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8/2/2019 Compartmentalized Versus Global Synaptic Plasticity... Makino, Malino
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Neuron
Article
Compartmentalized versus Global SynapticPlasticity on Dendrites Controlled by Experience
Hiroshi Makino1,2 and Roberto Malinow1,2,*1Center for Neural Circuits and Behavior, Section of Neurobiology, Division of Biology and Department of Neuroscience,
University of California, San Diego, La Jolla, CA 92093, USA 2Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
*Correspondence: [email protected]
DOI 10.1016/j.neuron.2011.09.036
SUMMARY
Synapses in the brain are continuously modified by
experience, but the mechanisms are poorly under-
stood. In vitro and theoretical studies suggest
threshold-lowering interactions between nearbysynapses that favor clustering of synaptic plasticity
within a dendritic branch. Here, a fluorescently
tagged AMPA receptor-based optical approach
was developed permitting detection of single-syn-
apse plasticity in mouse cortex. Sensory experience
preferentially produced synaptic potentiation onto
nearby dendritic synapses. Such clustering was
significantly reduced by expression of a phospho-
mutant AMPA receptor that is insensitive to
threshold-lowering modulation for plasticity-driven
synaptic incorporation. In contrast to experience,
sensory deprivation caused homeostatic synapticenhancement globally on dendrites. Clustered
synaptic potentiation produced by experience could
bind behaviorally relevant information onto dendritic
subcompartments; global synaptic upscaling by
deprivation could equally sensitize all dendritic
regions for future synaptic input.
INTRODUCTION
Cortical circuits display fine functional and structural organiza-
tion ( Feldmeyer et al., 2002; Lefort et al., 2009; Petreanu et al.,
2009 ) that is carefully established and tuned by sensory experi-ence ( Bender et al., 2003; Buonomano and Merzenich, 1998;
Feldman and Brecht, 2005; Stern et al., 2001 ). Modification of
synapses includes Hebbian plasticity mechanisms where corre-
lated (or uncorrelated) activity leads to structural as well as
functional alternations, such as changes in spine morphology
( Alvarez and Sabatini, 2007 ), or synaptic insertion or removal of
AMPA receptors ( Kessels and Malinow, 2009; Malenka and
Bear, 2004; Newpher and Ehlers, 2008; Nicoll et al., 2006 ). In
parallel to such Hebbian mechanisms, neurons are also equip-
pedwith homeostatic-scaling machinery that may serve to avoid
instability problems of network activity ( Turrigiano and Nelson,
2004 ). Such scaling can globally regulate synaptic strength by
altering the number of AMPA receptors in individual synapses
( Turrigiano et al., 1998 ). Although a number of molecular and
cellular mechanisms underlying these plasticity mechanisms
have been identified, how synapses on a dendritic branch coop-
erate with each other to drive such plasticity is not well
understood. Accumulating in vitro and theoretical evidence suggests that
there exists biochemical compartmentalization on dendrites
that leads to clustered synaptic plasticity ( Branco and Ha ¨ usser,
2010; Govindarajan et al., 2006; Ha ¨ usser and Mel, 2003; Iannella
and Tanaka, 2006; Larkum and Nevian, 2008 ). For example
NMDA receptor-dependent Ca2+ influx caused by a dendritic
spike ( Golding et al., 2002; Schiller et al., 2000; Wei et al.,
2001 ), spread of Ras activity during long-term potentiation
(LTP) ( Harvey et al., 2008 ), and exocytosis of AMPA receptors
into dendritic membrane during LTP ( Lin et al., 2009; Makino
and Malinow, 2009; Patterson et al., 2010; Petrini et al., 2009 )
all occur locally on short stretches of a dendrite and could
contribute to synaptic potentiation at nearby synapses. Indeed,
in hippocampus, LTP at one synapse reduces the threshold for
LTP induction at neighboring synapses ( Govindarajan et al.,
2011; Harvey and Svoboda, 2007 ). Moreover, there is a trend
that newly formed spines in hippocampal cultures appear in
close proximity to activated spines during LTP ( De Roo et al.,
2008 ), potentially leading to clustering of synaptic enhancement.
Such clustered synaptic potentiation could bind behaviorally
relevant inputs onto dendritic subcompartments and improve
storage capacity of individual neurons ( Poirazi and Mel, 2001 ).
Despite such studies, direct evidence for clustered synaptic
plasticity in vivo is still lacking, owing to difficulties in online or
retrospective identification of synaptic plasticity at individual
synapses. In this study, we have developed an AMPA
receptor-based optical approach to monitor recent history of synaptic plasticity induced in vivo through sensory experience
or deprivation. We show that synaptic potentiation, revealed by
experience-driven GluR1 incorporation into synapses, is clus-
tered on short stretches of dendrites. Such clustered synaptic
potentiation is effectively eliminated when animals are deprived
of sensory experience or by expressing AMPA receptors insen-
sitive to modulation for plasticity-driven incorporation into syn-
apses. In contrast, homeostatic plasticity, revealed by synaptic
GluR2 incorporation caused by sensory deprivation, occurs
globally on dendrites, showing little evidence for clustering.
Such coordinated modification of synapses could implement
a framework for circuit development and refinement.
Neuron 72, 1001–1011, December 22, 2011 ª2011 Elsevier Inc. 1001
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RESULTS
Identification of Experience-Driven Synaptic Plasticity
at Individual Spines
To examine experience-dependent plasticity at individual
synapses, we monitored the synaptic incorporation of fluores-
cently tagged AMPA receptor subunits, GluR1 and GluR2. To
achieve acute expression of recombinant genes in a smallnumber of neurons, we used a Cre/loxP-mediated inducible
expression system where the transcription of genes of interest
is regulated by a floxed stop cassette ( Matsuda and Cepko,
2007 ). In this system, Cre expression is dependent on 4-hydrox-
ytamoxifen (4-OHT). Once expressed, Cre drives removal of the
(floxed) stop cassettes, permitting expression of genes of
interest ( Figure 1 A). We used in utero electroporation to deliver
three DNA constructs into layer 2/3 pyramidal neurons of the
developing mouse barrelfield: (1)a floxed stop cassettefollowed
by thegene for GluR1 (or GluR2) taggedwith a pH-sensitive form
of green fluorescent protein (Super Ecliptic pHluorin, SEP) on the
N terminus; (2) a floxed stop cassette followed by the gene for
DsRed, a red cytoplasmic marker; and (3) the 4-OHT-dependent
Cre recombinase-expressing plasmid, pCAG-ERT2CreERT2.
Animals were injected intraperitoneally (i.p.) with 4-OHT at post-
natal day (P) 11, and coronal brain slices were prepared at P13
( Figure 1B). A small number of neurons (<1% of layer 2/3
neurons) displayed expression of SEP-GluR1 (or SEP-GluR2)
and DsRed ( Figure 1C). Animals that did not receive 4-OHT
showed no detectable expression (four animals; data notshown), indicating little leak in the expression system.
