Letter https://doi.org/10.1038/s41586-018-0165-4
The coding of valence and identity in the
mammalian taste system
Li Wang1,2,3
, Sarah Gillis-Smith1,2,3
, Yueqing Peng1,2,3
, Juen Zhang1,2,3
, Xiaoke Chen1,2,6
, C. Daniel Salzman3,4
, Nicholas J. P. Ryba5
& Charles S. Zuker1,2,3
*
The ability of the taste system to identify a tastant (what it tastes
like) enables animals to recognize and discriminate between the
different basic taste qualities1,2
. The valence of a tastant (whether
it is appetitive or aversive) specifies its hedonic value and elicits the
execution of selective behaviours. Here we examine how sweet
and bitter are afforded valence versus identity in mice. We show
that neurons in the sweet-responsive and bitter-responsive cortex
project to topographically distinct areas of the amygdala, with
strong segregation of neural projections conveying appetitive versus
aversive taste signals. By manipulating selective taste inputs to the
amygdala, we show that it is possible to impose positive or negative
valence on a neutral water stimulus, and even to reverse the hedonic
value of a sweet or bitter tastant. Remarkably, mice with silenced
neurons in the amygdala no longer exhibit behaviour that reflects
the valence associated with direct stimulation of the taste cortex,
or with delivery of sweet and bitter chemicals. Nonetheless, these
mice can still identify and discriminate between tastants, just as
wild-type controls do. These results help to explain how the taste
system generates stereotypic and predetermined attractive and
aversive taste behaviours, and support the existence of distinct
neural substrates for the discrimination of taste identity and the
assignment of valence.
The taste system is responsible for detecting and responding to the
five basic taste qualities: sweet, sour, bitter, salty and umami1,2
. Each
of these five tastes is detected by specialized taste receptor cells on the
tongue and palate epithelium, with different taste receptor cells dedicated
to each of the taste modalities1,2
. In rodents, taste information
travels from taste receptor cells in the oral cavity to primary gustatory
cortex (insular cortex) via four neural stations1,3
: taste receptor cells
to taste ganglia, then to the nucleus of the solitary tract, the parabrachial
nucleus, the thalamus and to insular cortex. Intrinsic4,5
and
two-photon6
imaging studies have shown that sweet and bitter taste are
represented in the cortex in topographically separate cortical fields; by
optogenetically activating these taste cortical fields in awake mice, it
is possible to evoke prototypical taste behaviours in the total absence
of taste stimuli7
.
The two most important sensory features of a taste stimulus are its
identity and its valence. We hypothesized that by examining the neural
targets of the sweet and bitter cortical fields it may be possible to
uncover the circuit logic for appetitive versus aversive tastes. To trace
the projections of neurons in the sweet and bitter cortex, we labelled
neurons in the sweet cortical field with enhanced green-fluorescent
protein (eGFP), those in the bitter cortex with red-fluorescent protein
(tdTomato), and then the whole brains were examined by clearing and
rapid 3D imaging with light-sheet microscopy using clear, unobstructed
brain imaging and computational analysis (CUBIC)8
. Our results show
that projections from the sweet and bitter cortical fields target multiple
brain areas, including contralateral taste cortex, amygdala, entorhinal
cortex, caudoputamen and thalamus (see Fig. 1). Notably, sweet and
bitter cortical projections exhibited strong segregation as separate lines
while navigating to the amygdala (Fig. 1b, c and Extended Data Fig. 1),
with neurons from the sweet cortical field terminating in the anterior
basolateral amygdala (BLA), whereas neurons from the bitter cortical
field predominantly projected to central amygdala (CEA), with some
1
Howard Hughes Medical Institute, Columbia University, New York, NY, USA. 2
Department of Biochemistry and Molecular Biophysics, Columbia College of Physicians and Surgeons, Columbia
University, New York, NY, USA. 3
Department of Neuroscience, Columbia College of Physicians and Surgeons, Columbia University, New York, NY, USA. 4
Department of Psychiatry and New York
State Psychiatric Institute, Columbia University, New York, NY, USA. 5
National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA. Present address:
6
Department of Biology, Stanford University, Stanford, CA, USA. *e-mail: cz2195@columbia.edu
A
P
L R
Objective
b
c
a GCsw
GCbt
BLAa
CEA BLAa CEA BLAa CEA
Bregma –0.8 mm –1.0 mm –1.2 mm –1.4 mm
BLAa
CEA
BLAa
CEA
BLAa
CEA
BLAp BLAp BLAp
–1.6 mm –1.9 mm –2.2 mm –2.5 mm
Coronal
Amy
Fig. 1 | Projections from the sweet cortex and the bitter cortex terminate
in distinct targets in the amygdala. a, Maximum-intensity z stack of
projections8
from the sweet cortical field labelled with eGFP (GCsw,
green) and the bitter cortical field labelled with tdTomato (GCbt, red).
A, anterior; P, posterior; L, left; R, right. Scale bar, 1 mm. b, Schematic
of whole-brain imaging with light-sheet fluorescence microscopy8
,
illustrating the coronal sections shown in c. Amy, amygdala. The
brain diagrams were rendered by the scalable brain composer (https://
scalablebrainatlas.incf.org/services/sba-composer.php?template=ABA_
v3) based on Allen Mouse Brain Common Coordinate Framework version
326,27
. c, Segregation of sweet and bitter projections. Sweet cortical neurons
project to the anterior BLA (BLAa, green), whereas bitter cortical neurons
predominantly innervate the CEA (red) and a portion of the posterior
BLA (BLAp, red; see also Extended Data Fig. 1). Scale bar, 0.5 mm. The
boundaries of amygdala nuclei were based on the Allen Brain Institute
atlas26
(http://brain-map.org/). Similar results were observed in six
animals.
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LetterRESEARCH
terminals in the posterior BLA. We extended these findings by performing
anterograde labelling experiments using adeno-associated
virus (AAV)-based transsynaptic transfer of Cre-recombinase9
from
sweet and bitter cortex to targets in the amygdala. These results substantiated
that the BLA was the target of sweet cortex projections, and
the CEA was the target of bitter cortex projections (Fig. 2; see Extended
Data Fig. 2 for activity-dependent labelling).
The amygdala is a key brain structure involved in processing emotions,
motivation and positive and negative stimuli10–19
. Previous studies
have shown that the BLA and CEA both contain distinct populations
of neurons that are activated by negative or positive stimuli10,13–19
. Our
finding of such strong segregation of appetitive (sweet) versus aversive
(bitter) projections to the amygdala immediately suggests an anatomical
division for the generation of valence-specific behavioural responses
to tastants.
