REPORT
◥
SLEEP RESEARCH
Homer1a drives homeostatic
scaling-down of excitatory synapses
during sleep
Graham H. Diering,1
Raja S. Nirujogi,2
* Richard H. Roth,1
* Paul F. Worley,1
Akhilesh Pandey,2
Richard L. Huganir1
†
Sleep is an essential process that supports learning and memory by acting on synapses
through poorly understood molecular mechanisms. Using biochemistry, proteomics,
and imaging in mice, we find that during sleep, synapses undergo widespread alterations in
composition and signaling, including weakening of synapses through removal and
dephosphorylation of synaptic AMPA-type glutamate receptors. These changes are driven
by the immediate early gene Homer1a and signaling from group I metabotropic
glutamate receptors mGluR1/5. Homer1a serves as a molecular integrator of arousal and
sleep need via the wake- and sleep-promoting neuromodulators, noradrenaline and
adenosine, respectively. Our data suggest that homeostatic scaling-down, a global form of
synaptic plasticity, is active during sleep to remodel synapses and participates in the
consolidation of contextual memory.
C
ognitive functions such as learning and
memory are supported by sleep, which
modifies synapses through poorly understood
mechanisms (1, 2). Information coding
during wake drives synapse strengthening,
which is offset by weakening of synapses during
sleep (3–5). Neurons use homeostatic scaling to
adjust overall synaptic weight and maintain neuronal
firing rates while protecting information
coding (6). Although homeostatic scaling-up has
been demonstrated in visual cortex in response
to sensory deprivation (7, 8), a clear physiological
function of scaling-down has not been defined.
Homeostatic scaling-down weakens excitatory
synapses through removal and dephosphorylation
of synaptic AMPA-type glutamate receptors
and is mediated by alterations in the signaling of
protein kinase A (PKA) and group I metabotropic
glutamate receptors (mGluR1/5) (9–11). We used
biochemical fractionation, proteomics, and in vivo
two-photon imaging to characterize the changes
that occur in synapse composition in mice through
the wake/sleep cycle to better understand the
molecular basis by which sleep promotes cognitive
processes and to test the hypothesis that the
physiological function of homeostatic scalingdown
is to remodel synapses during sleep.
We first examined whether previously described
synaptic changes during homeostatic scaling-down
in vitro also occurred in mouse forebrain synapses
during the dark/light cycle (12 hours dark/12
hours light). Four hours into the dark (wake) or
light (sleep) phases, forebrains were dissected,
followed by subcellular fractionation to isolate the
postsynaptic density (PSD) (Fig. 1, A and B). Livevideo
recording confirmed that mice spent the
majority of the first 4 hours of the dark or light
phase awake or asleep, respectively. PSD fractions
were analyzed by Western blot and quantitative
proteomics. Homeostatic scaling-down
involves a loss of synaptic AMPARs and AMPAR
dephosphorylation (6, 9, 11), major determinants
of synaptic strength (12). PSD samples collected
during sleep contained significantly less GluA1
and GluA2 AMPAR subunits, decreased phosphorylation
of GluA1 at S845 and S831, decreased
catalytic subunit of PKA and A-Kinase Anchor
Protein 5 (AKAP5) compared with PSDs prepared
from the wake phase (3) (Fig. 1, C and D).
There was no change in PSD-associated GluA3,
N-methyl-D-aspartate (NMDA)–type glutamate
receptor subunits, or core PSD scaffold molecule
PSD95 (Fig. 1, C and D). Total forebrain levels
of these proteins were not different, which suggests
that the observed changes are specific to
synapses (fig. S1).
Next, we used in vivo two-photon imaging to
examine wake/sleep synapse targeting of superecliptic
pHluorin (SEP)–tagged GluA1 in layer V
pyramidal neurons of primary motor cortex via
a cranial window (13). Synapses were repeatedly
imaged at 10 p.m. and 10 a.m. over multiple days.