Sensory experience, controlled by trimming or leaving intact
an animal’s whiskers ( Feldman and Brecht, 2005 ), can drive
GluR1 into synapses between layer 4 and layer 2/3 neurons
through an LTP-like process ( Clem and Barth, 2006; Takahashi
et al., 2003 ). We wished to determine whether synaptic incorpo-
ration of SEP-GluR1 can be monitored optically using dual-
channel two-photon microscopy. We measured SEP-GluR1
enrichment in dendritic spines, which is the spine SEP signal
normalized for spine area and for neuronal expression level of
the SEP-tagged protein (see Experimental Procedures ). We
focused on basal dendrites of layer 2/3 pyramidal neurons
0
0.8
1.2
1.6
CAG promoter ERT2 Cre ERT2 polyA
DsRed polyACAG promoter
loxP
CAG promoter DsRed polyA
loxP loxPSTOP
CAG promoter SEP polyA
loxP
AMPA receptorCAG promoter SEP polyA
loxP loxP
AMPA receptor
STOP
A
4-Hydroxytamoxifen(4OHT)
Constructs
pCALNL-SEP-GluR1pCALNL-SEP-GluR2
pCALNL-DsRed
pCAG-ERT2CreERT2
D
SEP-GluR1DsRed
m5
Enriched spineNon-enriched spine
SEP-GluR2DsRed
m5
E15In utero electroporation
P13Image
P114OHT Whisker manipulation
P11B C
L2/3
L4
L5
0
0.2
0.4
0.6
0.8
1
1.2
0 0.4 0.8 1.2 1.6
E Whisker intactWhisker trimmed
Enrichment value
C u m u l a t i v e p r o b a b i l i t y
GluR1p < 0.001
0
0.2
0.4
0.6
0.8
1
1.2
0 0.5 1 1.5 2 2.5 3
Enrichment value
C u m u l a t i v e p r o b a b i l i t y
Whisker intactWhisker trimmed
F
GluR2p < 0.001
G
E n r i c h
m e n t v a l u e
GluR1 GluR2
Whisker intactWhisker trimmed
Enriched spineNon-enriched spine
Figure 1. Identification of Experience-
Driven Synaptic Plasticity at Individual
Spines of L2/3 Pyramidal Cells in the Barrel
Cortex
(A) Expression system. Intraperitoneal injection of
4-OHT drives expression of Cre recombinase,which induces expression of AMPA receptor
subunits and DsRed (cytoplasmic marker) by
removing the transcriptional stop cassettes
located in front of the genes of interest.
(B) Experimental design. The DNA plasmids were
in utero electroporated at E15 in the right barrel
cortex. When animals reached P11, 4-OHT was
injected i.p., and whiskers were either left intact or
all trimmed daily. At P13, acute slices were
prepared, and basal dendrites of L2/3 pyramidal
cells were imaged with two-photon microscopy.
(C) Example of sparse expression of SEP-GluR1
and DsRed in L2/3 pyramidal cells in the barrel
cortex.
(D) Left panels show examples of SEP-GluR1 and
DsRed-expressing neurons in whisker-intact and
whisker-trimmed animals. Right panels are
examples of SEP-GluR2 and DsRed-expressing
neurons in whisker-intact and whisker-trimmed
animals. Arrowheads indicate enriched spines,
whereas arrows indicate nonenriched spines.
(E) Spine enrichment values for SEP-GluR1 in
whisker-intact (n = 2,701 spines, 23 cells, 11
animals) and whisker-trimmed (n = 1,878 spines,
17 cells, 7 animals) animals (p < 10À17, Kolmo-
gorov-Smirnov test).
(F) Spine enrichment values for SEP-GluR2 in
whisker-intact (n = 1,057spines,8 cells, 5 animals)
and whisker-trimmed (n = 1,226 spines, 8 cells, 4
animals) animals (p < 10À9, Kolmogorov-Smirnov
test).
(G) Spine enrichment values obtained from (E) and
(F) to illustrate the difference between SEP-GluR1
and SEP-GluR2 spine enrichment (mean ± SEM).
Neuron
Compartmentalized and Global Synaptic Plasticity
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because they receive the majority of synaptic inputs ( Feldmeyer
et al., 2002; Petreanu et al., 2009 ). Consistent with electrophys-
iological studies ( Clem and Barth, 2006; Takahashi et al., 2003 ),
following 2 days of 4-OHT-driven expression, SEP-GluR1 spine
enrichment was higher in animals with whiskers intact (0.84 ±0.005, n = 2701 spines) compared with animals with whiskers
trimmed (0.77 ± 0.006, p < 10À17, n = 1878 spines; Figures 1D,
1E, and 1G).
Although LTP is thought to depend on the GluR1 AMPA
receptor subunit, GluR2 is not required for LTP ( Hayashi et al.,
2000; Jia et al., 1996; Zamanillo et al., 1999 ) but is required for
homeostatic plasticity produced by deprivation of activity or
sensory input ( Gainey et al., 2009 ). We examined the synaptic
incorporation of SEP-GluR2 under similar (2 day expression)
conditions. In contrast to SEP-GluR1, following 2 days of 4-
OHT-driven expression, whisker-trimmed animals had increased
spine enrichment of SEP-GluR2 (1.43 ± 0.01, n = 1226 spines)
compared to whisker-intact animals (1.30 ± 0.01, p < 10À9, n =
1057 spines; Figures 1D, 1F, and 1G), consistent with the viewthat reduced input activity produces homeostatic synaptic
strengthening that is controlled by GluR2 ( Gainey et al., 2009 ).
Spine Enrichment of AMPA Receptors as an Indicator
for Plasticity
To test if spine enrichment of SEP-tagged AMPA receptors was
a good estimate of their synaptic incorporation, we used fluores-
cence recovery after photobleaching ( Makino and Malinow,
2009 ). Because synaptic receptors are relatively immobile ( Heine
et al., 2008; Makino and Malinow, 2009 ), the recovery of fluores-
cence after photobleaching a spine containing synaptic
SEP-tagged AMPA receptors is incomplete. Following 2 days
of 4-OHT-driven expression, the fraction of SEP-GluR1 spine
fluorescence that failed to recover (immobile fraction) correlated
well with the SEP-GluR1 spine enrichment (r = 0.58, p < 0.001,
n = 29 spines; Figures 2 A and2B). In contrast, immobile fractions
of spine SEP-GluR1 were not correlated with spine size (r = 0.12,
p = 0.53, n = 29 spines; Figure 2C), consistent with the view that
spine size is a consequence of plasticity integrated over a period
longer than the 2 day expression period of recombinant recep-
tors. Indeed, expression of SEP-GluR1 for longer periods (e.g.,
4 days) produced spines in which the SEP-GluR1 spine enrich-
ment was correlated with spine size (see Figures S1 A–S1D
available online). Similar to SEP-GluR1, following 2 days of
4-OHT-driven more expression, there was a strong relation
between SEP-GluR2 immobile fractions and SEP-GluR2 spine
enrichment (r = 0.66, p < 0.003, n = 19 spines; Figure 2D), butnot with spine size (r = 0.14, p = 0.56, n = 19 spines; Figure 2E).