If the amygdala imposes valence on tastants (that is, it represents the
hedonic value of a tastant to drive valence-specific behaviours), then
optogenetic activation of the terminals of sweet cortical neurons in
the BLA should elicit attractive responses, whereas activation of bitter
projections should evoke aversive behaviours. Therefore, we generated
mice expressing channelrhodopsin-2 (ChR2)20
in either the sweet or
bitter cortical field, implanted optical fibres over the amygdala, and
used a place-preference test to measure responses to photostimulation
of the cortico-amygdalar projections. Our results showed that mice
avoided the chamber linked to photostimulation of the bitter cortico-amygdalar
projections (Extended Data Fig. 3), but exhibited a strong
preference for the chamber associated with stimulation of the sweet
projections.
Next, we reasoned that optogenetic activation of the terminals
of sweet cortical neurons in the BLA would trigger appetitive taste
behaviours, whereas stimulation of the projections from bitter cortical
neurons in the CEA would instead impose a negative valence on the
stimulus. Therefore, we assayed whether ChR2 activation of sweetto-BLA
projections while a mouse is drinking a neutral stimulus (for
example, water) transforms it into a highly attractive one such as sugar,
and conversely, whether activation of the projections from bitter cortex
to CEA trigger strong laser-dependent suppression of licking, much like
the introduction of a bitter chemical would do.
We used a behavioural paradigm in which ChR2-expressing mice
were assayed for water drinking in a head-restrained setup7
. In these
experiments, the laser shutter was placed under lick-contact operation,
and thus the mouse has control of its own stimulation during
the light-on trials, and only self-stimulation would continue to trigger
appetitive responses (light stimulation on its own does not trigger
licking, or licking-like motor responses; see Methods)7
. By contrast, a
mouse would immediately terminate licking if contact-licking elicited
aversion. Indeed, optogenetic activation of the sweet cortex terminals in
BLA evoked a marked increase in licking (self-stimulation; Fig. 3b, c),
whereas activation of the bitter cortical projections to amygdala
strongly suppressed licking responses (Fig. 3b, d). To confirm that
these light-triggered behaviours were not caused by back-propagation
of action potentials from the stimulation in the amygdala (that is, back
to the taste cortex and thus to other potential taste cortical targets),
we repeated the experiment, however, this time we pharmacologically
silenced synaptic activity locally in the amygdala by infusion of the
AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)
receptor antagonist NBQX21
. Our results demonstrate that silencing
synaptic transmission in the amygdala abolished all light-evoked
responses (Fig. 3e, f). As expected, responses fully recovered after washout
of the drug (Fig. 3e, f). Taken together, these data demonstrate that
activation of sweet or bitter cortico-amygdalar pathways is sufficient to
impose a positive or a negative valence on a neutral taste cue.
We hypothesized that strong activation of the bitter and sweet cortico-amygdalar
projections might override the hedonic response elicited
by sweet and bitter tastants. Therefore, we predicted that optogenetic
activation of the bitter cortical terminals in the CEA may impose an
GC
AAV1-hsyn-Cre
Ai9 mice
BLA CEA
AAV1-Cre in sweet cortex
BLA
CEA
AAV1-Cre in bitter cortex
Anterograde
AAV1-hsyn-Cre Ai9-tdTomato tdTomato tdTomato
a cb
Fig. 2 | Segregation of sweet and bitter targets in the amygdala. a,
Schematic illustrating anterograde transsynaptic labelling of neurons in
the amygdala following AAV1-hsyn-Cre injection9
in the sweet or bitter
taste cortex of mice expressing a tdTomato reporter. b, c, Representative
confocal images of tdTomato expression in amygdala following AAV1
injection in the bitter cortex (b) or sweet cortex (c; pseudocoloured green).
Scale bars, 200 μm. Similar results were obtained in three animals for each
experiment. See also Extended Data Fig. 2.
a bLight stim.
Amy
GC
ChR2
c
e
d
f
2
1
Lickratio
Pre NBQX Post
2
1
Pre Saline Post
40
30
20
10
0
Licksper5s
Water LightWater
+ light
40
20
0
Licksper5s
100
Trial number
100
Sweet projection Bitter projection
30
20
10
0
Licksper5s
Water LightWater
+ light
1
0
Pre Saline Post
1
0
Lickratio
Pre NBQX Post
Fig. 3 | Activation of sweet and bitter cortical terminals in the
amygdala drives appetitive and aversive behaviours. a, Optogenetic
stimulation strategy. Sweet neurons in the anterior gustatory cortex (GC)
or bitter neurons in posterior gustatory cortex were transduced with
AAV-ChR2. Stimulating optical fibres were placed above BLA or CEA.
For coupling the photostimulation to drinking behaviour, laser pulses
were triggered by licking. b, Representative histograms showing licking
events in the presence (blue) or absence (grey) of photostimulation of
sweet cortico-amygdalar projections (left) or bitter cortico-amygdalar
projections (right). Note the marked enhancement or suppression of
licking, respectively. c, d, Quantification of licking responses with and
without light stimulation. c, Sweet cortical terminals in the BLA. n = 24
mice, two-tailed paired t-test, P < 0.0001. d, Bitter cortical terminals
in the CEA. n = 21 mice, two-tailed paired t-test, P < 0.0001. See also
Extended Data Fig. 4. e, f, Pharmacological silencing demonstrated that
the light-dependent licking behaviours are due to activity in the amygdala;
the panels show quantification of lick ratios before and after infusion of
NBQX (top) or control saline (bottom) in the amygdala. e, Stimulation
of sweet projections. n = 6 mice. f, Stimulation of bitter projections.
n = 7 mice. Note that NBQX abolishes the light-dependent changes in
licking responses. Values are mean ± s.e.m. Repeated-measures oneway
analysis of variance (ANOVA) followed by Bonferroni post hoc test
(Supplementary Table 1).
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Letter RESEARCH
aversive response to an orally applied sweet tastant, whereas strong
stimulation of sweet terminals in BLA might suppress aversion to an
orally applied bitter tastant. We used a behavioural test in which thirsty
mice expressing ChR2 in the bitter or sweet cortex were exposed to
random presentations of water, a bitter chemical or a sweet solution
(Fig. 4a). Next, we examined the effect of photoactivating bitter cortico-amygdalar
projections by placing the stimulating optical fibre over
the amygdala of mice that expressed ChR2 in the bitter cortex (Fig. 4b).
Stimulation of bitter targets in the amygdala is indeed sufficient to
transform the appetitive nature of a sweet tastant into an aversive one
(Fig. 4c). Conversely, by photoactivating the amygdala targets of the
sweet cortex it was possible to change the perceived valence of a bitter
tastant (Fig. 4d, e). These results highlight the key role of the amygdala
in imposing valence on a taste cue. To examine the effect of taste stimulation
in the absence of amygdala function, we carried out a number of
studies in which the neurons of the amygdala were reversibly silenced.