Spine GluA1 content and spine size were highly
correlated (fig. S2) (13). Average spine GluA1
signal over all synapses imaged was reduced
during the sleep phase compared with the wake
phase (Fig. 1, E and F). Although some spines
showed an increase in spine GluA1 during sleep,
a greater number of spines showed reduced
spine GluA1 during sleep (Fig. 1G). We separated
synapses into two groups, “high” or “low” GluA1expressing
synapses, which contain greater or
less than average spine GluA1 levels during wake,
respectively. “High” synapses showed a disproportionate
loss of GluA1, as well as reduced spine
size during sleep, whereas “low” synapses showed
no change in GluA1 or size (Fig. 1G and fig. S2).
The correlation between wake spine GluA1 levels
and the change in spine GluA1/size during sleep
(sleep/wake ratio) showed a negative slope, suggesting
that spines with more GluA1 during wake
were most likely to lose GluA1 and shrink in size
during sleep (fig. S2).
Next, we used quantitative proteomics to measure
changes in PSD protein/phosphorylation
levels (Fig. 1, H and I, and fig. S3). We quantified
more than 4500 PSD proteins and close to
10,000 unique phosphopeptides. About 20% of
PSD proteins and phosphorylation sites showed
regulation during wake or sleep, including GluA1
phosphorylation, and signaling molecules PKA, classic
protein kinase C isoforms (PKCb/g), calciumand
calmodulin-dependent kinase II (CaMKIIa),
and type-1 inositol triphosphate receptor (IP3R)
(Fig. 1, H and I, and tables S1 and S2). Kinase
prediction algorithms suggest that during wake,
more PSD proteins are targeted by PKA/PKC,
whereas during sleep, more PSD proteins are
targeted by CDK5 (fig. S4). Motif analysis revealed
that the most highly enriched motifs during
wake or sleep corresponded with consensus
target sequences for PKA or CDK5, respectively
(fig. S4).
We next attempted to dissect the underlying
molecular mechanisms driving the described
changes. Group I metabotropic glutamate receptors
(mGluR1/5) form a signaling complex with
PKC and IP3Rs linked by long forms of the scaffold
Homer (14). Neurons can express an endogenous
dominant-negative short variant, Homer1a, which
disrupts mGluR1/5-IP3R complex formation (15).
Homer1a and mGluR5 are important for the regulation
of sleep and wakefulness (16–20) and homeostatic
scaling-down in vitro and in response
to electroconvulsive therapy in vivo (10). We therefore
hypothesized that Homer1a drives remodeling
of the mGluR1/5 complex during sleep.
Whereas mGluR5, PKCg, and IP3R showed a
concerted decrease in PSD during sleep, PSD
Homer1a levels drastically increased (Fig. 2, A and
C), despite a decrease in Homer1a protein from
whole-forebrain homogenate (fig. S1). Wake/sleep
regulation of PSD-associated mGluR5, PKCg, and
IP3R was absent in Homer1a knock-out (KO)
mice (Fig. 2, B and D). Using coimmunoprecipitation,
we observed significantly less interaction
between Homer1L, mGluR5, and IP3R during the
sleep phase, whereas interaction between Homer1L
and Shank (21) were unchanged (Fig. 2, E and F).
Unlike in wild-type mice, wake/sleep regulation of
PSD-associated AMPAR subunits and PKA/AKAP5
was abolished in Homer1a KO mice (Fig. 2, G and
H, and fig. S5). These results agree with previous
Diering et al., Science 355, 511–515 (2017) 3 February 2017 1 of 5
1
Solomon Snyder Department of Neuroscience, Kavli
Neuroscience Discovery Institute, Johns Hopkins University,
Baltimore, MD, USA. 2
Department of Biological Chemistry,
Institute of Genetic Medicine, Johns Hopkins University,
Baltimore, MD, USA.
*These authors contributed equally to this work.