These results indicate that experience- or deprivation-driven
synaptic plasticity can be detected using fluorescently tagged
AMPA receptors.
To test further the view that spine enrichment of SEP-tagged
AMPA receptors serves as an indication of their synaptic incor-
poration, we performed glutamate uncaging onto spines that
had various levels of SEP-GluR1 enrichment. We obtained
whole-cell recordings from neurons expressing recombinant
receptors and measured AMPA receptor-mediated responses
from focally applied glutamate on spines ( Figure 2F; see Exper-
imental Procedures ). We recorded responses at positive
(VH = +40mV) and negative (VH = À60mV) holding potentials;
their ratio (current at VH = +40 mV/current at VH = À60 mV) is
the rectification index. Because recombinant receptors form
homomeric receptors, they display little outward current at posi-
tive potentials and, thus, a low rectification index. We founda correlation between rectification indices and enrichment
values for different spines (r = À0.59, p < 0.03, n = 14spines; Fig-
ure 2G), consistent with the view that enrichment value is a good
measure for synaptic incorporation of recombinant SEP-tagged
AMPA receptors.
Experience-Dependent Clustering of Synaptic
Potentiation on Dendrites
To examine if nearby spines on individual dendrites displayed
similar levels of plasticity, we calculated the correlation coeffi-
cient of SEP-GluR1 spine enrichment for neighboring spines
(see Experimental Procedures; Figure 3 A) following 2 day tran-
sient expression. Neighboring spines showed a significant posi-
tive correlation value (0.14 ± 0.03, p < 10À5, n = 95 dendrites) indendrites from animals with whiskers intact ( Figures 3B–3D and
S2 A). This correlation value between neighboring spines was
significantly greater than that observed in whisker-trimmed
animals (0.003 ± 0.03, p < 0.009 with Bonferroni correction,
n = 68 dendrites; Figures 3D and S2 A). These results indicate
that sensory experience drives coordinated potentiation onto
nearby synapses.
It is possible that some of the dendritic segments examined
received little plasticity during the period of SEP-GluR1 expres-
sion (see below). Thus, we wished to determine what fraction of
dendriticsegmentsshowed a significantcorrelation in the enrich-
ment values of neighboring spines. For each dendritic segment
we calculated the correlation coefficient of neighboring enrich-
ment values and compared this to a value obtained by random
shuffling of the enrichment values for that dendritic segment. If
the correlation coefficient for the dendritic segment was greater
than 95% of the correlation coefficients obtained from randomly
shuffled enrichment values, that dendrite was deemed to have
a significant correlation of nearby enrichmentvalues. In dendrites
obtained from animals with whiskers intact, 28 of 95 (29%)
dendrites displayed a significant correlation between neigh-
boring spine enrichment values ( Figure 3E). The correlation coef-
ficient for enrichment values in neighboring spines in dendrites
with significant correlation was 0.36 ± 0.04 ( Figure 3F). In
dendrites obtained from animals with whiskers trimmed, only 5
of 68 (7%) were significant. The fraction of dendrites with signifi-
cant correlation with nearby spines was greater in those obtainedwith whiskers intact (p < 0.0007, Fisher’s exact test).
Sensory Deprivation Causes Global Synaptic Upscaling
on Dendrites
Inactivity or sensory deprivation produces homeostatic synaptic
upscaling that is global throughout a cell and depends on GluR2
( Gainey et al., 2009; Turrigiano, 2008 ). We, thus, testedthe effect
of sensory deprivation on the correlation of enrichment values
in spines from cortical neurons expressing SEP-GluR2, using
the same temporally regulated expression system. In animals
with whiskers trimmed for 2 days, nearby spines failed to
show significant positive correlation (0.02 ± 0.03, p = 0.46,
Neuron
Compartmentalized and Global Synaptic Plasticity
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n = 45 dendrites; Figures 3D and S2B); this value was signifi-cantly different from that found in animals with whiskers intact
expressing SEP-GluR1 (p < 0.05 with Bonferroni correction;
n = 95 dendrites) but not different from that observed in animals
with whiskers intact expressing SEP-GluR2 ( À0.05 ± 0.03, p =
0.11, n = 44 dendrites; Figures 3D and S2B). These results
indicate that synaptic incorporation of GluR2 caused by
homeostatic plasticity occurs globally on dendrites with little
compartmentalization.
Reconstruction of Single Neurons
To gain more insight into the distribution of clustered plasticity in
a whole neuron, we measured enrichment values for all identifi-
able spines in individual neurons ( Figures 4 A, 4B, S3 A, andS3B). For a neuron expressing SEP-GluR1 in a whisker-intact
animal, of the 1,078 spines we considered the spines with the
highest 15% of enrichment values. Spines with these values
appeared not to be randomly distributed. Many of the highly en-
riched spines were seen at the very tip of dendrites (p < 0.0003,
n = 161 spines, compared to nonenriched spines, n = 917 spines;
Figures 4 A and 4C), suggesting that terminal dendritic segments
were particularly sensitive to plasticity. Indeed, when we exam-
ined all of the data obtained from individual dendritic segments
expressing GluR1, we noted an increase in enrichment as a
function of distance from cell body ( Figure S3C). We wished
to test if the occurrence of highly enriched spines was more
-0.4
-0.2
0
0.2
0.4
0.6
0.8
0 0.5 1 1.5 G l u
R 2 i m m o b i l e f r a c t i o n
Spine size
-0.4
-0.2
0
0.2
0.4
0.6
0.8
0 0.5 1 1.5 2 2.5Enrichment value
G l u
R 2 i m m o b i l e f r a c t i o n
Whisker intactWhisker trimmed
Whisker intactWhisker trimmed
D E
-0.4
-0.2
0
0.20.4
0.6
0.8
0 0.5 1 1.5-0.4
-0.2
0
0.20.4
0.6
0.8
0 0.5 1 1.5 2 2.5 G l u R 1 i m m o b i l e f r a c t i o n
Enrichment value
Whisker intactWhisker trimmed
Spine size
G l u R 1 i m m o b i l e f r a c t i o n
Whisker intactWhisker trimmed
B C
p < 0.001r = 0.58
p = 0.53r = 0.12
p < 0.003r = 0.66
p = 0.56r = 0.14
+25 min+3 min-10 min
m5
A SEP-GluR1 DsRed
ab
0
0.1
0.2
0.3
0.2 0.4 0.6 0.8 1
Enrichment value
a
b
G l u R 1 i m m o b i l e
f r a c t i o n
Uncaging pulse
m5
F
+40 mV
-60 mV
a b
0
0.1
0.2
0.3
0.4
0.5
0.6
0 1 2 3
Enrichment value
R e c t i f i c a t i o n
( + 4 0 m V / - 6 0 m V )
b
a
20 ms
10 pA
G
m
Dodt imageSEP-GluR1DsRed
50
p < 0.03r = -0.59
Figure 2. Spine Enrichment of AMPA Receptors as an Indicator for Their Synaptic Localization
(A) Exampleof fluorescencerecovery after photobleachingof spines expressing SEP-GluR1.Top panels showtwo spines (‘‘a’’ and ‘‘b,’’ indicatedas arrowheads)that were simultaneously photobleached, and recovery of the SEP-GluR1 fluorescence was monitored at 25 min to measure the immobility of the receptor.