First, we used a behavioural assay that relies on direct stimulation
of the taste cortex. In one group of mice, we introduced ChR2 into
neurons in the sweet cortical field (Fig. 5a), bilaterally injected an
AAV encoding inhibitory designer receptors exclusively activated by
designer drugs (DREADD) into amygdala neurons for chemogenetic
silencing22
(see Methods for details), and tested the mice before and
after clozapine N-oxide (CNO) injection. Importantly, ChR2 and the
stimulating fibre are both in the sweet cortex (Fig. 5a), and because
sweet neurons project to many targets (see Fig. 1a), the full repertoire
is likely to be co-activated upon stimulation of the sweet cortical field.
Notably, silencing of the amygdala was sufficient to abolish all attractive
responses associated with activation of the sweet cortex (Fig. 5b);
equivalent results were obtained using pharmacological inhibition of
the amygdala with NBQX rather than inhibitory DREADD (Fig. 5c).
We repeated similar studies but this time examined the activation of the
bitter cortex (Fig. 5d). Our results showed that silencing of the amygdala
is also sufficient to abolish aversive responses associated with the
activation of the bitter cortex (Fig. 5e, f). Finally, we reasoned that the
valence associated with sweet and bitter tastants delivered to the tongue,
rather than direct stimulation of the taste cortex, could also be compromised.
As predicted, the results shown in Fig. 5g–i demonstrate that
silencing the amygdala impairs the behavioural preference for sweet
chemicals and the aversion to bitter chemicals.
Previously, we showed that silencing the sweet or bitter cortex prevented
the recognition of sweet or bitter tastants, whereas optogenetic
activation of those same cortical fields triggered prototypical sweetand
bitter-associated behaviours7
. We reasoned that if tastant identity
and valence are encoded in separate neural substrates, with the taste
cortex responsible for imposing identity to a tastant, and the amygdala
for affording its valence, then mice with silenced amygdala should still
recognize the identity of a sweet or bitter taste stimulus, even if blind
to its hedonic value.
We trained mice to report the identity of a tastant by using two different
behavioural assays: a three-port test and a go/no-go assay. In
the three-port test, mice learned to sample a taste cue from a centre
spout (random presentations of water, a sweet or a bitter chemical),
and then report its identity either by going to the right or left port;
cb
Water
Quinine
AceK
Oral stim.
Light stim.
CEA
GC
a
40
30
20
10
0
Licksper5s
W
ater
BitterSw
eet
40
30
20
10
Licksper5s
Water Bitter Sweet Sweet
+ light
d e
Light stim.
BLA
GC
Water
Quinine
AceK
Oral stim.
30
20
10Licksper5s
Water Sweet Bitter Bitter
+ light
Fig. 4 | Activation of cortico-amygdalar circuits overrides the
hedonic valence of orally delivered tastants. a, Licking responses (no
photostimulation) to water, bitter (0.5 mM quinine) and sweet (4 mM
AceK) stimuli. n = 18 mice; data are mean ± s.e.m. b, Schematic of
optogenetic stimulation strategy; AAV-ChR2 was injected into the bitter
cortex and the optical fibre was placed above the CEA. c, Quantification
of licking response to water, bitter and sweet stimuli and sweet stimuli
plus light. n = 6 mice. Stimulation overrides the attractive responses to
the sweet stimulus. d, Schematic of photostimulation of sweet terminals.
e, Quantification of licking response to water, sweet and bitter stimuli and
bitter stimuli plus light. n = 12 mice. Stimulation overrides the aversive
responses to the bitter stimulus. Repeated-measures one-way ANOVA
followed by Bonferroni post hoc test; see Supplementary Table 1.
4
3
2
1
0
Lickratio(AceK/water)Pre-saline
Pre-N
BQ
XSalineN
BQ
X
Post-saline
Post-N
BQ
X
0.8
0.6
0.4
0.2
0
Lickratio(Qui/water)
Post-N
BQ
X
Post-saline
N
BQ
X
Saline
Pre-N
BQ
X
Pre-saline
a b
d e f
Light
stim.
BLA
GC
hM4Di
NBQX
ChR2
c
Light
stim.
CEA
GC
NBQX
ChR2
h ig
NBQX
Sweet
Bitter
Water
Sweet Bitter
Pre CNO Post
20
10
0
Licksper5s
W L W L W L
Pre NBQX Post
30
20
10
0
Licksper5s
W L W L W L
30
20
10
0
Licksper5s
W L W L W L
Pre NBQX Post
30
20
10
0
Licksper5s
W L W L W L
Pre Saline Post
Fig. 5 | Silencing neurons in the amygdala impairs taste valence.
a, Schematic of optogenetic stimulation and the chemogenetic/
pharmacological silencing strategy. AAV-ChR2 was injected
unilaterally into the sweet cortex, and an optical fibre was implanted for
photostimulation. AAV-hM4Di28
was targeted bilaterally to the BLA for
chemogenetic silencing (alternatively, cannulas were implanted bilaterally
over the amygdala for pharmacological silencing). b, Chemogenetic
silencing of the amygdala with inhibitory DREADD (hM4Di) and
CNO abolished the strong appetitive behaviour observed following
photostimulation of sweet cortex (compare pre and post with CNO).
n = 4 mice, water (W) versus water plus light (L), two-tailed paired ttest;
pre, P = 0.0054; CNO, P = 0.8900; post, P = 0.0265. See Extended
Data Fig. 5 for controls. c, Pharmacological silencing of the amygdala
with NBQX similarly abolished the appetitive behaviour associated with
photostimulation of the sweet cortex. n = 6 mice, two-tailed paired t-test;
pre, P = 0.0049; NBQX, P = 0.9458; post, P = 0.0042. See Extended Data
Fig. 5 for controls. d, e, NBQX silencing of the amygdala abolished aversive
responses to photostimulation of the bitter cortex. n = 3 mice, two-tailed
paired t-test; pre, P = 0.0047; NBQX, P = 0.9125; post, P = 0.0261.
f, Saline controls for NBQX silencing following photostimulation of
the bitter cortex. n = 3 mice, two-tailed paired t-test; pre, P = 0.0261;
saline, P = 0.0230; post, P = 0.0005. g–i, NBQX silencing of the amygdala
diminished preference for sweet chemicals (n = 5 mice) and aversion to
bitter chemicals (n = 8 mice); the small remaining responses to the orally
applied sweet and bitter tastants probably reflect brain-stem-dependent
immediate reactions to taste observed in decerebrated animals29
.
Repeated-measures one-way ANOVA followed by Bonferroni post hoc test
(Supplementary Table 1). Values are mean ± s.e.m.
