 antago-
nist:
AM630: 30 (3-
30) mg/kg, i.p.
Bl6/6JJmsSlc
mice
MK-801 (0.5 mg/kg, i.p.)
methamphetamine (2 mg/kg,
i.p.)
disruption of PPI in
MK-801 mice
no effect on PPI in methamphetamine
pretreated
mice
not determined [41]
FAAH Inhibi-
tor:
URB597: 0.3 mg/kg,
i.p.
Wistar rats PCP (5 mg/kg/day, i.p.) twice a
day for 7 days
cognitive deficit not determined [110]
CBD: 0.5 mg/kg, i.m. Rhesus mon-
keys
THC (0.2-0.5 mg/kg, i.m.) cognitive deficit not determined [138]Non-
psychotropic
cannabinoid:
CBD: 5 (1-15) mg/kg, i.p Swiss mice MK-801 (1 mg/kg, i.p.) disruption of PPI clozapine (4 mg/kg, i.p.) [154]
Table 2. The table summarizes the effects of direct pharmacological manipulation of the endocannabinoid signalling on cognitive/attention deficits in rodent models of schizophrenia.
Acronyms: THC: 9
-tetrahydrocannabinol, CBD: cannabidiol, FAAH: fatty acid amide hydrolase, FST: forced swim test, i.c.v.: intracerebroventricular, i.m.: intramuscular, i.p.:
intraperitoneal, i.v.: intravenous, LI: latent inhibition, PCP: phencyclidine, PND: postnatal day, PPI: prepulse inhibition, RIM: rimonabant (SR141716), s.c.: subcutaneous, SI: social
interaction, WIN: WIN55,212-2.
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20 Recent Patents on CNS Drug Discovery, 2014, Vol. 9, No. 1 Kucerova et al.
Acute administration of the main pharmacologically active
principle of the Cannabis sativa, THC, as well as the
CB1 agonists such as WIN55,212-2, CP55,940 or AEA induce
in animals and healthy humans memory deficits similar
to those seen in SCZ, which could be mediated through a
disruption of prefrontal and hippocampal functions [124,
125]. However, in the PCP-induced animal model controversial
data have been obtained following CB1 activation.
While Vigano and colleagues found that chronic THC treatment
in juvenile rats worsened cognitive impairment [126],
by contrast the CB1 agonist WIN55, 212-2 attenuated the
PCP cognitive deficits in adult rats [86]. Although the
authors used the same experimental model, the discrepancies
between these studies could be due to either physiochemical
differences between CB1 agonists or the different age of
pharmacological treatment (juvenile vs. adult). Furthermore,
Seillier and colleagues found that indirect activation of CB1
receptors through the use of FAAH inhibitor URB597
caused working memory deficits in saline treated rats comparable
to those after PCP treatment, which may arise due to
perturbing the endocannabinoid tone [110]. In contrast, other
evidence from animal studies suggests that pharmacological
CB1 receptor blockade could exert promnesic effects. In this
context, it has been seen that the memory disruptive
effects induced by CB1 agonists such as THC, AEA or
WIN55,212-2 were counteracted by rimonabant treatment
[124, 127, 128]. Furthermore, the CB1 receptor antagonist/inverse
agonist AM251 reversed memory impairment in
pharmacological and neurodevelopmental models of SCZ
[110, 114, 129, 130]. Despite potential pro-cognitive effects
of CB1 antagonists described above, in the few clinical studies
assessing its role on cognitive functioning in human, rimonabant
worsened ketamine induced deficits [131] or did
not improve global cognitive functioning. In this later study
just a specific learning deficit in schizophrenic patients based
on response to positive feedback was recorded [132]. In a
recent clinical trial assessing the potent and selective CB1
antagonist AVE1625 for improving cognitive deficits in
schizophrenic, there was an insufficient efficacy of the
treatment (Clinical Trials.gov identifier: NTC00439634).