Bottom graph shows enrichment values and GluR1 immobile fractions of the two spines (‘‘a’’ and ‘‘b’’).
(B) Correlation between SEP-GluR1 spine enrichment values and immobile fractions of spine SEP-GluR1 (r = 0.58, p < 0.001, n = 16 spines, 4 cells, 4 animals for
whisker-intact; and n = 13 spines, 3 cells, 2 animals for whisker-trimmed animals).
(C) No correlation between spine size and immobile fractions of spine SEP-GluR1 (r = 0.12, p = 0.53, n = same as B).
(D) Correlation between SEP-GluR2 spine enrichment values and immobile fractions of spine SEP-GluR2 (r = 0.66, p < 0.003, n = 10 spines, 3 cells, 3 animals for
whisker-intact; and n = 9 spines, 2 cells, 2 animals for whisker-trimmed animals).
(E) No correlation between spine size and immobile fractions of spine SEP-GluR2 (r = 0.14, p = 0.56, n = same as D).
(F) Example of a whole-cell recording and glutamate uncaging at a spine with high SEP-GluR1 enrichment. Uncaging-evoked AMPA receptor-mediated post-
synaptic currents are shown in (G) spine b.
(G) Left view shows correlation between SEP-GluR1 spine enrichment values and rectification indices (r = À0.59, p < 0.03, n = 14 spines, 9 cells, 5 animals).
Rectification indices were measured as amplitude of AMPA current at +40mV/amplitude of AMPA current at À60mV. Right view shows example traces of
glutamate uncaging-evoked AMPA receptor-mediated postsynaptic currents at spines with different SEP-GluR1 enrichment values.
Neuron
Compartmentalized and Global Synaptic Plasticity
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likely to occur in neighboring spines. In this neuron, of the 161
spines showing the highest 15% enrichment, 50 were neigh-
boring spines. When the enrichment values were randomly
shuffled, there was on average 24 pairs of neighboring spines
with enrichment values in the top 15% (p < 0.001; Figure 4D).
We conducted similar analysis considering the top 5% or 10%
of enrichment values. In all cases the number of neighboring
spines with highly enriched values in the analyzed neuron was
significantly greater than what was observed when the enrich-
ment values were randomly shuffled ( Figures S3 A and S3B).
We next examined SEP-GluR2 on a fully reconstructed neuron
from a whisker-trimmed animal. In this case highly enriched
spines were not found on distal regions, as was the case for
SEP-GluR1 ( Figure4B).Therewas a tendency forhighly enriched
spines (n = 150) to be proximal relative to nonenriched spines
(p < 0.005, n = 851 spines; Figures 4C, S3B, and S3C). We
also noted that neighboring spines were no more likely to have
high enrichment values than randomly shuffled values (p =0.29; Figures 4E, S3 A, and S3B). Taken together, these results
suggest that there are distinct trafficking patterns produced by
experience-driven synaptic potentiation and deprivation-driven
synaptic upscaling.
Clustered Synaptic Potentiation with Heteromeric
AMPA Receptors
The data above suggest that the clustering of plasticity is
observed for GluR1, but not GluR2, consistent with their depen-
dence on LTP and experience ( Hayashi et al., 2000; Takahashi
et al., 2003; Zamanillo et al., 1999 ). However, when expressed
alone, these AMPAreceptorsubunitsform homomericreceptors,
which normally comprise a small proportion of endogenously
expressed receptors ( Wenthold et al., 1996 ).To examine thetraf-
ficking of heteromeric receptors, which constitute the predomi-
nant species of receptors ( Wenthold et al., 1996 ), we transiently
coexpressed SEP-GluR1 with untagged-GluR2, or untagged-
GluR2 and SEP-GluR3 (see Experimental Procedures ). We first
confirmed, using electrophysiological measures, that hetero-
meric receptors were formed when expressing SEP-GluR1 with
GluR2. We obtained whole-cell recordings from neurons ex-
pressing recombinant receptors and measured responses from
focally applied glutamateon spines ( Figure 5 A; see Experimental
Procedures ). Homomeric receptors display inward rectification,
which was observed in neurons expressing SEP-GluR1 (0.28 ±
0.02, n = 15 spines; Figure 5B). However, no such inward rectifi-
cation was observed from neurons expressing SEP-GluR1 and
GluR2 (0.49 ± 0.03, p < 0.00003, n = 13 spines; Figure 5B), indi-
cating that heteromeric receptors were formed.
We examined in animals with whiskers intact the spine enrich-ment values in neurons transiently expressing SEP-GluR1 and
GluR2 ( Figures 5C and 5E). Spine enrichment of SEP-GluR1/
GluR2 heteromeric receptors (0.84 ± 0.006, n = 1865 spines)
did not differ from that of SEP-GluR1 homomeric receptors
(0.84 ± 0.005, p = 0.70, n = 2701 spines; Figures 5C, 5E, S4 A,
and S4C). Similarly, spine enrichment of GluR2/SEP-GluR3
(1.29 ± 0.01, n = 1390 spines) was not different from that of
SEP-GluR2 (1.30 ± 0.01, p = 0.08, n = 1057 spines; Figures
5D, 5E, S4B, and S4C). As with homomeric receptors, the immo-
bile fractions of heteromeric receptors correlated with spine
enrichment (r = 0.72, p < 10À5, n = 31 spines; Figure 5F), but
not with spine size (r = 0.04, p = 0.85, n = 31 spines; Figure 5G).
00.20.40.60.8
11.2
1.41.6
0 5 10 15 20 25 30 35Spine number
E n r i c h m e n t v
a l u e
CA
Potentiated spine
Distributed Clustered
or
B
Enriched spine
SEP-GluR1DsRed
m5
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0 2 4 6 8 10Neighboring spine
C o r r e l a i t o n c o e f f i c i e n t
F
0
5
10
15
20
Correlation coefficient
at 1st neighbor
F r e q u e n c y
0-0.6 0.6
p < 0.05p > 0.05
ED
-0.1
0
0.1
0.2ns**
C o r r e l a i t o n c o e f f i c i e n t
I n t a c t
T r i m m e d
I n t a c t
T r i m m e d
GluR1 GluR2
*
Figure 3. Experience-Dependent Clustering
of Synaptic Potentiation and Global Syn-
aptic Upscaling Driven by Sensory Depriva-
tion
(A) Schematic of distributed synaptic potentiation
and clustered synaptic potentiation.(B) Example of clustered synaptic SEP-GluR1
enrichment in a basal dendrite of a L2/3 pyramidal
cell in a whisker-intact animal.
(C) Profile of SEP-GluR1 spine enrichment along
the dendrite shown in (B). Line indicates a running
average.