†Corresponding author. Email: rhuganir@jhmi.edu
RESEARCH
onMarch16,2017http://science.sciencemag.org/Downloadedfrom
findings that scaling-down was absent in Homer1a
KO neurons (10) and suggest that PSD targeting of
Homer1a during sleep results in a loss of synaptic
mGluR5 and associated signaling molecules, disassembly
of the mGluR5-Homer1L-IP3R complex,
and removal of synaptic AMPARs (summarized
in Fig. 2I). We asked if these same changes occurred
at synapses during scaling-down in vitro.
Bicuculline-induced scaling-down (6) resulted in a
loss of PSD mGluR5, PKCg, and IP3R, an increase
in PSD Homer1a (fig. S6), and decreased association
of mGluR5 and IP3R (fig. S6).
In addition to uncoupling mGluR1/5 from
IP3R (15), Homer1a binding activates constitutive
ligand-independent mGluR1/5 signaling that
drives homeostatic scaling-down (10, 22). Because
Homer1a displaces mGluR1/5 from major signaling
effectors IP3R/PKCg (15), other downstream pathways,
such as extracellular signal–regulated kinase
1/2 (ERK1/2), may be relevant during scaling-down
and sleep (23, 24). Treatment of cultured neurons
with the mGluR1/5-specific agonist dihydroxyphenylglycine
caused a significant increase in
phosphorylated ERK1/2, which was completely
occluded by bicuculline (fig. S6). Levels of phosphorylated
ERK1/2 were significantly higher in
forebrain homogenate and synaptosome fractions
during the sleep phase (fig. S7) (23).
Next, we investigated whether mGluR1/5 signaling
contributes to synaptic AMPAR removal
and memory consolidation during sleep. Mice
were treated by intraperitoneal injection during
the wake or sleep phases with MTEP and
JNJ6259685 (MJ), noncompetitive mGluR5/1 inhibitors.
MJ injection during the sleep phase resulted
in a small but significant increase in PSD-associated
GluA1 and a decrease in phospho-ERK1/2 levels
in synaptosomes, whereas treatment during the
wake phase had no effect (fig. S7). Next, we trained
mice in contextual fear conditioning (CFC), which
is sleep sensitive (2), using a single uncued footshock.
Training occurred either at the beginning
of the sleep phase or immediately before the
wake phase, followed by MJ injection (Fig. 3, A
and D). Mice were tested for fear responses at
the beginning of the sleep phase, 24 or 36 hours
after training, to avoid the strong time-of-day
effects of memory recall (25). Compared with vehicle,
MJ injection significantly increased freezing
responses in both trained (fearful) and novel
(nonreinforced) contexts when injected during
the sleep phase (Fig. 3, A to C), whereas injection
immediately before the wake phase had no effect
(Fig. 3, D to F). Conventional Homer1a KO mice
did not show a contextual memory phenotype
(fig. S5), likely due to developmental compensation.
We suspect that blocking scaling-down
by MJ injection during sleep resulted in a more
intense fear memory but also impaired the ability
of the mice to appropriately discriminate between
the trained and untrained context.
In our final experiments, we wished to determine
the mechanisms controlling Homer1a synapse
targeting. Homer1a gene expression is strongly
linked with learning and neuronal activity (15).