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LetterRESEARCH
a correct response was rewarded with 4 s of water (Fig. 6a). We initially
focused on attractive responses as they represent the expression
of a selective, positive behavioural response. After 20–30 sessions of
training over 10–14 days (see Methods for details), trained mice were
able to report the identity of each tastant in hundreds of randomized
trials with over 90% accuracy (see Fig. 6b), showed correct behavioural
responses to other sweet and bitter tastants that were not used in the
training set (Fig. 6b, compare training to testing), and appropriately
reported direct optogenetic activation of the sweet taste cortex as sweet,
with approximately 90% of the water plus light trials producing correct
responses (Fig. 6c, pre-silencing). Next, we assayed whether silencing
of the amygdala using inhibitory DREADD in the BLA affected the
ability of these mice to correctly identify a sweet stimulus. Our results
(Fig. 6c) demonstrated that loss of amygdala function, while abolishing
the ability of the sweet cortex to evoke appetitive responses (Fig. 5a–c),
has no impact on the ability of the mice to properly identify sweet
tastants (or to recognize light activation of the sweet cortex as sweet).
As anticipated, inhibitory DREADD expression in the sweet cortex (just
as has previously been shown using NBQX7
), severely impairs sweet
taste recognition (Extended Data Fig. 6).
The second behavioural platform relied on go/no-go behavioural
assays, and examined both sweet and bitter recognition. Thirsty mice
were trained to sample a test tastant from a spout, and then to report its
identity either by licking (go) or withholding licking (no-go; Fig. 6d).
We trained animals to report ‘go’ when tasting bitter tastants and ‘no-go’
in response to sweet tastants, exactly the opposite of the innate drive.
After 15–20 sessions of training, mice reported tastant identity with
over 90% accuracy (Fig. 6e). Indeed, silencing of the amygdala, just as
observed in the three-port tests, has no effect on recognition of sweet
or bitter tastants (Fig. 6f). Notably, these experiments used the same
mice that exhibited loss of sweet and bitter valence after NBQX infusion
into the amygdala (Fig. 5). Taken together, these studies show that the
amygdala is necessary and sufficient to drive valence-specific behaviours
to taste stimuli and that the cortex can independently represent
taste identity.
The senses of taste and smell function as the principal gateways for
assessing the attraction to, and palatability of, food cues. In its most
fundamental state, taste mediates innate consummatory and rejection
behaviours, while also allowing an animal to learn the association
of food sources with hardwired tastant-dependent actions. Here we
studied the neural basis for innate responses to sweet and bitter, and
showed that the taste cortex and the amygdala function as two essential,
but distinct, neural stations for identifying tastants and for imposing
valence on sweet and bitter.
Recent molecular studies have identified distinct populations of
neurons in the amygdala that may serve as neural substrates for a wide
range of positive and negative hedonic responses10,13–19
. In this study,
we show that sweet and bitter cortical fields exhibit separate projection
targets in the amygdala, and that photoactivation of these cortico-amygdalar
projections evokes opposing responses. However, these
can be experimentally dissociated from the cortex, such that animals
may recognize a ‘taste stimulus’ but remain oblivious to its valence.
Together, these results provide an anatomical substrate for imposing
hedonic value to sweet and bitter, and the basic logic for the generation
of hardwired, stereotypic attractive and aversive taste responses.
The amygdala is known to provide representations of Pavlovian asso-
ciations11,23,24
, such that innately rewarding and aversive tastants may
also function as unconditioned stimuli in conditioning protocols25
.
Therefore, in addition to imposing valence on tastants, the amygdala
probably links taste valence to other stimuli so that associative memories
can be formed, and thereby appropriate valence-specific behaviour
may be elicited by previously neutral cues from other modalities that
would now predict a bitter or sweet tastant. Notably, the sweet and
bitter cortex project to several additional brain areas, including those
involved in feeding, motor systems, multisensory integration, learning
and memory (Fig. 1). In the future, it will be exciting to unravel how
these circuits come together to drive innate and learned responses.
Online content
Any Methods, including any statements of data availability and Nature Research
reporting summaries, along with any additional references and Source Data files,
are available in the online version of the paper at https://doi.org/10.1038/s41586-
018-0165-4.
Received: 3 October 2017;Accepted: 26 March 2018;
Published online xx xx xxxx.
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+ light
100
50
0
Leftchoice(%)
Water Water Bitter Sweet
100
0
100
Choice(%)
AceKQ
uinineW
ater
Sucralose
C
yx
Training Testing
LeftRight
100
50
0
Go(%)
AceK
Q
uinine
C
yx
Sucralose
Training Testing
100
50
0
Go(%)
PreInfusion
Post
PreInfusion
Post
d e f
Sweet
(No-go)
Water
reward
Cue
Bitter
(Go)
Lick
No lick
No lick
Lick
No action
Air puff
No action
Cue
3 sDelay 3 s
Trial
start
Reward/air puff
Left or
Right
Sample port
Left
Right
RewardCorrect
TimeoutWrong
Cue
a b c
Light
BLA
GC
hM4Di
Amy
Pre
CNO
Post
Saline
NBQX
Sweet (No-go)
Bitter (Go)
Fig. 6 | Silencing the amygdala does not prevent tastant recognition. a,
Schematic and flow chart for the three-port taste-recognition task. In each
trial, a mouse had 0.5 s to lick a randomly presented taste cue from the
middle port, and then go to either the left or right to report the identity of
the tastant (in this example mice were trained to go left for sweet and right
for bitter or water); correct responses were rewarded with 4 s of water,
whereas incorrect ones led to a 5-s timeout penalty. b, Quantification of
results from three-port recognition sessions, demonstrating highly reliable
recognition of the stimulus identity (>90% accuracy). n = 6 mice. Tastant
concentrations: 10 mM AceK, 1 mM quinine, 3 mM sucralose, 10 μM
cycloheximide (Cyx). c, The mice used in Fig. 5b were assayed for the
effect of silencing the amygdala. n = 4 mice for pre, CNO; n = 3 mice for
post. Mice with a silenced amygdala can still identify the different tastes
with normal accuracy. Importantly, photostimulation of the sweet cortex
is recognized as a sweet-tasting stimulus7
, and remains so after CNO
silencing of the amygdala (compare water with water plus light). The graph
only presents the responses to the left port (sweet identity). d, The animals
used in Fig. 5e, h, i were assayed using go/no-go tastant recognition tests7
.
e, Mice show highly reliable recognition of the stimulus identity after
training (>90% accuracy). n = 8 mice. Tastant concentrations: 4 mM
AceK, 1 mM quinine, 3 mM sucralose, 10 μM Cyx. f, Pharmacological
silencing of amygdala has no significant effect on either sweet or bitter
recognition. n = 8 mice. Two-way or repeated-measures one-way ANOVA
followed by Bonferroni post hoc test (Supplementary Table 1). Values are
mean ± s.e.m.
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© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Letter RESEARCH
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Acknowledgements We thank M. Tessier-Lavigne, N. Renier and P. Ariel for
help with CUBIC; members of the Zuker laboratory and R. Axel for helpful
discussions. We also acknowledge the Bio-Imaging Resource Center at
Rockefeller University. Research reported in this publication was supported by
the National Institute on Drug Abuse of the National Institutes of Health under
award number R01DA035025 (C.S.Z). N.J.P.R. is supported by the Intramural
Research Program of the NIH, NIDCR; and C.D.S. was supported by R01
MH082017 from NIMH. C.S.Z. is an investigator of the Howard Hughes Medical
Institute and a Senior Fellow at Janelia Farm Research Campus.