(D) Correlation coefficientsat neighboring synapses
of dendrites expressing SEP-GluR1 in whisker-
intact (n = 95 dendrites, 23 cells, 11 animals) and
whisker-trimmed (n = 68 dendrites, 17 cells, 7
animals) animals (**p < 0.009, t test with Bonferroni
correction; mean ± SEM), and SEP-GluR2 in
whisker-intact (n = 44 dendrites, 8 cells, 5 animals)
and whisker-trimmed (n = 45 dendrites, 8 cells, 4
animals) animals (*p < 0.05, t test with Bonferroni
correction; ns, nonsignificant; mean ± SEM).
(E) Histogram of correlation coefficients at neigh-
boring synapses for dendrites expressing SEP-
GluR1in whisker-intactanimals(n = 95 dendrites,23
cells, 11 animals). For each dendrite, p value for its
correlation coefficient was obtained by calculating
the likelihood of obtaining such a correlation coeffi-
cient from randomly shuffled spinesof thatdendrite.
(F)Autocorrelation of dendriteswithp < 0.05in (E)as
a function of spine lag (mean± SEM).
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For neurons expressing SEP-GluR1 and GluR2, there was
a significant positive correlation in enrichment values between
neighboring spines in animals with whiskers intact (0.12 ± 0.03,
p < 0.0005, n = 59 dendrites; Figures 5C, 5H, 5I, and S2C). Of
59 dendrites, 12 (20%) showed significant near-neighborcorrela-
tions ( Figure 5J), which reached a value of0.32 ± 0.04( Figure5K).
For neurons expressingGluR2 andSEP-GluR3,the distribution of enrichment values mirrored that found in neurons expressing
SEP-GluR2: neighboring spines displayed no significant correla-
tion in enrichment values ( À0.005 ± 0.02, p = 0.85, n = 47
dendrites; Figures 5I and S2C). These results indicate that the
effect of experience on the distribution of heteromeric SEP-
GluR1/GluR2 and GluR2/SEP-GluR3 receptors is similar to that
observed in homomeric SEP-GluR1 or SEP-GluR2 receptors.
Receptor Modulation Sensitivity Controls Clustered
Synaptic Potentiation
The results presented above indicate that neural activity
patterns onto cortical neurons driven by sensory experience
13
2
4
3
0
0.1
0.2
0.3
0.4
0.5
Shuffled
0 50# Enriched spine pair
Raw data
P r o b a b i l i t y
p = 0.29
E
GluR2
D
P r o b a
b i l i t y
0
0.1
0.2
0.3
0.4
0.5
50# Enriched spine pair
Shuffled
Raw data
p < 0.001
GluR1
C
0
0.2
0.40.6
0.8
1
1.2
0 100 200 C u m u l a t i v e
p r o b a b i l i t y
GluR1 enriched ***GluR1 non-enriched
GluR2 non-enrichedGluR2 enriched **
Distance from soma ( )m
GluR2
GluR1
SEP-GluR2 & DsRed from whisker-trimmed animalB
SEP-GluR1 & DsRed from whisker-intact animalA
m5
m5 m50
m50
1
4
2
1 3
2 4
1
2
3
4
0
Figure 4. Reconstruction of Single Neurons
(A) Left, reconstruction of a neuron expressing
SEP-GluR1 from a whisker-intact animal. Insets
are examples of dendritic segments. Right, spines
with the highest 15% enrichment are shown in red
(n = 161 spines) and the rest in gray (n = 917spines).
(B) Left, reconstruction of a neuron expressing
SEP-GluR2from a whisker-trimmed animal. Insets
are examples of dendritic segments. Right, spines
with the highest 15% enrichment are shown in red
(n = 150 spines) and the rest in gray (n = 851
spines).
(C) Absolute distance from the soma of enriched
and nonenriched spines for the GluR1 (***p <
0.0003, Kolmogorov-Smirnov test) and GluR2-
expressing neurons (**p < 0.005, Kolmogorov-
Smirnov test).
(D) Histogram of the number of neighbor pairs with
high SEP-GluR1 enrichment obtained from shuf-
fled spines. There are 50 such pairs in the original
data (dashed line; p < 0.001).
(E) Histogram of the number of neighbor pairs with
high SEP-GluR2 enrichment obtained from shuf-
fled spines. There are 24 such pairs in the original
data (dashed line; p = 0.29).
produce clustered potentiation of nearby
synapses. Such patterning could be
produced by LTP-like processes, which
have been shown in in vitro systems to
lower threshold of nearby spines for plas-
ticity ( Govindarajan et al., 2011; Harvey
and Svoboda, 2007; Harvey et al.,
2008 ). One model to explain such nearby
threshold lowering is the following: nor-
mally, an individual synapse is potenti-
ated (and accumulates GluR1) when it
receives sufficient presynaptic activity
paired with postsynaptic depolarization
(the latter provided by close or distant synapses). Such point
potentiation would activate intracellular signal transduction
pathways (e.g., Ras; Harvey et al., 2008 ) that could activate
downstream kinases leading to phosphorylation of GluR1 at
nearby regions (within $5 mm). Receptors at these nearby
regions would now have lower threshold for becoming incorpo-
rated into synapses (for as long as GluR1 maintains a phosphor-ylated status). To test for this possibility, we expressed SEP-
GluR1 with mutations at two phosphorylation sites (S831A and
S845A) in the cytoplasmic segment (designated GluR1AA; Fig-
ure 6 A). These mutations on GluR1 render the receptor insensi-
tive to modulation by protein kinases at these sites. Phosphory-
lation at these sites is known to lower the threshold for GluR1
incorporation into synapses during LTP ( Hu et al., 2007 ). We
examined the distribution of spine enrichment values in animals
with whiskers intact transiently expressing SEP-GluR1AA. The
average spine enrichment of SEP-GluR1AA (0.84 ± 0.007, n =
1584 spines) was similar to that of SEP-GluR1 (0.84 ± 0.005,
p = 0.14, n = 2701 spines; Figure 6B). This is consistent with
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the previous observation that mice in which GluR1 has been re-
placed with GluR1AA have the same number of synaptic AMPA
receptors as wild-type mice ( Lee et al., 2003 ). Apparently, the
reduced synaptic incorporation resulting from the lost
threshold-lowering effects of phosphorylation is offset by the
reduced synaptic receptor removal produced by the absent
LTD also described for this GluR1 mutant ( Lee et al., 2003 ).
Immobile fractions of SEP-GluR1AA were well correlated with
its enrichment in spines (r = 0.87, p < 0.00003, n = 15 spines; Fig-
ure 6C), but not with spine size (r = 0.29, p = 0.29, n = 15 spines;
Figure 6D). Unlike SEP-GluR1, the enrichment values at neigh-
boring spines were not positively correlated (0.03 ± 0.03,
p = 0.41, n = 62 dendrites), and were significantly different from
the correlation value displayed by neighboring spines in animals
with whiskers intact expressing SEP-GluR1 (p < 0.04 with
Bonferroni correction, n = 95 dendrites; Figures 6E and S2D).
These data suggest that removing trafficking modulation signals
on GluR1 effectively eliminates the dendritic clustering of
synaptic potentiation displayed by SEP-GluR1.