Accordingly, Homer1a mRNA is low during sleep
and increases during wake, especially after sleep
deprivation (19, 20). However, Homer1a protein is
targeted to the PSD in the sleep phase (Fig. 2, A
and C) and during scaling-down (fig. S6), suggesting
that wake-related neuronal activity drives the
expression of Homer1a mRNA but limits targeting
of Homer1a protein to the PSD. We reasoned
that release of the neuromodulator noradrenaline
(NA), strongly linked with wake and arousal (26),
may limit Homer1a synapse targeting during
wake. Indeed, acute NA treatment reversed
Diering et al., Science 355, 511–515 (2017) 3 February 2017 2 of 5
C
PSD95
tubulin
S1
wake 1μg
S2
P2
Syn
PSD
S1
S2
P2
Syn
PSD
sleep 1μg
Wild-type 8-10 weeks
A
D
B
H
I
E
0
1
2
3
4
-1
-2
-3
-4
PSDratiowake/sleep(Log2)
0 2K 4K 6K 8K 10K
number of phosphorylations
PSD: phosphorylations
AKAP5 S29
GluA1 S831
GluA1 S845
CamKIIα T286
6pm 6am
10pm: wake
dark light
10am: sleep
PSD prep
(awake: 81%) (awake: 31%)
0
1
2
3
4
-1
-2
-3
-4
PSDratiowake/sleep(Log2)
IP3R
0 1K 2K 3K 4K
number of proteins
AKAP5
PKCγ
PKCβ
PKA
PSD95 GluN1
PSD: total proteins
wake
sleep
150
125
100
75
50
25
0
sleepPSDlevel(%wake)
GluA1GluA2
GluA3GluA1p845GluA1p831
GluA2p880
GluN1GluN2BGluN2APSD95AKAP5
PKA
* *
*
* *
*
F
RelativespineGluA1
1.5
1.0
0.5
0
all spines
high GluA1
low GluA1
wake
sleep
wake
sleep
wake
sleep
****
** ns
G
sGluA1
dsRed
0.00
0.05
0.10
0.15
0.20
# of spines
increasing:
162 (42%)
# of spines
decreasing:
221 (58%)
1.0
0.8
0.6
0.4
0.2
0.0
1.2
1.4
1.6
1.8
2.0
Probability
Normalized spine GluA1 sleep/wake ratio
CaMKIIα T310
PSD95 S600
mGluR5 S870
GluA1
GluA2
GluA3
GluA1p845
GluA1p831
GluA2p880
wake
wake
sleep
sleep
WB: PSD
100kDa
100kDa
100kDa
100kDa
100kDa
100kDa
wake
wake
sleep
sleep
GluN1
GluN2B
GluN2A
PSD95
AKAP5
PKA
WB: PSD
100kDa
250kDa
100kDa
150kDa
37kDa
250kDa
150kDa
150kDa
100kDa
50kDa
75kDa
5K
Fig. 1. Forebrain postsynaptic densities are remodeled during the wake/sleep cycle. (A) Experimental
design. Time spent awake was measured during the first 4 hours of the dark or light phase by video
recording as a fraction of total time. (B) Subcellular fractionation to yield S1, S2, P2, synaptosome (Syn),
and postsynaptic density (PSD) fractions. (C and D) Western blot analysis of wake/sleep PSD fractions.
N = 8 brains for each condition; *P < 0.05, Student’s t test. (E) Wake/sleep phase in vivo two-photon
imaging of SEP-tagged GluA1 in dendritic spines (sGluA1, green) and dsRed (magenta) in layer V
pyramidal neurons of the primary motor cortex. Scale bar, 2 mm. (F) Relative spine GluA1 is significantly
reduced during sleep compared with wake. Spines that contain greater-than-average spine GluA1 during
wake (high GluA1) show a greater change than spines with lower-than-average spine GluA1 (low GluA1).
N = 383 spines from 4 mice; “high GluA1” N = 168 spines; “low GluA1” N = 215 spines; **P < 0.01; ****P <
0.0001; ns, not significant, Student’s t test. (G) Change in spine GluA1 during sleep (sleep/wake ratio)
histogram. More spines show a decrease in spine GluA1 during sleep than an increase. N = 383 spines
from 4 mice, significantly different from a randomized distribution, Kolmogorov-Smirnov test, P < 0.05.
(H and I) Quantitative proteomics of PSD total proteome (H) or phosphoproteome (I). Positive and
negative values indicate PSD enrichment during wake or sleep, respectively. Data obtained from 5 mice
per condition. Error bars, mean ± SEM.