Reviewer information Nature thanks I. de Araujo and P. Kenny for their
contribution to the peer review of this work.
Author contributions L.W. and S.G.-S. designed the study, carried out
the experiments and analysed data, Y.P. and J.Z. performed behavioural
experiments. C.S.Z., X.C., N.J.P.R. and C.D.S. designed the study and analysed
data. C.S.Z., L.W. and N.J.P.R. wrote the paper.
Competing interests The authors declare no competing interests.
Additional information
Extended data is available for this paper at https://doi.org/10.1038/s41586-
018-0165-4.
Supplementary information is available for this paper at https://doi.
org/10.1038/s41586-018-0165-4.
Reprints and permissions information is available at http://www.nature.com/
reprints.
Correspondence and requests for materials should be addressed to C.S.Z.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.
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LetterRESEARCH
Methods
Animals and surgery procedures. All procedures were carried out in accordance
with the US National Institutes of Health (NIH) guidelines for the care and use of
laboratory animals, and were approved by the Columbia University Institutional
Animal Care and Use Committee. Seven- to nine-week-old male C57BL/6J mice
and B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze
/J mice (Ai9)30
were used for viral
injections.
Animals were anaesthetized with ketamine and xylazine (100 mg kg−1
and
10 mg kg−1
,intraperitoneal),placedintoastereotaxicframewithaclose-loopheating
system to maintain body temperature, and unilaterally injected with 20–50 nl of
AAV carrying ChR2 (AAV9-CamKIIa-hChR2(H134R)-EYFP-WPRE-SV40, Penn
VectorCore)eitherinthesweetcorticalfield(bregma1.7 mm;lateral3.1 mm;ventral
1.8 mm) or the bitter cortical field (bregma −0.35 mm; lateral 4.2 mm; ventral
2.7 mm). The location of the taste cortex was verified by anatomical and optogenetic
assays. Anterograde tracing6
and retrograde tracing (Extended Data Fig. 7)
showed that these cortical areas receive input from the taste thalamus (VPMpc).
Photostimulation of these sweet and bitter cortical fields evokes prototypical attractive
and aversive taste behaviours, respectively7
. We also examined behavioural data
from 14 ChR2 injections in the middle (Extended Data Fig. 8); six of the animals
showed a modest increase in lick responses, 3 exhibited no change and 5 showed
a small range of aversion. We believe this variability probably reflects the spread
of the injection site.
Following viral injections, a customized implantable fibre (core diameter
200 μm, NA 0.39) was implanted 300–500 μm above the injection site, and guide
cannulas (26 gauge, PlasticsOne) were unilaterally or bilaterally implanted above
the anterior BLA (bregma −1.0 mm; lateral 3.2 mm; ventral 3.7 mm) or CEA
(bregma −1.2 mm; lateral 3.0 mm; ventral 3.7 mm). These guide cannulas were
used both for photostimulation of cortical projections and intracranial infusion
in pharmacological silencing experiments. A metal head post was attached for
head fixation during behavioural tests. All implants were secured onto the skull
with dental cement (Lang Dental Manufacturing). For chemogenetic silencing
experiments, 150–250 nl of AAV carrying hM4Di (AAV8-hSyn-hM4Di-mCherry,
UNC Vector Core) was injected bilaterally into the BLA (bregma −1.0 mm; lateral
3.2 mm; ventral 4.2 mm) or sweet cortical field (bregma 1.7 mm; lateral 3.1 mm;
ventral 1.8 mm) at a slow rate (15 nl min−1
). All ventral coordinates listed above
are relative to the pial surface. Mice were allowed to recover for at least 2–3 weeks
before the start of behavioural experiments. For anterograde transsynaptic tracing,
AAV1–hSyn-Cre9
(20–50 nl) was injected into the sweet or bitter cortical field of
mice carrying a Cre-dependent tdTomato reporter (Ai930
). Mice were examined
four weeks after the injection. Placements of viral injections, guide cannulas and
implanted fibres were histologically verified at the termination of the experiments
using DAPI (1:5,000, Thermo Fisher Scientific) or TO-PRO-3 (1:1,000, Thermo
Fisher Scientific) staining of 100-μm coronal sections. A confocal microscope
(FV1000, Olympus) was used for fluorescence imaging.
Whole-brain clearing and imaging. For whole-brain tracing of the projections of
cortical neurons, we unilaterally injected a small volume (10–20 nl) of mixed AAVs
carrying Cre-recombinase and Cre-dependent eGFP (AAV1-CamKII0.4-Cre-SV40
and AAV1-CAG-Flex-eGFP-WPRE-bGH, 1:100, Penn Vector Core) in the sweet
cortical field, and the same volume of mixed AAVs carrying Cre-recombinase
and Cre-dependent tdTomato (AAV1-CamKII0.4-Cre-SV40 and AAV9-CAGFlex-tdTomato-WPRE-bGH,
1:100, Penn Vector Core) in the bitter cortical field.
Four weeks after AAV injection, mice were transcardially perfused with 5–10 ml
of phosphate-buffered saline (PBS) containing 10 U ml−1
heparin, followed by
20 ml 4% paraformaldehyde; brains were post-fixed in 4% paraformaldehyde for
an additional 3 h at room temperature. Whole brains were then treated following
the CUBIC clearing protocol8,31
. To prevent sample deformation caused by temperature
fluctuation and to minimize fluorescence loss during clearing, all clearing
procedures were performed at room temperature. CUBIC clearing reagents were
prepared as previously described8,31
. Reagent 1 contained 25 wt% urea (SigmaAldrich),
25 wt% N,N,N′,N′-tetrakis(2-hydroxypropyl)ethylenediamine (SigmaAldrich)
and 15 wt% Triton X-100 (Nacalai Tesque). Reagent 2 contained 50 wt%
sucrose (Sigma-Aldrich), 25 wt% urea, 10 wt% triethanolamine (Sigma-Aldrich)
and 0.1% (v/v) Triton X-100. The fixed brains were washed three times with PBS,
immersed in reagent 1 (diluted 1:2 in water) overnight with gentle shaking and then
incubated in reagent 1 for 7–10 days with gentle shaking. Brains were washed with
PBS, degassed in PBS overnight and were then transferred into 5 ml of reagent 2
diluted 1:2 in PBS for 6–24 h before immersion in reagent 2 for 3–7 days for further
clearing and reflection index matching. TO-PRO-3 (1:5,000, Thermo Fisher
Scientific) was added to reagent 2 for counterstaining. During the immersion in
reagent 2, tubes were not shaken to avoid bubbles. Samples were kept in reagent 2
for up to one week at room temperature before imaging.