Finally, we examined if clustering of GluR1 synaptic delivery
could be observed in older animals ( Figures S5 A–S5C). In this
group of animals, electroporation was conducted in utero, and
the induction (injection with 4-OHT) was initiated at P34 or
P35. Two days later, brain slices were prepared and neurons
0
0.2
0.4
0.6
0.8
1
1.2
0 0.5 1 1.5 2 2.5 3
+40 mV
-60 mV
GluR1 GluR1/2
20 ms
10 pA1 1 1 2
A
Uncaging pulse 0
0.1
0.20.3
0.4
0.5
0.6
GluR1/1 GluR1/2
***
15 13
D E
B
m5
SEP-GluR1GluR2DsRed
m50Dodt image
m5
SEP-GluR3GluR2
DsRed
C u m u l a t i v e p r o b a b i l i t y
GluR1/2GluR2/3
Enrichment value
p < 0.001
SEP-GluR1
GluR2DsRed
C
m10
-0.4
-0.2
0
0.2
0.4
0.6
0.8
0 0.5 1 1.5 2 2.5Enrichment value
I m m o b i l e f r a c t i o n
GluR1/2GluR2/3
p < 0.001r = 0.72
-0.4
-0.2
0
0.2
0.4
0.6
0.8
0 0.2 0.4 0.6 0.8 1
I m m o b i l e f r a c t i o n
Spine size
GluR1/2GluR2/3
p = 0.85r = 0.04
F G
R e c t i f i c
a t i o n
( + 4 0 m V /
- 6 0 m V )
H
00.20.40.60.8
11.21.41.6
0 10 20 30 40 50 60
E n r i c h m e n t v a l u e
Spine number
-0.1
0
0.1
0.2
GluR1/2 GluR2/3
**
C o r r e l a i t o n c o e f f i c i e n t
I J
0
5
10
15
20
0-0.6 0.6
p < 0.05p > 0.05
F r e q u e n c y
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0 2 4 6 8 10Neighboring spine
C o r r e l a i t o n c o e f f i c i e n t
K
Correlation coefficient
at 1st neighbor
EP-GluR1lu 2
DsRed
m0Dodt image
Figure 5. Clustered Synaptic Potentiation with Heteromeric AMPA Receptors
(A) Example of a whole-cell recording and glutamate uncaging at a spine containing GluR1/2 heteromeric AMPA receptors.
(B) Left views show example traces of glutamate uncaging-evoked AMPA receptor-mediated postsynaptic currents at spines expressing GluR1/1 and GluR1/2.
Right views illustrate rectification indices for GluR1/1 homomeric (n = 15 spines, 10 cells, 8 animals) and GluR1/2 heteromeric (n = 13 spines, 8 cells, 5 animals)
AMPA receptorsat singlespines(***p< 0.00003, t test;mean± SEM).Rectificationindicesweremeasured asamplitude of AMPA current at+40 mV/amplitude of AMPA current at À60mV.
(C) Example of clustered synaptic potentiation with GluR1/2 heteromeric AMPA receptors in a whisker-intact animal. Arrowheads indicate enriched spines.
(D) Example of a GluR2/3 heteromeric AMPA receptor-expressing neuron in a whisker-intact animal.
(E) Spine enrichment values for GluR1/2 (n = 1,865 spines, 11 cells, 8 animals) and GluR2/3 (n = 1,390 spines, 14 cells, 5 animals) heteromeric AMPA receptors
(p < 10À166, Kolmogorov-Smirnov test).
(F) Correlation between spineenrichmentvalues and spine immobile fractions of heteromeric AMPAreceptors (r = 0.72,p < 10À5, n = 16spines,3 cells,3 animals
for GluR1/2; and n = 15 spines, 3 cells, 3 animals for GluR2/3).
(G) No correlation between spine size and spine immobile fractions of heteromeric AMPA receptors (r = 0.04, p = 0.85, n = same as F).
(H) Profile of SEP-GluR1/2 spine enrichment along the dendrite shown in (C). Line indicates a running average.
(I) Correlation coefficients at neighboring synapsesof dendritesexpressingGluR1/2 (n = 59 dendrites, 11 cells, 8 animals)and GluR2/3 (n = 47 dendrites, 14 cells,
5 animals) heteromeric AMPA receptors (**p < 0.005, t test; mean ± SEM).
(J) Histogram of correlation coefficients at neighboring synapses for dendrites expressing SEP-GluR1/2 in whisker-intact animals (n = 59 dendrites, 11 cells,
8 animals). p values are calculated as in Figure 3E.
(K) Autocorrelation of dendrites with p < 0.05 in (J) as a function of spine lag (mean ± SEM).
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were imaged ( Figure S5 A). Spine enrichment values were signif-
icantly higher (1.27 ± 0.01, n = 996 spines) than those seen in
younger animals (0.84 ± 0.005, n = 2701 spines, p < 10À148; Fig-
ure S5B), due to a large reduction in SEP-GluR1 on dendritic
membrane (data not shown). Correlation of enrichment values
between neighboring spines was significantly different from
zero (0.16 ± 0.04, p < 0.002, n = 24 dendrites; Figure S5C). Of
24 dendritic segments, 10 (42%) displayed significant near-
neighbor correlations, which reached a value of 0.27 ± 0.04.
These observations indicate that experience-driven clustering
of synaptic potentiation also occurs in older animals.
DISCUSSION
In this study, we have examined the spatial distribution of plas-
ticity on neuronal dendrites produced as a result of sensory
experience. We used temporally restricted expression of SEP-tagged glutamate receptors to identify individual synapses that
had recently undergone plasticity in vivo. The spine enrichment
correlated well with the immobile fraction as well as the electro-
physiological property of tagged receptor, indicating that spine
enrichment corresponds to synaptically incorporated receptors.
Experience increased the synaptic enrichment of SEP-GluR1,
whereas deprivation increased the synaptic enrichment of
SEP-GluR2, supporting their use as indicators of plasticity. The
trafficking of SEP-GluR1, which forms homomeric receptors,
mirrored that of heteromeric SEP-GluR1/GluR2 receptors.
Similarly, the trafficking of SEP-GluR2 paralleled that of hetero-
meric SEP-GluR3/GluR2. These findings, in addition to previous
results indicating that the level of overexpression of transiently
expressed recombinant AMPA receptors in dendritic regions isless than $50% above endogenous levels ( Kessels et al.,
2009 ), suggest that our optical approach to measure receptor
incorporation into synapses can be used to analyze endogenous
synaptic plasticity mechanisms.
A number of in vitro and theoretical studies have examined
the role of compartmentalized plasticity in neuronal function
( Govindarajan et al., 2006; Larkum and Nevian, 2008; Poirazi
and Mel, 2001; Polsky et al., 2004 ). Clustered plasticity could
bind functionally relevant inputs onto dendrites and enhance
storage capacity of individual neurons by locally recruiting
nonlinear voltage-gated conductances ( Poirazi and Mel, 2001 ).