The withdrawal of rimonabant, due to the psychiatric side
effects in the metabolic syndrome treatment, interrupted the
entire industrial development of CB1 antagonists/inverse
agonists. However, several CB1 receptor inverse agonist
compounds have been patented for the treatment of cognitive
impairment associated with SCZ [133, 134]. Thus, a possible
solution for the safe use of this class of compounds could be
to determinate which patients are at high risk of psychiatric
side effects through detailed phenotypic assessment and genetic
testing [135] or change to the use of neutral CB1 antagonist
[136, 137].
Clinical and preclinical data suggest that CBD which is
the most extensively investigated phytocannabinoid for potential
use in psychiatric disorders, is also able to ameliorate
cognitive deficits. More specifically, it was able to reverse
the THC-induced deficits in rhesus monkeys [138], as well
as THC induced cognitive impairment in human [139].
Moreover, CBD effects on cognitive function in schizophrenic
patients are currently under investigation in a
phase II clinical trial (Clinical Trials.gov identifier:
NCT00588731). Patients with SCZ exhibit deficits in an
operational measure of sensorimotor gating: pre-pulse inhibition
(PPI) of startle reaction. Similar deficits in PPI are produced
in animals by pharmacological or developmental manipulations.
These experimentally induced PPI deficits in
rats clearly do not represent animal models of schizophrenia
per se, but provide us an investigative tool with high face,
predictive, and construct validity for sensorimotor gating
deficits in SCZ patients [140]. To confirm that younger animals
have different vulnerability to cannabinoid treatment in
development of SCZ-like symptoms, rats treated at adulthood
with CB1 receptor agonist WIN55,212-2 have not
shown disruption of PPI [141]; in comparison, the prepubertal
CB1 agonist treatment induced PPI deficits in adult
age [142]. However, at adulthood, WIN55,212-2 and THC
improved and impaired the PPI of the startle response in
psychosocially stressed rodents, respectively [143, 144].
Discrepancies between these studies could be due to interspecies
(rat vs. mice) dissimilarities in response to treatments
(e.g. pharmacokinetic issues), to physiochemical characteristics
of the specific compounds (THC vs. WIN) and to the
different experimental procedures. Nevertheless, in an experimental
model of SCZ, CB1 agonist WIN 55,212-2 was
able to attenuate the PCP-induced deficit in PPI [145]. Controversial
data were obtained following enhancement of
AEA level through the use of AM404, an AEA reuptake
inhibitor. While in mice AM404 disrupts sensorimotor gating
[146]; in contrast it was ineffective in the PPI test in rats [147],
suggesting an interspecies (rats vs. mice) difference in the
response to the pharmacological modulation of AEA levels.
The potential antipsychotic properties of CB1 antagonists
have also been explored in the impaired sensorimotor gating,
as a model of perceptual distortion [148]. It has been seen
that the antagonists/inverse agonists rimonabant and AM251
reversed the disrupted PPI in several experimental models of
SCZ, similarly as the conventional neuroleptics [114, 149,
150]. On the other hand, the genetic blockade of CB1 signalling
resulted in unaltered PPI response, as shown by the phenotype
of mice with a complete deletion of CB1 receptors;
however, they have shown a decreased parvalbumin
immunoreactivity in the cortex and striatum, which is typical
in schizophrenic human subjects [151, 152]. Again it is still
unknown the exact mechanisms underling these discrepancies,
but compensatory mechanisms in knock out mice could
be involved. Given recent attention has been drawn to the
role of CB2 receptors in psychiatric disorders, preclinical
and clinical data indicate that a reduced CB2 signalling elicited
a sensorimotor gating and an increased risk of SCZ in
human, respectively [41, 42]. The potential antipsychoticlike
property of CBD have also been supported by its ability
to reverse the sensorimotor gating deficits in different experimental
models, similarly to that induced by clozapine
[153, 154].