On the day of imaging, samples were gently wiped to remove reagent 2 residue
and transferred into an oil mix (mineral oil and silicone oil 1:1, final refraction
index 1.48–1.49) at least 1 h before imaging. Light-sheet fluorescence microscopy
(UltraMicroscope, LaVision BioTec) with a 2× objective lens (0.5 NA, working
distance 10 mm) or 4× objective lens (0.3 NA, working distance 6 mm) was used
for rapid image acquisition of the whole brain. The samples were sequentially
illuminated with a unidirectional light sheet produced by 488-nm, 561-nm and
640-nm lasers and scanned with a z-step size of 8.13–13 μm from ventral to dorsal.
Exposure time was 50–200 ms per channel per z step. To cover the whole brain,
each sample was imaged either via 4 × 4 tile scans with the 4× lens or using multi-position
scans with the 2× lens (three manually assigned positions to cover two
hemispheres and brainstem).
Whole-brain image tiles were scaled to 1/8 of the original size and stitched
in three dimensions using ImageJ 1.51n (Fiji distribution). The 640-nm channel
of the whole-brain data was registered to a reference atlas (Allen Brain Institute,
25-μm resolution volumetric data with annotation map, http://www.brain-map.
org) using ANTs (advanced normalization tools 1.9.x) with affine transforma-
tion26,32
. The same transformation was applied to the other two channels using the
WarpImageMultiTransform function of ANTs. z projections of maximum intensity
and virtual sections were processed in ImageJ (noise was filtered with the remove
outlier function). Because of the high dynamic range of the fluorescent intensity
between the soma and the fine processes, the gamma value of the images shown
in Fig. 1 was set to 0.5 for display purposes.
Fos induction and immunohistochemistry. Mice expressing ChR2 in the sweet
or bitter cortex were habituated by performing mock stimulations (see below)
once a day for three days before Fos induction. On the day of the experiment,
mice were photostimulated for 30 min (473 nm, 20 Hz, 20-ms pulses, 5 s on and
5 s off, 5–10 mW per mm2
). Mice were then allowed to rest for 1 h and were
processed for immunostaining as previously described7
. Tissue sections were
incubated with goat anti-Fos antibody (1:500, Santa Cruz, sc-52-G) for 24 h at
4 °C. Fluorescently tagged secondary antibodies (Alexa-594 donkey anti-goat
or Alexa-647 donkey anti-goat, 1:1,000, Thermo Fisher Scientific) were used to
visualize Fos expression. All sections were imaged using an Olympus FV-1000
confocal microscope.
Head-restrained lick preference assays. Head-restrained lick preference assays
were performed as previously described7
. Mice expressing ChR2 in the sweet or
bitter cortex were initially water-deprived for 24 h to motivate drinking in headrestrained
assays and then acclimated to drinking from a motor-positioned spout
(two sessions per day for at least two days) before testing. Mice were weighed daily
during the behavioural assays and supplied with necessary water to maintain at
least 85% of their initial body weight. Each trial began with a light cue, followed
1 s later by the spout swinging into position and a tone cue to indicate the onset
of tastant delivery; after 5 s (during which the mouse could lick) the spout rotated
out of position. To measure attractive responses, mice were mildly water restricted
(water-deprived for 24 h, and then provided with water until they exhibited an
average of 5–15 licks per 5-s window); mice were supplied with 2–5 μl water at the
beginning of each trial. To measure aversion, mice were water-deprived for 24 h,
and supplied with 5–10 μl water per trial; mice normally exhibited active licking
over the full five seconds (average 30–40 licks per 5 s as a sign of thirst). Training
sessions consisted of 60 trials with water; testing sessions shown in Fig. 3 consisted
of 15 trials with water, 4 of which were coupled to photostimulation of cortical
terminals in the amygdala; testing sessions in Fig. 5b–f consisted of 20 trials with
water, 10 of which were pseudo-randomly coupled to photostimulation of the
sweet cortex; testing sessions in Fig. 4 consisted of 60 trials, 20 trials with water, 20
trials with bitter taste (0.5 mM quinine), 20 trials with sweet taste (4 mM AceK).
To examine the effect of amygdalar nuclei on taste preference, 50% of sweet trials
in Fig. 4c or 50% of bitter trials in Fig. 4e were pseudo-randomly coupled to photostimulation
of the CEA (Fig. 4c) or BLA (Fig. 4e). The delivery of tastants was
triggered by licking actions such that mice could consume as much or little as they
chose during the 5 s. To minimize the influence of thirst and satiety on the assessment
of taste palatability, for each test session in Fig. 4 we included consecutive
trials satisfying two criteria: (1) licks to bitter less than 20 (otherwise mice were too
thirsty); (2) more than 5 licks to water (otherwise mice were already satiated). The
licking behaviour was videotaped during the entire session and licking events were
identified by a custom-written code in MATLAB. For photostimulation, 473-nm
light stimuli (diode-pumped solid-state laser, Shanghai Laser & Optics Century Co.
or fibre-coupled LED, Thorlabs) were delivered via an optical fibre inserted into a
guide cannula over the amygdala or via an implantable fibre over the taste cortex.
Light stimulation was controlled by contact of the tongue with the metal spout;
one lick triggered a train of light pulses (10–20 Hz, 20 ms per pulse, 20 pulses,
5–15 mW per mm2
). Licks during the light stimulation extended the stimulus until
1 s after the last lick. Light/tone cues, the delivery of tastants and light stimuli were
controlled using a MATLAB program via a microcontroller board (Arduino Mega
2560, Arduino)7
. Each point in Fig. 3c–f and Fig. 4 indicates data averaged from
multiple test sessions for an individual mouse. In Fig. 3e, f, the lick ratio refers to
the number of licks in the presence of light stimulation over the number of licks
in water-only trials.
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Letter RESEARCH
Free-moving lick preference assays. Taste preference (Fig. 5g–i) was also measured
in free-moving animals by using a custom-built gustometer33
. Prior to testing,
mice were water-restricted for 24 h, and then provided with water until they
exhibited an average of <20 licks per 5-s window (to test attractive responses).
Alternatively, after 24 h of water-restriction mice were provided with unrestricted
water access for 5–10 min, and then assayed 18 h later (that is, to test aversive
responses animals need to be sufficiently thirsty to be motivated to sample an
unattractive cue). For testing, mice were presented with water versus 4 mM AceK
or water versus 1 mM quinine as previously described33
.
Place-preference assays. Mice expressing ChR2 in sweet or bitter cortex were
tested in a custom-built two-chamber arena placed inside a sound-attenuating
cubicle; the arena (30 cm × 15 cm), was designed with two chambers, one with
alternating black and white vertical stripes, and the other with alternating black
and white squares. Animal locations were tracked in real-time by videotaping7
.