RESEARCH | REPORT
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bicuculline-induced PSD targeting of Homer1a
in cultured neurons (fig. S6), suggesting that NA
is sufficient to limit Homer1a access to the PSD
during scaling. To determine the role of NA in
controlling Homer1a PSD targeting in vivo, we
increased NA by injection of catecholamine reuptake
inhibitor D-amphetamine during the sleep
phase (8 a.m.), when NA is normally low, or
blocked NA signaling with the a and b adrenergic
receptor inhibitors, prazosin and propranolol,
during the wake phase (8 p.m.), when NA is
high (26). Homer1a PSD levels were examined
2 hours after injection. D-amphetamine treatment
significantly reduced Homer1a PSD levels
(Fig. 4, A and B), whereas inhibition of NA
receptors resulted in a significant increase in
Homer1a PSD levels (Fig. 4, C and D). Neither
of the drug treatments affected total cellular
Homer1a protein levels (fig. S8).
To directly test whether Homer1a targeting to
synapses is gated by sleep, mice were sleep deprived
(SD) at the start of the sleep phase for 4
hours, with or without 2.5 hours of recovery sleep
(SD+R). SD significantly increased Homer1a PSD
levels, which was reversed after recovery sleep
(Fig. 4, E and F). AMPAR PSD levels during SD
were the same as control sleep, whereas mGluR5
and AKAP5 levels showed a further decrease (fig.
S8). GluN1/GluN2B NMDA receptor (NMDAR)
subunits also showed a significant decrease in
response to SD (fig. S8). These results suggest
that Homer1a PSD targeting is acutely regulated
by increased sleep need, not strictly by time-ofday
effects, and that the scaling-down pathway
is engaged after prolonged wakefulness. Increased
adenosine signaling mediates sleepiness during
periods of increased sleep need (27). Mice were
treated with vehicle or adenosine A1 receptor–
selective antagonist DPCPX during 4 hours of
SD. DPCPX completely blocked up-regulation of
PSD Homer1a by SD (Fig. 4, G and H). Further
confirming this finding, we found that acute
treatment of cultured neurons with adenosine
or A1 receptor–selective agonist CCPA significantly
increased Homer1a PSD levels (fig. S6),
showing that adenosine, through the A1 receptor,
can promote Homer1a PSD trafficking. Together,
these data suggest that Homer1a serves
as a molecular integrator of arousal and sleep
need through the opposing actions of NA and
adenosine.
Sleep is governed by circadian and homeostatic
components, such that sleep deprivation leads
to accumulated sleep pressure, or sleep need, resulting
in enhanced amplitude of delta oscillations
during subsequent recovery sleep (5, 27).
Mild sleep deprivation or activation of adenosine
A1 receptors caused enhanced targeting of
Homer1a protein to the PSD. Adenosine is a wellknown
mediator of sleep need (27), and polymorphisms
in the Homer1a gene are genetically
associated with the accumulation of sleep need
(19, 20). Adenosine has also been shown to weaken
excitatory synapses (28), which may occur in
coordination with Homer1a (29). Thus, regulated
targeting of Homer1a to synapses by adenosine
provides a molecular mechanism linking the
previously described genetic and chemical basis
for the homeostatic accumulation of sleep need
(5). Based on our data, we propose the following
model. During waking life, learning-related synaptic
activity drives the expression of Homer1a
(15, 16, 19, 20), which is excluded from synapses
due to high levels of NA. At the onset of sleep or
after prolonged wakefulness, NA levels decline
(26) and adenosine increases (27); Homer1a is
then targeted to synapses where it binds mGluR1/5
and remodels/activates mGluR1/5 signaling to
drive synapse weakening (Fig. 4I). This mechanism
links wake-related learning and gene expression
with remodeling of synapses during
sleep. In neurons that underwent long-term
potentiation (LTP) during waking life, scalingdown
during sleep may facilitate memory consolidation
by enhancing signal-to-noise ratios
(5, 6), consistent with a recent study showing
that neurons with the highest firing rates
Diering et al., Science 355, 511–515 (2017) 3 February 2017 3 of 5
Fig. 2. Wake/sleep
remodeling of the
mGluR5 signaling complex
by Homer1a. (A to
D) Western blot analysis
of mGluR5 signaling proteins
in PSD fractions
collected from wild-type
or Homer1a KO mice during
wake or sleep. N = 8
brains for each condition;
*P < 0.05, **P < 0.01,
Student’s t test. (E and F)
Coimmunoprecipitation
of mGluR5, IP3R, Shank,
and Homer1L in forebrain
P2 fractions during wake
or sleep. N = 8; **P <
0.01, Student’s t test.