4. CURRENT & FUTURE DEVELOPMENTS
As outlined above, preclinical and clinical evidence
strongly suggest a dysregulation of the ECS in schizophrenia,
such as abnormalities in cannabinoid (CB1 and/or CB2)
receptor function and endocannabinoid (AEA and/or 2-AG)
levels in different cerebral areas. However, so far, the full
picture on the role of the endocannabinoid system in this
253
Endocannabinoid System and Schizophrenia Recent Patents on CNS Drug Discovery, 2014, Vol. 9, No. 1 21
pathology has yet to emerge. To date, the pharmacotherapy
of negative symptoms and cognitive deficits of SCZ has
been disappointing; as antipsychotics have not met the expectations
and the development of more effective therapies
have been inadequate [155]. Thus, the ability of cannabinoids
to modulate schizophrenia-like symptoms is extremely
attractive for the development of novel antipsychotics
agents. Although use of CB1 antagonists/inverse agonists is
hampered by unwanted psychiatric side effects and that the
possibly safer direct modulation of CB2 receptors still lacks
sufficient experimental evidence to justify its use, the use of
CBD has produced very promising results in animal models
with a pharmacological profile resembling that of atypical
antipsychotics. Clinical evidence also suggests that CBD,
being devoid of psychotropic activity, could represent a reliable
compound for psychosis in schizophrenia especially in
view of its lack of extrapyramidal side effects [96]. Further
clinical studies will determine if CBD treatment would be
the novel pharmacotherapy for the disturbances in the social
and cognitive functions in schizophrenic patients.
AUTHOR CONTRIBUTIONS
Jana Kucerova has collected the literature sources, was
involved in the discussion of the manuscript structure and
wrote a substantial part of the text.
Katarina Tabiova was responsible for cross-checking the
literature, preparation of the tables and reference collection.
Filippo Drago was involved in the discussion of the
structure and revised both the draft and final version of the
manuscript.
Vincenzo Micale has organized the structure of the whole
text and wrote a substantial part of the manuscript.
CONFLICT OF INTEREST
The authors confirm that this article content has no conflict
of interest.
ACKNOWLEDGEMENTS
This work was supported by the project financed from
the SoMoPro II programme (to Vincenzo Micale). The research
leading to this result has acquired a financial grant
from the People Programme (Marie Curie action) of the Seventh
Framework Programme of EU according to the REA
Grant Agreement No. 291782. The research is further cofinanced
by the South-Moravian Region; and by the project
“CEITEC - Central European Institute of Technology”
(CZ.1.05/1.1.00/02.0068) from European Regional Development
Fund. The authors are grateful to Tony Fong (Toronto,
Canada) for his kind help with manuscript preparation
and proof reading.
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Act Nerv Super Rediviva 2010; 52(4): 263–267
O R I G I N A L A R T I C L E
Activitas Nervosa Superior Rediviva Volume 52 No. 4 2010
Aripiprazole does not influence methamphetamine
I.V. self-administration in rats
Jana Kucerova1,2, Jana Pistovcakova1,2, Dagmar Vrskova1,2, Alexandra Sulcova1,2
1Masaryk University, Brno – Central European Institute of Technology (CEITEC); 2Faculty of Medicine,
Department of Pharmacology, Brno, Kamenice 5, 625 00 Brno, Czech Republic.
Correspondence to: Jana Kucerova, Department of Pharmacology, Faculty of Medicine, Masaryk University
Brno, Kamenice 5, 625 00 Brno, Czech Republic. phone: +420 549 494 238; fax: +420 549 492 364.
e-mail: jkucer@med.muni.cz
Submitted: 2010-11-12 Accepted: 2010-12-01 Published online: 2011-01-25
Key words: methamphetamine; aripiprazole; IV Self-administration; behavioural sensitization;
Wistar rats
Act Nerv Super Rediviva 2010; 52(4): 263–267 ANSR520410A06 ©2010 Act Nerv Super Rediviva
Abstract OBJECTIVE: Effects of an atypical antipsychotic and antidepressant aripiprazole on methamphetamine
(MET) I.V. self-administration (IVSA) under conditions of behavioural
sensitization to MET in rats was investigated in the present study.