Mice were tested in the arena for 30 min with photostimulation of the sweet or
bitter cortico-amygdalar projections via an optical fibre above the BLA or CEA,
respectively. The last 15 min of each testing session was used to calculate the preference
index (PI); PI = (t1 − t2)/(t1 + t2), where t1 is the fraction of time a mouse
spent in the chamber 1 (stimulating chamber), and t2 is the time spent in chamber
2 (non-stimulating chamber). For photostimulation of the sweet cortico-amygdalar
projections, light was delivered for 5 s, with a 3-s interval (20 Hz, 20-ms pulses,
5–10 mW per mm2
) to avoid over-stimulation or phototoxicity; for photostimulation
of the bitter cortico-amygdalar projection, light (20 Hz, 20-ms pulses, 2–5 mW
per mm2
) was delivered for 1 s with a 3-s interval; a sound cue was used to mark the
onset of each stimulation15,34,35
. For each cohort (8 mice for sweet and 5 animals
for bitter), half the animals were tested with light on in the baseline-preferred
chamber, and half with light on in the baseline unpreferred chamber15,34
. When
the mouse crossed to the non-stimulating chamber, the light was automatically
turned off immediately.
Three-port taste-recognition assays. Mice deprived of water for 24 h were trained
to perform a taste-recognition task in a customized three-port behaviour chamber
in which they sampled taste cues from the middle port and then reported the taste
identity of the cue by choosing to lick from either the left or right port. Taste cues
(AceK, quinine or water) were pseudo-randomly delivered through a metal spout
in the middle port. Each trial began with the shutter opening in the middle port.
Mice were given (up to) 15 s to initiate a trial by licking the middle spout (failure to
initiate a trial resulted in the shutter closing and a new trial starting). The shutter
in the middle port closed 0.5 s after the first lick allowing animals 0.5 s to sample
tastants cues (2–3 μl); 0.5 s after the middle port closed, the shutters of the left
and right ports opened simultaneously. Mice were given 4 s to make a left or right
choice and obtain the water reward (total ~6 μl). For a given mouse, reward from
side ports was assigned to taste cues (for example, left for sweet, right for bitter and
water). A wrong choice triggered a penalty of a 5-s timeout. The inter-trial interval
was 1 s. Mice were trained for two sessions per day, with 80–100 pseudo-randomized
trials per session until they could effectively discriminate the tastants with
approximately 90% accuracy (2–3 weeks). To test the effect of photostimulation
of sweet cortical neurons, mice expressing ChR2 in the sweet cortex were trained
to discriminate sweet from bitter and water (for example, left for sweet, right for
either bitter or water) and then tested with sweet, bitter, water and water with light
(473 nm, 20 Hz, 20 ms per pulse, 20 pulses triggered by one lick of the middle
spout). A testing session consisted of 20 sweet trials, 20 bitter trials, 10 water-only
trials and 10 water with light trials. To avoid mice using photostimulation light as a
visual cue, the connection between implantable fibre and patch cable was properly
shielded. To prevent learning during the test, no time-out penalties were given
and no reward was provided for water with light trials. Performances were calculated
as the percentage of correct choices for a given taste cue. The lick behaviour
was detected by a capacitive touch sensor (MPR121, SparkFun). The delivery of
tastants, shutter position and light stimuli were controlled by a custom-written
program in MATLAB via an Arduino board.
Go/no-go taste-recognition assays. Go/No-go taste-recognition assays were performed
as previously described7
. Mice were trained until they could effectively
discriminate the tastants with approximately 90% accuracy (over 1–2 weeks).
On the ‘probe’ sessions, no punishment was applied for ‘no-go’ tastants to avoid
re-learning; neither reward nor punishment were delivered to novel tastants.
Pharmacological inhibition. The selective AMPA receptor antagonist NBQX
(2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide,
5 mg ml−1
in 0.9% NaCl, 100–300 nl, Tocris Bioscience) was unilaterally (Fig. 3e,
f) or bilaterally (Figs. 5, 6) infused into the amygdala using a 1-μl microsyringe
(Hamilton) and an internal cannula (PlasticsOne) inserted into the guide cannula
above the amygdala. The infusion rate was approximately 100 nl min−1
. After
intracranial infusion, mice were allowed to rest in their home cage for 1–1.5 h
before re-test. A post-test was performed after a recovery period of 24 h. As a
control, the same experiment was conducted using isotonic saline (0.9% NaCl)
in the same animals.
Chemogenetic inhibition. Mice injected with ChR2 in the sweet cortex and
hM4Di in the BLA were first tested in the head-restrained lick preference assay as
described above. To effectively examine inhibition using hM4Di, we determined
(and used) the minimum light intensity for photostimulation that produced significant
attractive responses (pre-test). On the following day, CNO was injected
(10 mg kg−1
, intraperitoneal) and the behavioural test with the same level of photostimulation
was repeated between 1 and 2 h after CNO injection. Mice were
allowed to rest and recover in their home cage and a post-test was performed at
least 24 h later.
The same mice were then trained in the three-port assay for taste recognition.
After achieving at least 90% accuracy in training sessions, mice were tested for
the ability to recognize tastants and optogenetic stimulation of the sweet cortex
in the three-port taste-recognition assay before and after chemogenetic silencing
(pre: 24 h before CNO injection; CNO: between 1–2 h after injection; post: at least
24 h after injection). To confirm that amygdala was indeed efficiently silenced in
the experiments presented in Fig. 6c, mice were tested for attractive responses to
photostimulation of the sweet cortex in a lick preference assay; this was performed
after each three-port session before and after chemogenetic silencing.
Statistics. No statistical methods were used to predetermine sample size, and investigators
were not blinded to group allocation. No method of randomization was
used to determine how animals were allocated to experimental groups. Animals in
which post hoc histological examination showed that viral targeting or the position
of implanted fibre/cannulas was in the wrong location were excluded from analysis.
Statistical methods are indicated when used, and statistical analyses for all figures
are provided in Supplementary Table 1. Multiple comparisons were analysed using
repeated-measures one-way or two-way ANOVAs followed by the Bonferroni correction.
All analyses were performed in MATLAB 2016a (MathWorks), Prism 7.0a
(GraphPad) and Igor Pro 6.37 (WaveMetrics). Data are presented as mean ± s.e.m.
Reporting summary. Further information on experimental design is available in
the Nature Research Reporting Summary linked to this paper.
Code availability. Custom code for behavioural assays is available from the corresponding
author upon reasonable request.
Data availability. All data supporting the findings of this study are available from
the corresponding author upon reasonable request.
	30.	 Madisen, L. et al. A robust and high-throughput Cre reporting and
characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140
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	31.	 Susaki, E. A. et al. Advanced CUBIC protocols for whole-brain and whole-body
clearing and imaging. Nat. Protoc. 10, 1709–1727 (2015).
	32.	 Avants, B. B. et al. A reproducible evaluation of ANTs similarity metric
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sharing similar signaling pathways. Cell 112, 293–301 (2003).