(G and H) Western blot
analysis of AMPARs in
PSD fractions from
Homer1a KO mice during
wake or sleep. N = 8 brains
for each condition; **P <
0.01, Student’s t test.
(I) Model of Homer1adependent
remodeling of
mGluR5 signaling
complex. Error bars, mean
± SEM.
G
Homer1L
mGluR5
PKCγ
IP3R
Shank
150
125
100
75
50
25
0
sleepPSDlevel(%wake)
Homer1L
mGluR5
PKCγ
IP3R
Shank
300
250
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50
0
sleepPSDlevel(%wake)
*
**
H
A C
E F
I
B D
wake
wake
sleep
sleep
Homer1a -/-
PKCγ
IP3R
Homer1L
mGluR5
pan-Shank
wild type Homer1a -/-
150
100
*
Homer1a
PKCγ
IP3R
Homer1L
mGluR5
wake
wake
sleep
sleep
wild type
pan-Shank
Homer1a
mGluR5
IP3R
Input 2% IP: Homer1
conIP
Homer1L
pan-Shank
wake
wake
sleep
sleep
wake
wake
sleep
sleep 120
100
80
60
40
20
0
120
100
80
60
40
20
0
IP3RcoIP
mGluR5coIP
** **
120
100
80
60
40
20
0
ShankcoIP
140
wake
sleep
wake
sleep
wake
sleep
Homer1a -/-
GluA1
GluA2
GluA3
wake
wake
sleep
sleep
PSD95
wake
sleep
150
125
100
75
50
25
0
PSDlevel(%wake)
PKCγ
mGluR5
IP3R
H1L
+
PSD
wake
PKCγ
mGluR5
IP3R
H1a -
sleep
PSD
GluA1 GluA2 GluA3
Homer1a
**
250kDa
75kDa
50kDa
25kDa
150kDa
150kDa
250kDa
250kDa
50kDa
150kDa
150kDa
250kDa
100kDa
100kDa
100kDa
100kDa
RESEARCH | REPORT
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during wake show the greatest decrease in
activity during sleep (30).
The synapse homeostasis hypothesis (5) (SHY)
posits that synapses undergo a net strengthening
during waking through information coding via
LTP-type mechanisms, followed by a global weakening
during sleep (3–5). Consistent with SHY, we
find that synapses undergo changes in the levels of
synaptic AMPARs and AMPAR phosphorylation
through the wake/sleep cycle. Synaptic changes
during sleep and homeostatic scaling-down are
remarkably similar, and both processes require
Homer1a (10), indicating a shared molecular mechanism.
Our results further suggest that scalingdown
may be up-regulated by adenosine during
sleep deprivation. Sleep significantly improves,
whereas sleep deprivation impairs, memory performance,
suggesting that scaling-down may interact
with sleep-specific patterns of neuronal
activity to facilitate memory consolidation (1, 5).