METHODS: Adult male Wistar rats were randomly divided into 2 groups: the first group
received MET (0.05 mg/kg, intraperitoneally for 14 days); the other received vehicle as
a control. During following 14 days of wash-out period the surgery required for IVSA
including recovery was realized. The IVSA of MET paradigm was performed in operant
chambers under fixed ratio (FR) schedule of reinforcement. When a defined stable intake
of MET at FR3 was reached water was given to the animals orally until they achieved stable
intake again. Since then aripiprazole (3 mg/kg/day) was administered orally by tube 30
minutes before each self-administration session. For statistic analysis the nonparametric
ANOVA Kruskal-Wallis test with Dunn‘s Multiple Comparisons post test was applied.
RESULTS: A significant decrease of the drug intake was recorded in animals sensitized by
MET (MET pretreated) comparing to non-sensitized corresponding group. Aripiprazole
did not significantly affect the baseline of MET intake.
CONCLUSION: The behavioural sensitization to MET induced by 14 day intraperitoneal
administration of MET decreased the number of the drug doses self-administered per
session. This finding might result from increasing efficacy of the drug after sensitization
caused by repeated MET intermittent administration. However, aripiprazole did not influence
MET intake neither in the behaviourally MET sensitized nor in the non-sensitized
rats.
Abbreviations:
MET – methamphetamine; IVSA – IV self-administration
Introduction
The atypical antipsychotic aripiprazole acts as a partial
agonist on dopamine (D2 and D3) receptors. At the
same time aripiprazole influences serotonin receptors
(agonism at 5-HT1A and antagonism at 5-HT2A
receptors). So far it has been approved for therapy of
schizophrenia (so called 3rd generation antipsychotic)
(Mailman & Murthy 2010) and manic episodes in
bipolar disorder (De Fazio et al 2010). Moreover, it has
also been studied and recommended for treatment of
resistant depressions (Pae et al 2011; Pistovcakova &
Sulcova 2008) and anxiety disorders (Pae et al 2008;
Patkar et al 2006). As a partial D2/D3 agonist aripiprazole
is believed to be able to normalize dopaminergic
tone by acting similarly as antagonists during high
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Jana Kucerova, Jana Pistovcakova, Dagmar Vrskova, Alexandra Sulcova
dopaminergic firing and as agonists in dopaminergic
deficit during withdrawal (Rung et al 2008). Due to its
mechanism of action aripiprazole seems to be a promising
drug in addiction treatment (Backstrom et al 2011)
as the partial agonism is considered to be useful in
treatment of dependence. There is a number of other
partial agonists e.g. selective partial nicotine receptor
agonist varenicline in nicotine addiction (Vasic et al
2011; Walter & Wiesbeck 2009) or partial opioid agonist
buprenorphine in substitution therapy of opioid
addiction (Frei 2010).
Behavioural sensitization to drugs of abuse and the
related adaptations in striatal neurotransmission are
thought to play an important role in certain aspects
of addiction such as tendency to relaps to drug use
in abstaining individuals (Ohmori et al 2000). The
aim of this study was to utilize the intravenous drug
self-administration (IVSA) model, known as a reliable
model for testing dependence potential and abuse
liability of drugs (Collins et al 1984), for quantitative
comparison of the methamphetamine (MET) intake
of rat males subjected to a repeated drug pretreatment
proven (Landa et al 2005) to induce behavioural sensitization
and drug naïve animals. We expected a decrease
of the total number of MET doses self-administered by
rats per experimental session based on the increasing
response to MET administered due to behavioural sensitization
recorded earlier in our laboratory (Kucerova
et al 2009).
Aripiprazole is able to attenuate locomotor activity
induced by behavioural sensitization probably by the
antagonistic action on 5-HT1A serotonin receptors
(Futamura et al 2011). This pharmacological mechanism
could be responsible for the decrease of MET
intake induced by behavioural sensitization (Kucerova
et al 2009). Therefore, the rats were treated repeatedly
with aripiprazole before IVSA sessions to evaluate its
possible impact on spontaneous MET intake.
