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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

mailto:[email protected]

http://dx.doi.org/10.1016/j.neuron.2011.09.036

http://dx.doi.org/10.1016/j.neuron.2011.09.036

mailto:[email protected]



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

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1

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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

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1

1.2

0 0.5 1 1.5 2 2.5 3

Enrichment value


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


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

1002 Neuron 72, 1001–1011, December 22, 2011 ª2011 Elsevier Inc.



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





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

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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

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G l u

R 2 i m m o b i l e f r a c t i o n



D E

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Enrichment value


Spine size

G l u R 1 i m m o b i l e f r a c t i o n


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





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

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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**


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).

Neuron





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

Neuron





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


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

**


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


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).

Neuron





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


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

*


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.

Neuron





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.

Neuron





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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