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© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
LetterRESEARCH
Extended Data Fig. 1 | Projections from the sweet and the bitter cortex
terminate in distinct targets in the amygdala. a, The cartoon illustrates
the imaging planes of the optical horizontal sections at different depths
of the amygdala. The brain diagrams were rendered by the scalable brain
composer (https://scalablebrainatlas.incf.org/services/sba-composer.
php?template=ABA_v3) based on the Allen Mouse Brain Common
Coordinate Framework version 326,27
. b, Segregation of sweet and bitter
cortical projections in the amygdala. Sweet cortical neurons project to
anterior BLA (green), whereas bitter cortical neurons predominantly
innervate the CEA (red) and a portion of the posterior BLA (red). Top,
optical horizontal sections at different dorsal–ventral positions are shown
(sections 1–4; see a). Bottom, the boundaries of amygdala nuclei were
determined by aligning fluorescence images to the Allen Brain Institute
atlas26
(http://brain-map.org/). Scale bar, 500 μm. Similar results were
observed in six independently labelled and imaged animals.
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Letter RESEARCH
Extended Data Fig. 2 | Activity-dependent labelling of sweet and
bitter cortical targets in the amygdala. Fos expression16
in response to
optogenetic activation of the sweet and bitter cortical fields was used to
label activated neurons. a, Schematic of optogenetic stimulation strategy
in the bitter cortex for Fos induction. b, Fos expression in the amygdala
in response to photostimulation of the bitter cortex. The majority of Fos+
neurons are localized in the CEA. c, Fos expression in a control mouse
without light stimulation. d–f, Photostimulation of the sweet cortex
induces Fos expression in the amygdala. Note that the CEA, as a major
local output of the BLA11,36
, also shows strong Fos labelling in response
to photostimulation. Scale bars, 200 μm. Similar results were obtained in
three animals for each experiment.
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
LetterRESEARCH
Extended Data Fig. 3 | Place preference by photostimulation of corticoamgydalar
projections. a, Representative tracking of a mouse during the
15-min place-preference test in a two-chamber arena; the left chamber
in the diagram was coupled to stimulation of sweet cortico-amygdalar
projections (see Methods for details); this animal spent over 80% of the
test time in the chamber linked to stimulation of sweet projections to the
amygdala. b, Quantification of preference index before (Pre) and during
light stimulation. n = 8 mice, two-tailed paired t-test, P = 0.0156.
c, d, Place-preference test with stimulation of bitter cortical projections in
the amygdala. n = 5 mice, two-tailed paired t-test, P = 0.0207. e, f, Animals
used in d were also tested in a licking assay with similar light stimulation
intensity, demonstrating strong suppression of licking responses. n = 5
mice, two-tailed paired t-test, P = 0.0056. Values are mean ± s.e.m.
We note that we have examined multiple independent behavioural
experiments activating sweet projections to the BLA and have never
observed the induction of motor patterns or consummatory behaviour.
Strong stimulation of bitter cortico-amygdalar projections (20 Hz,
10–15 mW) often elicited prototypical orofacial rejection behaviour
(Supplementary Video 1).
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Letter RESEARCH
Extended Data Fig. 4 | Activation of bitter cortical projections to
posterior BLA is aversive. As shown in Fig. 1 and Extended Data Fig. 1,
a fraction of the bitter cortico-amygdalar projections terminate in the
posterior BLA. As expected, stimulation of these projections elicits
aversive responses. a, Representative histograms showing licking events in
the presence (blue) or absence (grey) of photostimulation of bitter cortical
projections to the posterior BLA. AAV-ChR2 was injected into the
bitter cortex, and the stimulating fibre was targeted above the posterior
BLA (coordinates: bregma −2 mm; lateral 3.4 mm; ventral 4.3 mm).
b, Quantification of licking responses. n = 3 mice, two-tailed paired t-test,
P = 0.0121. Values are mean ± s.e.m.
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
LetterRESEARCH
Extended Data Fig. 5 | Control experiments for silencing amygdala with
DREADD and NBQX. a, Quantification of licking response before and
after saline administration in mice that expressed inhibitory DREADD
(hM4Di; see Fig. 5). n = 6 mice, two-tailed paired t-test; pre, P = 0.0011;
saline, P < 0.0001; post, P = 0.0014. b, Quantification of licking response
before and after CNO administration (10 mg kg−1
) in wildtype
(WT)
non-DREADD-expressing animals. n = 6 mice, two-tailed paired t-test;
pre, P = 0.0025; CNO, P = 0.0008; post, P = 0.0021. c, Controls with
saline infusion instead of NBQX for Fig. 5c. n = 6 mice, two-tailed paired
t-test; pre, P = 0.0080; saline, P = 0.0054; post, P = 0.0046. Values are
mean ± s.e.m.
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Letter RESEARCH
Extended Data Fig. 6 | Chemogenetic silencing of neurons in the taste
cortex impairs tastant recognition. a, Quantification of the error rate
for sweet taste recognition (2 mM AceK) in a three-port assay before and
after silencing neurons in the sweet taste cortex with inhibitory DREADD
(hM4Di) and CNO (10 mg kg−1
). n = 5 mice, repeated-measures one-way
ANOVA followed by Bonferroni post hoc test, F2,8 = 46.84, P < 0.0001.
Pharmacological silencing data using NBQX can be found in a previously
published study7
. b, Quantification of the error rate of sweet taste
recognition (1 mM AceK) before and after silencing of the amygdala with
inhibitory DREADD (hM4Di) and CNO (10 mg kg−1
). n = 4 mice, twotailed
paired t-test; pre versus CNO, P = 0.7888. See also Fig. 6. Note that
sweet recognition was only affected by silencing the taste cortex, but not
the amygdala. All tested animals recognized sweet taste with the correct
behavioural choice (before silencing) in at least 90% of the trials. Values
are mean ± s.e.m. See Supplementary Table 1 for detailed statistics.
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
LetterRESEARCH
Extended Data Fig. 7 | Retrograde labelling of gustatory thalamic
neurons. a, b, Injection of the retrograde tracer cholera toxin subunit B–
Alexa Fluor 594 in the taste cortex (bitter cortical field; shown in red in a)
selectively labels neurons in the taste thalamus (VPMpc; b). Similar results
were observed in two animals. c, d, Diagrams of the corresponding brain
regions, adapted from the Allen Brain Institute atlas. Scale bars, 500 μm.
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Letter RESEARCH
Extended Data Fig. 8 | Licking responses to photostimulation of
intermediate regions between sweet and bitter cortex. Behavioural
responses (see Fig. 3) in water-only trials linked to contact-driven selfstimulation
are shown for mice expressing ChR2 between the sweet and
bitter cortical fields. Note that a positive index means attraction, whereas
a negative index means aversion to light stimulation. n = 14 mice. Data
points indicate the behavioural test of individual animals at different
stimulation sites relative to bregma position.
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
1
natureresearch|reportingsummaryMarch2018
Corresponding author(s): Charles Zuker
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