Whereas homeostatic scaling-down is engaged
in cultured neurons in vitro by bicucullineinduced
hyperactivity (6), scaling-down during
sleep is likely under the control of a coordinated
interplay of neuromodulators, such as NA and
adenosine (26, 27). Recently, it was found that
homeostatic scaling-up in visual cortex (7) only
Diering et al., Science 355, 511–515 (2017) 3 February 2017 4 of 5
50
40
30
20
10
0
vehicle
MTEP/JNJ**
trained
%freezing
20
10
0
5
15 *
vehicle
MTEP/JNJ
novel
%freezing
MTEP/JNJ injection
CFC test novel
Light LightDark
A
B C
50
40
30
20
10
0
vehicle
MTEP/JNJ
trained
%freezing
20
10
0
5
15
vehicle
MTEP/JNJ
novel
%freezing
MTEP/JNJ injection
CFC test novel
LightDark LightDark
D
E F
Fig. 3. mGluR1/5 signaling during the sleep
phase affects memory consolidation. (A to
C) Mice were trained in contextual fear conditioning
at the beginning of the sleep phase,
followed by intraperitoneal injection of vehicle or
MJ. Mice were tested 24 hours later for freezing
responses in the trained context (B) or a novel
context (C). N = 19 vehicle, 19 MJ; *P < 0.05, **P <
0.01, Student’s t test. (D to F) Mice were trained
and injected, as above, before the wake phase.
Mice were tested for freezing responses >36 hours
later at the same time of day as above in the trained
context (E) or a novel context (F). N = 12 vehicle,
12 MJ; P > 0.05, Student’s t test. Error bars,
mean ± SEM.
A C
Homer1PSDlevel
H1a
100
120
80
60
40
20
0
vehicle
α + β
300
250
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150
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50
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Homer1PSDlevel
* 300
250
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0
Homer1PSDlevel
Homer1a
Homer1L
Con
Con
SD
SD
SD+RSD+R
WB: PSD
vehicle
Amph.
E
B D F
G
H
I
H1L
*
H1a
H1L
** **
* con
SD
SD+R
Homer1LHomer1a
Light Dark
4hrs SD
2.5hrs recovery
Con
Con
SD+A1SD+A1
SD+vehSD+veh
Light Dark
4hrs SD
injection
Homer1LHomer1a
300
250
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150
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50
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Homer1PSDlevel
* con
SD+veh
SD+A1
WB: PSD
Homer1a
Homer1L
50kDa
25kDa
50kDa
25kDa
vehiclevehicleAmph.Amph.
WB: PSD
Homer1a
Homer1L
8am injection
Light Dark
10am PSD
50kDa
25kDa
PSD95 100kDa
Homer1a
Homer1L
WB: PSD
vehiclevehicleα+β
α+β
8pm injection
Light Dark
10pm PSD
50kDa
25kDa
PSD95 100kDa
PSD95
PSD95
100kDa
100kDa
Time
wake sleep
Homer1a mRNA expression
PSD targeted Homer1a
Noradrenaline
Homer1a
AMPAR
mGluR5
PSD
arousal: high sleep need: high
wake (10pm) sleep (10am)
Adenosine
Fig. 4. Homer1a PSD targeting is controlled by NA and adenosine signaling. (A and B) Mice were
injected with D-amphetamine (Amph.), 2 mg per kg of weight (mg/kg) at 8 a.m., and forebrain PSD was
prepared 2 hours later at 10 a.m. Amphetamine treatment resulted in a significant decrease in Homer1a
PSD targeting. N = 5; *P < 0.05, Student’s t test. (C and D) Mice were injected with a cocktail of a and b
adrenergic receptor inhibitors, prazosin and propranolol (a + b) (2 mg/kg and 20 mg/kg) at 8 p.m., and
forebrain PSD was prepared 2 hours later at 10 p.m. a + b treatment resulted in a large increase in
Homer1a PSD targeting. N = 4; *P < 0.05, Student’s t test. (E and F) Mice were sleep deprived (SD) for
4 hours (6 to 10 a.m.) by placing mice into a new cage with or without recovery sleep (2.5 hours, SD+R)
in their original cage, followed by forebrain PSD preparation. SD resulted in a significant increase in
Homer1a PSD targeting that was reversed by recovery sleep. N = 8; *P < 0.05, **P < 0.01, Student’s
t test with Bonferroni correction. (G and H) Mice were left in their home cage (con) or subjected to
4 hours SD (6 to 10 a.m.), with two injections, spaced 2 hours apart, of vehicle or adenosine A1
receptor inhibitor DPCPX (A1) (0.5 mg/kg), followed by PSD isolation and Western blot. A1 blockade
prevented up-regulation of Homer1a in the PSD during SD. N = 6; *P < 0.05, Student’s t test with
Bonferroni correction. (I) Model. Homer1a targeting to the PSD is inhibited by noradrenaline and
promoted by adenosine. Homer1a in the PSD binds to mGluR5 and activates signaling to promote
AMPAR removal. Black arrows indicate signaling pathways; green arrows indicate protein trafficking/
translocation. Homer1a mRNA is expressed during wake after synaptic plasticity such as LTP and
learning. Homer1a mRNA expression is low during sleep. The amount of scaling-down during sleep
will be a function of the amount of Homer1a expressed during wake. Error bars, mean ± SEM.