The working hypotheses on results of aripiprazole
effects in the experimental model used were the following:
a) decrease of MET intake in non-sensitized
rat group due to D2 partial agonism; b) drug induced
suppression of behavioural sensitization in pre-treated
(sensitized) animals by normalizing (increasing) MET
intake.
Material and methods
Animals
Adult male albino Wistar rats weighting 180-220 g at
the beginning of the study were purchased from Biotest
Ltd. (Konarovice, Czech Republic). The animals were
housed in groups of five in standardized rat plastic
cages. After the catheter implantation surgery was
performed, the rats were housed individually. Environmental
conditions during the whole study were constant:
relative humidity 50-60 %, temperature 23 ºC ±
1 ºC, inverted 12-hour light-dark cycle (5 a.m. to 5 p.m.
darkness). Food and water were available ad libitum. All
experiments were conducted in accordance with relevant
laws and regulations of animal care and welfare.
The experimental protocol was approved by the Animal
Care Committee of the Masaryk University Faculty of
Medicine, Czech Republic and carried out under the
European Community guidelines for the use of experimental
animals.
Drugs and treatments
Methamphetamine from Sigma Chemical, Co., St Louis,
MO, USA was used for both initial drug pretreatment
and the IVSA model. The administration of MET prior
to IVSA was according to the following dosing regimen,
which was successfully used in previous studies carried
out at our laboratory (Kucerova et al 2006; Landa et al
2005, 2008) to induce behavioural sensitization: 0.5 mg/
kg/day, intraperitoneally, for 14 days, administered in
home cages. The identical volume and route of administration
of saline solution (SAL) was used for all control
treatments. MET dose available in the operant cage
for IVSA was 0.08 mg per infusion with the maximum
number of infusions obtainable in one session set to 50
which was a procedure producing reinforcing effects in
the same model of IVSA in our laboratory (Vinklerova
et al 2002).
Aripiprazole was used in a dissolved tablet form
(ABILIFY 15  mg oral tablets, Bristol-Meyers Squibb
S.r.L., Anagni, Italy) at the dose of 3 mg/kg/day administered
orally by tube 45 minutes before the self-administration
session.
I.V. self-administration surgery and procedures
Under the general anaesthesia with ketamine 50 mg/kg
and xylazine 8 mg/kg given intraperitoneally (NARKAMON
5% and ROMETAR 2%, Bioveta a.s., Czech
Republic, in combination with isoflurane inhalation
for induction to anesthesia) a permanent intracardiac
silastic catheter was implanted through the external
jugular vein to the right atrium. The outer part of the
catheter exited the skin in the midscapular area. A small
nylon bolt was fixed on the skull with dental acrylic
to stainless-steel screws embedded in the skull; this
served as a tether to prevent the catheter from being
pulled out while the rat was in the self-administration
chamber. The catheters were flushed daily before all
the sessions with 0.2  ml of heparinized cephalosporine
(VULMIZOLIN 1.0 inj sicc, Biotika a.s., Slovak
Republic) solution (0.05 mg/kg in saline with 2.5 I.U./
kg) and 0.05  ml of heparin (HEPARIN LECIVA inj.
sol. 1x10ml/50 I.U., Zentiva a.s., Czech Republic) solution
(5 I.U.) to prevent infection and occlusion of the
catheter. During this procedure the blood was aspired
daily to assess the patency of the catheter, and changes
in general behaviour, weight and other circumstances
were recorded. When a catheter was found blocked the
animal was excluded from the analysis.