Furthermore, clustered plasticity can increase the probability of
local spike initiation by enhancing excitability of dendrites ( Frick
et al., 2004 ), which in turn strengthens the coupling between
a dendritic branch and the soma ( Losonczy et al., 2008 ). Such
branch strength potentiation permits temporally precise and
robust somatic output, which is generally believed to be impor-
tant for information processing by single neurons ( Koch and
Segev, 2000 ). Clustered synaptic plasticity could complement
plasticity of dendritic excitability as mechanisms of experi-
ence-driven information storage ( Makara et al., 2009 ).
What cellular mechanisms could underlie such clustered
synaptic plasticity? Based on simple simulations (see Figure S6 ),
we found that our data with SEP-GluR1 (and GluR1/2) are
consistent with a model in which the cluster of synaptic potenti-
ation spans on average approximately four synapses, corre-
sponding to $8 mm of dendrite. Notably, such a spatial scale issimilar to the biochemical compartmentalization of dendritic
plasticity machinery in vitro ( Harvey et al., 2008; Makino and
Malinow, 2009; Patterson et al., 2010; Schiller et al., 2000; Wei
et al., 2001 ) as well as in vivo ( Jia et al., 2010 ), suggesting that
the local spread of intracellular signaling factors is important
for the coordinated potentiation among nearby synapses. In
this respect the GluR1AA mutant, which should be insensitive
to heterosynaptic biochemical signals (e.g., Ras-driven protein
kinase activation) and, thus, the effect of the heterosynaptic
threshold reduction, showed no clustered spine enrichment.
Our data cannot fully rule out the possibility that groups of
presynaptic fibers with similar activity patterns, thereby driving
0
0.2
0.4
0.6
0.8
1
1.2
0 0.4 0.8 1.2 1.6
B
C u m u l a t i v e p r o b a b i l i t y
Enrichment value
GluR1AAGluR1wt
p = 0.14
E
-0.4
-0.2
0
0.2
0.4
0.6
0.8
0 0.5 1 1.5 G l u R 1 i m m o b i l e f r a c t i o n
D
Spine size
GluR1AA
GluR1wt
p = 0.29r = 0.29
-0.4
-0.2
0
0.2
0.4
0.6
0.8
0 0.5 1 1.5 2 G l u R 1 i m m o b i l e f r a c t i o n
Enrichment value
CGluR1AAGluR1wt
p < 0.001r = 0.87
HighLow
Modulation sensitive regionA
PotentiationGluR1wt
831 S845 S
GluR1AA
831 S->A845 S->A
Potentiation
-0.1
0
0.1
0.2
GluR1wt GluR1AA
*
C o r r e l a i t o n c o e f f i c i e n t
Figure 6. Modulation Insensitivity Controls Clustered Synaptic
Potentiation
(A) Schematic of heterosynaptic threshold reduction for plasticity by single-
spine potentiation. GluR1AA is insensitive to heterosynaptic biochemical
signals, and thus, heterosynaptic threshold reduction is minimized.
(B) Spine enrichment values for SEP-GluR1AA (n = 1,584 spines, 11 cells,
5 animals) and SEP-GluR1 (n = 2,701 spines, 23 cells, 11 animals) in whisker-
intact animals (p = 0.14, Kolmogorov-Smirnov test). The ‘‘GluR1wt’’ data are
from Figure 1E.
(C) Correlation between SEP-GluR1AA spine enrichment values and immobile
fractions of spine SEP-GluR1AA (r = 0.87, p < 0.00003, n = 15 spines, 3 cells,
2 animals). The ‘‘GluR1wt’’ data are from Figure 2B. The regression line was
fitted for both conditions.
(D) No correlation between spine size and immobile fractions of spine SEP-
GluR1AA (r = 0.29, p = 0.29, n = same as C). The ‘‘GluR1wt’’ data are from
Figure 2C. The regression line was fitted for both conditions.
(E) Correlation coefficients at neighboring synapses of dendrites expressing
SEP-GluR1wt(n = 95dendrites,23 cells, 11animals)andSEP-GluR1AA(n = 62
dendrites, 11 cells, 5 animals, *p < 0.04, t test with Bonferroni correction;
mean ± SEM). The ‘‘GluR1wt’’ data are from Figure 3D.
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similar levels of plasticity, make synapses on nearby regions of
dendrites. However, a recent study in the auditory cortex argues
against simple sensory activity providing such clustered inputs
( Chen et al., 2011 ). Furthermore, a model in which the clustering
is solely due to afferent coactivity is difficult to reconcile with theresults observed with GluR1AA. Our data suggest that natural
stimuli engage postsynaptic mechanisms leading to locally clus-
tered enhancement of synapses.
In conclusion our results support the view that experience can
drive clustered synaptic enhancement onto neuronal dendritic
subcompartments, providing fundamental architecture to circuit
development and function. Sensory deprivation drives cell-wide
synaptic enhancement that globally sensitizes a neuron.
EXPERIMENTAL PROCEDURES
Experiments were conducted according to National Institutes of Health
guidelines for animal research and were approved by the Institutional Animal
Care and Use Committee at Cold Spring Harbor Laboratory and Universityof California, San Diego.
DNA Constructs
SEP-GluR1, SEP-GluR1(S831A,S845A), SEP-GluR2(R586Q), untagged-
GluR2(edited), and SEP-GluR3 from rat were PCR amplified and subcloned
into an expression vector with a ubiquitous promoter CAG, pCALNL.
pCALNL-DsRed and pCAG-ERT2CreERT2 were obtained from Addgene.
All the DNA plasmids were amplified with the endotoxin-free Maxiprep kit
(QIAGEN). For the formation of homomeric GluR2, SEP-GluR2(R586Q) was
expressed. Heteromeric AMPA receptors were formed by coexpressing
untagged-GluR2(edited) with either SEP-GluR1 or SEP-GluR3 at a 1:1 molar
ratio.
In Utero Electroporation
L2/3 progenitor cells were transfected by in utero electroporation. E15 time
pregnant C57BL/6J mice (Charles River) were anesthetized with an isoflur-
ane-oxygen mixture (Lei Medical). Approximately 0.5 ml of DNA solution con-
taining fast green was pressure injected through a pulled-glass capillary
tube by mouth into the right lateral ventricle of each embryo. The head
of each embryo was placed between tweezers electrodes with the anode
contacting the right hemisphere. Electroporation was achieved with five
square pulses (duration = 50 ms, frequency = 1 Hz, voltage = 25V; Harvard
Apparatus).
Cre Recombinase Activation by 4-OHT
4-OHT (Sigma-Aldrich) was dissolved in ethanol at a concentration of
20 mg/ml and diluted with 9 vol of corn oil (Sigma-Aldrich). Diluted 4-OHT
(2 mg/ml) was i.p. injected into each mouse at P11 (100 ml per animal) or
P34–P35 (300–450 ml per animal).
Whisker Manipulation
For sensory deprivation all the major whiskers were trimmed daily from P11.
Whisker-intact animals were handled similarly to whisker-trimmed animals.