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occurs during wake and is actually inhibited by
sleep (8). These findings suggest that homeostatic
scaling up and down fulfill distinct physiological
functions and are engaged in vivo
by distinct mechanisms, such as wake/sleeppatterned
neuronal activity or chemical modulators.
Our characterization of the large-scale
remodeling of the synaptic proteome during
the wake/sleep cycle will form a foundation to
explore additional mechanisms by which sleep is
able to support cognitive function. Finally, we provide
mechanistic evidence that one physiological
function of homeostatic scaling-down, previously
described only in cultured neurons, is to remodel
synapses during sleep.
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ACKNOWLEDGMENTS
All proteomics data obtained in this study are available in the PRIDE
repository with the data set identifier PXD004537. The authors
thank members of the Huganir laboratory, especially I. Hong, S. Heo,
and N. Hussain, for insightful comments and critical reading of the
manuscript. Image analysis software was developed by R. Cudmore
and D. Linden, Department of Neuroscience, Johns Hopkins
University. G.H.D. is a recipient of a fellowship award from the
Canadian Institutes for Health Research. Funding was provided by an
NIH grant (P50MH100024, RO1NS036715, to R.L.H.), an NIH shared
instrumentation grant (S10OD021844, to A.P.), and the Center for
Proteomics Discovery at Johns Hopkins University. Data are stored
and curated on a secure server located in the Department of
Neuroscience at Johns Hopkins University. Data will be made available
upon request.
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/355/6324/511/suppl/DC1
Materials and Methods
Figs. S1 to S8
Tables S1 and S2
References (31–35)
18 August 2016; accepted 8 December 2016
10.1126/science.aai8355
Diering et al., Science 355, 511–515 (2017) 3 February 2017 5 of 5
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(6324), 511-515. [doi: 10.1126/science.aai8355]355Science
2017)
Worley, Akhilesh Pandey and Richard L. Huganir (February 2,
Graham H. Diering, Raja S. Nirujogi, Richard H. Roth, Paul F.
synapses during sleep
Homer1a drives homeostatic scaling-down of excitatory
Editor's Summary
, this issue p. 457, p. 507; see also p. 511Science
synapse weakening.
to move to excitatory synapses and driveHomer1asleep, noradrenaline levels decline, allowing
accumulates in neurons but is excluded from synapses by high levels of noradrenaline. At the onset of
Homer1aIn awake animals,Homer1a.sleep-wake cycle, driven by the immediate early gene
found that synapses undergo changes in synaptic glutamate receptors during theet al.stable. Diering
sleep. The largest relative changes occurred among weak synapses, whereas strong ones remained
and spine head volume in the mouse brain. They observed a substantial decrease in interface size after
used serial scanning electron microscopy to reconstruct axon-spine interfaceet al.Acsády). De Vivo
strengthening. This is counterbalanced by weakening of synapses during sleep (see the Perspective by
General activity and information processing while an animal is awake drive synapse
Synapse remodeling during sleep
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