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Aripiprazole does not influence methamphetamine I.V. self-administration in rats
IV self-administration protocol
Standard experimental cages with two nose-poke holes
allocated on one side of the cage were programmed by
software L2T2 (Coulbourn Instruments, USA) and the
IVSA sessions were conducted under the fixed ratio
(FR) schedule of reinforcement starting at FR1 (each
correct response reinforced). Fixed-ratio requirements
were raised (e.g. FR2 - two correct responses required,
etc.); when the animal fulfilled the following conditions
for three consecutive sessions:
a) at least 70% preference of the drug-active
nose-poke;
b) minimum intake of 10 infusions per session;
c) stable intake of the drug (maximum 10 %
deviation) in three consecutive sessions.
Active nose-pokes led to the activation of the infusion
pump and administration of a single infusion followed
by 30 sec time-out, while the other nose-pokes were
recorded but not rewarded. The cage was illuminated
by a house light which was twinkling when administering
infusion and off in the time-out. The daily IVSA
sessions lasted 90  minutes and took place regularly
between 7 a.m. and 4 p.m. during the dark period of
the inverted light cycle.
After reaching stable baseline intake each animal
was subjected to water administration (control) orally
by tube 45 minutes before the self-administration session
and the possible effect on the MET intake was
recorded. When the rat developed stable MET intake
on oral water administration, it was replaced by aripiprazole
solution (3  mg/kg/day) administered by the
same manner as water before.
Experimental groups
There were 14 rats at the beginning of the experiment.
However, due to complicated surgical procedures 1 of
the subjects was lost.
The final groups as introduced to the first IVSA session
follow:
d. SAL group (n=7): 14 days of saline (SAL)
intraperitoneal pretreatment, the experiment was
completed by 7 animals
e. MET group (n=7): 14 days of MET (0.5 mg/kg/
day) intraperitoneal pretreatment, the experiment
was completed by 6 animals
Statistical Data analysis
The means of MET intake were compared when the
animals reached a stable intake of MET at FR2 and FR3
protocol. For statistical analysis of differences in MET
IVSA the nonparametric ANOVA - Kruskal-Wallis
test with Dunn’s Multiple Comparisons post test - was
used. Level of statistical significance was determined to
p<0.05.
Results
The Figure 1 shows that both groups of rats, pretreated
with saline or MET, demonstrated significant (p<0.001)
prevalence of active nosepokes, i.e. those associated
with MET reward.
Spontaneous intake of MET defined as a mean
number of infusions significantly differed between
groups. Chronic intermittent MET pretreatment
led to decrease of MET intake in the IVSA model as
expected due to behavioural sensitization (Figure 2).
This effect was statistically significant in comparison
to non-treated baseline intake (p<0.001) as well as in
comparison to intake during oral water pretreatment
administration (p<0.05).
Figure 3 exhibits the effect of aripiprazole oral
administration on the MET intake in IVSA. The significant
difference between experimental groups induced
by behavioural sensitization was abolished by aripiprazole
administration. There was no significant effect
of oral pretreatment with water/aripiprazole on MET
intake in neither previously drug naïve group (SAL)
nor behaviourally sensitized group (MET).
Discussion
According to results of our earlier studies in the same
paradigm (Kucerova et al 2009) in the present study
the significantly higher responding to nosepokes associated
with drug administration in all animals tested
confirmed the rewarding effect of methamphetamine
as expected.
This study correlates with our previous findings
that Wistar male rats repeatedly pre-treated with MET
(“MET” group) self-administer significantly lower
Figure 1: Graph displays mean (± SEM) numbers of nosepokes
during baseline intake. Both, methamphetamine (MET)
and saline solution (SAL) pretreated rats performed
significantly higher number of the nosepokes associated with
methamphetamine (p<0.001).
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Jana Kucerova, Jana Pistovcakova, Dagmar Vrskova, Alexandra Sulcova
tion paradigm induced by amphetamine. This result
was recorded after acute drug administration as well as
after chronic intermittent treatment leading to behavioural
sensitization to amphetamine which indicates
the possible intensified firing in the dopaminergic
reward pathway (Mavrikaki et al 2010). However, in IV
self-administration paradigm 3  mg/kg dose aripiprazole
(equal dose as in our experiment) was recorded
to decrease intake of d-amphetamine. This suggests an
attenuation of reinforcing effects of d-amphetamine
in Wistar rats (identical strain as in our experiment).