Preparation
Acute coronal brain slices (350 mm thick) from in utero electroporated mice at
P13 or P36–P37 were prepared. Slices were cut in gassed (95% O2 and 5%
CO2 ) ice-cold solution containing 25 mM NaHCO3, 1.25 mM NaH2PO4,
2.5 mM KCl, 0.5 mM CaCl2, 7 mM MgCl2, 25 mM D-glucose, 110 mM choline
chloride, 11.4 mM sodium ascorbate, and 3.1 mM sodium pyruvate. Slices
were then incubated in artificial cerebrospinal fluid (ACSF) containing
118 mM NaCl, 2.5 mM KCl, 26 mM NaHCO3, 1.2 mM NaH2PO4, 11 mM
D-glucose, 4 mM MgCl2, and 4 mM CaCl2 at 35C for 30 min and then at
room temperature until used. All experiments were performed at 30C.
Imaging
We used a two-photon laser-scanning microscope (Prairie) to image L2/3
pyramidal cells of the mouse barrel cortex (403 0.8 NA objective lens and
1.4 NA oil condenser; Olympus) in a perfusion chamber containing ACSF.
SEP and DsRed were excited at 910 nm with a Ti:sapphire laser (Coherent).
Green and red fluorescencesignalswere separated by a setof dichroic mirrorsand filters (Chroma).Both epifluorescence and transfluorescencesignalswere
collected by photomultiplier tubes (PMTs), and they were summed. Individual
spines were photobleached by scanning a single plane 50 times with higher
intensity of the laser power, which took $0.5 s.
Electrophysiology
Whole-cell voltage-clamp recordings were obtained from L2/3 pyramidal cells
expressing SEP-GluR1 (homomeric receptors) or SEP-GluR1 and untagged-
GluR2(edited) (heteromeric receptors) for 4–6 days. Patch recording pipettes
( $3–6 MU ) were filled with internal solution containing 115 mM Cs-methane-
sulfonate, 20 mM CsCl, 10 mM HEPES, 2.5 mM MgCl2, 4 mM Na2 ATP,
0.4 mM Na3GTP, 10 mM Na-phosphocreatine, 0.6 mM EGTA (pH 7.2), and
0.1 mM Spermine (Sigma-Aldrich). A total of 2.5 mM MNI-caged-L-glutamate
(Tocris), 1 mM tetrodotoxin (Ascent Scientific), and 0.1 mM APV (Tocris) was
added to ACSF, and recordings were obtained at 30C. Glutamate uncag-
ing-evoked AMPA receptor-mediated postsynaptic currents were measured
at individual spines located in basal dendrites in response to test stimuli
(1 ms, 0.05 Hz) at À60mV and +40mV holding potentials (5–20 sweeps aver-
aged). The intensity of the uncaging laser (Ti:sapphire laser tuned at 720 nm)
was controlled with electro-optical modulators (Pockels cells; Conoptics).
Data Analysis
SEP and DsRed fluorescence in spines and dendrites was measured as inte-
grated green and red fluorescence, respectively, after background and leak
subtraction. To measure the density of spine surface AMPA receptors as an
enrichment value, spine SEP fluorescence was normalized to:
ð4 Ã pÞ1=3Ãð3 Ã RSpineÞ
2=3;
where RSpine represents spine DsRed fluorescence (i.e., spine volume was
converted to spinearea assumingthat spine heads are spherical). To compare
across different cells, these values were then normalized to the fluorescencesignal of common dendritic regions. Thus, spine enrichment values were
calculated as:(GSpine
ð4 Ã pÞ1=3Ãð3 Ã RSpineÞ
2=3
),(GDendrite
ð4 Ã pÞ1=3Ãð3 Ã RDendriteÞ
2=3
);
where GSpine and GDendrite represent spine and dendrite SEP fluorescence,
respectively, and RDendrite dendrite DsRed fluorescence.
Fluorescencerecoveryof spine SEP wasmeasuredat +25and +30min after
photobleaching and compared to baseline fluorescence obtained atÀ10 and
À5 min prior to photobleaching, and averaged. Immobility of AMPA receptors
was calculated as: immobility = 1 À fluorescence recovery.
To measure autocorrelation functions, two factors were considered: fluctu-
ations in spine enrichment values independent of distance-dependent
changes and the distance-dependent changes in spine enrichment values.
The fluctuations were obtained by subtracting regression lines (linear compo-
nent) fitted for each dendrite as a function of spine lag. This allowed us tomeasure autocorrelation functions without contributions from the distance-
dependent changes we observed ( Figure S3C). Autocorrelation coefficients
of spine SEP enrichment were then calculated for each dendrite by the
following equation, averaged across dendrites, and normalized so that the
correlation coefficients at zero lag corresponds to 1.0.
Cð mÞ =
XN À mÀ1
n=0
x n À
1
N
XN À1
i =0
x i
! x ð n+ mÞ À
1
N
XN À1
i =0
x i
!:
Here,xn representsthe spine SEPenrichmentvaluesat thenth spine,withN
the number of spines for the dendrite, and m, the spine lag. The regression
lines for spine enrichment values were used to examine distance-dependent
changes.
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Compartmentalized and Global Synaptic Plasticity
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Simulation
To determine the number of synapses in clusters that would produce the
autocorrelation values we obtained, we performed simulations (depicted in
Figure S6 ). The following procedure was conducted to generate dendrites
with simulated enrichment values satisfying different cluster distributions.
We considered a series of 40 spines per dendritic segment and assigned aninitial enrichment value to each spine that varied randomly from 0 to 1. On
top of these values, a cluster of enrichment-potentiated spines was added.
Two cluster parameters were varied: cluster size and potentiation value of
enrichment. A cluster size was characterized by Gaussian-distributed enrich-
mentpotentiationvalues along a dendrite withSD s = 0.8, 1.2, and1.6.Enrich-
ment potentiation p varied from 2 to 5.5. To simulate a dendrite with s cluster
size and potentiation factor p, a Gaussian distribution with SD = s and
maximum value p was multiplied by a random number (between 0 and 1) at
each spine lag and added at a random location within the initial 40 spine
enrichment values. By calculating an autocorrelation coefficient for each
dendrite and repeating the same procedure 10,000 times, we derived an
average autocorrelation curve for each parameter combination. By fitting the
true data with the simulated data, we determineds and the potentiation factor
p (i.e., the number of potentiated synapses) in the cluster. Simulations were
carried out using MATLAB (MathWorks).
SUPPLEMENTAL INFORMATION
Supplemental Information includes six figures and can be found with this
article online at doi:10.1016/j.neuron.2011.09.036.
ACKNOWLEDGMENTS
We thank C. Cepko for pCALNL-DsRed (Addgene 13769) and pCAG-ERT2
CreERT2 (Addgene 13777), W. Guo for cloning the DNA constructs, D. Bortone
for technical advice, and J. Isaacson, T. Komiyama and M. Scanziani for crit-
ical comments on the manuscript. This study was supported by National Insti-
tutes of Health (to R.M.) and Elizabeth-Sloan Livingston Fellowship (to H.M.).
Accepted: September 28, 2011
Published: December 21, 2011
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