The opposite effect was recorded in the same paradigm
when aripiprazole increased d-amphetamine intake at
the dose of 1 mg/kg (Backstrom et al 2011). In preclinical
experimental paradigm of relapse to drug of abuse
intake, aripiprazole exhibited capacity to reduce cocaine
relapse rate in two different rat strains (Feltenstein et al
2007; Roman & Gyertyan 2009).
Present experiments did not confirm the efficacy of
aripiprazole in management of MET intake in the rat
model of IVSA of methamphetamine. Nevertheless, the
literature available refers a certain potential of aripiprazole
in treatment of psychostimulant addiction such
as amphetamine, MET or cocaine dependency. Summarizing
the currently available data on aripiprazole,
we can speculate that aripiprazole might be useful in
reduction of the relapse rate in addicted individuals,
rather than influencing the overall MET intake. Efficacy
of aripiprazole seems to be largely influenced by
the choice of appropriate (probably rather high) dose
and the treatment paradigm itself. Thus, despite of the
contradictory data obtained, it is too early to discard
Figure 3: Graph displays influence of aripiprazole pretreatment on
the mean (± SEM) numbers of MET infusions self-administered
by the animals when stable intake was reached. There is
apparent significant difference between the experimental
groups at conditions of oral water administration (p<0.05). When
aripiprazole (3 mg/kg/day) was administered 45 minutes before
the session orally, there was neither statistically significant
difference between the experimental groups anymore nor
significant alteration of MET intake in each group.
Figure 2: Graph displays mean (± SEM) numbers of MET infusions
self-administered by the animals after reaching stable baseline
intake and influence of water pretreatment 45 minutes before
session orally. There is a statistically significant difference
between the sensitized (MET group) and non-sensitized
(SAL group) experimental groups in the baseline MET intake
(p<0.001) as well as in stable intake with control oral water
administration before the session (p<0.05). However, there was
not recorded any significant alteration of MET intake induced by
oral administration procedure (water).
number of MET infusions under a fixed ratio schedule
compared to animals pretreated with saline (“SAL”
group) (Kucerova et al 2009). Higher MET rewarding
effects decreased drug-seeking behavioural signs in
previously sensitized animals what can be considered
as behavioural sensitization in this model. At the dose
which did not influence rat locomotor activities in the
open field test (Pistovcakova & Sulcova 2008) aripiprazole
did not elicit any influence on the MET intake
in drug naïve nor in sensitized rats. The observed
significant difference in drug intake between experimental
groups induced by behavioural sensitization
was abolished by aripiprazole treatment. However, this
cannot be interpreted as a suppression of behavioural
sensitization, because MET intake was not significantly
increased in sensitized animals by aripiprazole
administration.
Clinically, aripiprazole was examined for possible
attenuation of drug intake in MET addict volunteers,
where it seemed to increase rewarding effects of MET.
However, the main limitation of this study was a small
sample size (8 in each arm) and authors themselves
recognize a low probability of aripiprazole potential to
increase MET rewarding effects (Newton et al 2008).
Clinical studies with alcohol addicts show inconsistent
results but in some cases they demonstrate ability of
aripiprazole to reduce chronic alcohol consumption in
co-administration (Vergne & Anton 2010).
In the preclinical study by Mavrikaki et al 2010 a
completely opposite result was shown as aripiprazole
attenuated rewarding effects of amphetamine in the
intracranial self-stimulation model and hyperlocomo-
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Aripiprazole does not influence methamphetamine I.V. self-administration in rats
aripiprazole from suggested indication. Further studies
are needed to evaluate its benefit in treating drug
addiction.
Acknowledgements
This work was supported by the Czech Ministry of
Education - research project: MSM0021622404.
Conflict of interest statement
The authors have no financial interest in this manuscript
and no affiliations (relationships) to disclose.
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