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Synapse-specific regulation of AMPA receptorfunction by
PSD-95Jean-Claude Béı̈que, Da-Ting Lin, Myoung-Goo Kang, Hiro
Aizawa, Kogo Takamiya, and Richard L. Huganir*
Department of Neuroscience, Johns Hopkins University School of
Medicine and Howard Hughes Medical Institute, 725 North Wolfe
Street,Baltimore, MD 21205
Contributed by Richard L. Huganir, October 2, 2006 (sent for
review September 15, 2006)
PSD-95 is a major protein found in virtually all mature
excitatoryglutamatergic synapses in the brain. Here, we have
addressed therole of PSD-95 in controlling glutamatergic synapse
function bygenerating and characterizing a PSD-95 KO mouse. We
found thatthe �-amino-3-hydroxy-5-methylisoxazole-4-propionic acid
(AMPA)subtype of glutamate receptor (AMPAR)-mediated synaptic
trans-mission was reduced in these mice. Two-photon (2P) uncaging
ofMNI-glutamate onto individual spines suggested that the
decreasein AMPAR function in the PSD-95 KO mouse stems from an
increasein the proportion of ‘‘silent’’ synapses i.e., synapses
containingN-methyl-D-aspartate (NMDA) receptors (NMDARs) but no
AM-PARs. Unexpectedly, the silent synapses in the KO mouse
werelocated onto morphologically mature spines. We also
observedthat a significant population of synapses appeared
unaffected byPSD-95 gene deletion, suggesting that the functional
role ofPSD-95 displays synapse-specificity. In addition, we report
that thedecay of NMDAR-mediated current was slower in KO mice:
Thecontribution of NR2B subunit containing receptors to the
NMDAR-mediated synaptic current was greater in KO mice. The
greateroccurrence of silent synapses might be related to the
greatermagnitude of potentiation after long-term potentiation
inductionobserved in these mice. Together, these results suggest a
synapse-specific role for PSD-95 in controlling synaptic function
that isindependent of spine morphology.
glutamate receptors � hippocampus � spines � synaptic
transmission �two-photon uncaging
G lutamate, the major excitatory neurotransmitter in thebrain,
activates ionotropic glutamate receptors of
the�-amino-3-hydroxy-5-methylisoxazole-4-propionic acid(AMPA),
N-methyl-D-aspartate (NMDA), and kainate sub-types. There is
considerable interest in elucidating the molecularmechanisms that
controls synaptic targeting and trafficking ofthese receptors, in
part because of their role in the induction andexpression of
various forms of synaptic plasticity (1). Thesereceptors are
imbedded in an electron-dense structure, thepostsynaptic density
(PSD), which is believed to contain keymolecules involved in the
regulation of glutamate receptortargeting and trafficking. PSD-95,
a member of the membrane-associated guanylate kinase (MAGUK)
superfamily of proteins,is a core component of the PSD and is
thought to be importantin the control of excitatory synapse
function (2, 3). Because ofits interaction with the cytoplasmic
domains of NMDA receptor(NMDAR) subunits, it has long been
suspected that PSD-95might control the synaptic targeting of NMDARs
(4, 5). How-ever, more recent studies based on sustained or
transient over-expression of PSD-95 in neurons have forced a
reassessment ofthis view by suggesting that the primary role of
PSD-95 isrestricted to controlling AMPAR synaptic expression
(6–10).Intriguingly, previous work on a PSD-95 KO mouse, reported
noapparent changes in either AMPAR or NMDAR function (11).The
interpretation of these data are, however, complicated bythe fact
that these PSD-95 KO mice still express, albeit at lowlevels, a
functional truncated form of PSD-95 (7, 10).
Here, we have reexamined the role of PSD-95 in
glutamatergicneurotransmission in the hippocampus by carrying out
electro-physiological recordings in a mouse line carrying a
completePSD-95 gene deletion. Our results outline a defect in
AMPAR-mediated transmission in the hippocampus of the KO mice.
Thiseffect can fully be accounted for by the greater proportion
of‘‘silent’’ synapses found in KO mice as determined by two-photon
(2P) uncaging of MNI-glutamate (MNI-GLU). Interest-ingly, the
defect in AMPAR function induced by PSD-95 genedeletion appeared to
be restricted to only a subpopulation ofsynapses, thereby
suggesting that PSD-95 displays synapse spec-ificity in its
actions. In addition, these silent synapses found inKO mice were
observed on morphologically mature spines,suggesting that PSD-95
plays a distinct role in controllingglutamatergic synapse function
and spine morphology.
ResultsA previous study using gene targeting of the PSD-95
generesulted in the expression of a truncated form of PSD-95
thatcontained the N-terminal portion of PSD-95 including PDZdomains
1 and 2 (11). This truncated form has been shown tofunctionally
mimic the effect of full length PSD-95 on AMPARsynaptic expression
(7). To generate a complete KO and mini-mize the possibility of
generating a functional PSD-95 deletion,a targeting construct was
designed to delete PDZ 1 and 2 andgenerate an out-of-frame PSD-95
transcript (Fig. 5A, which ispublished as supporting information on
the PNAS web site). Byusing this targeting construct, the PSD-95
gene was disrupted byhomologous recombination in embryonic stem
cells. Propertargeting was confirmed by Southern blot analysis of
genomicDNA using the probe depicted in Fig. 5B and by PCR
analysis.Western blot analysis confirmed that PSD-95 protein was
notexpressed in PSD-95�/�(KO) mice (Fig. 5C). Similar results
wereobtained with several PSD-95 antibodies (data not shown).
Nogross anatomical or obvious behavioral abnormalities wereapparent
in these mice.
The AMPAR to NMDAR Ratio of Excitatory Postsynaptic Currents
IsReduced in PSD-95�/� Mice. To examine the effect of a
completePSD-95 KO on glutamatergic transmission, we first
determined
Author contributions: J.-C.B. and D.-T.L. contributed equally to
this work; J.-C.B., D.-T.L.,K.T., and R.L.H. designed research;
J.-C.B., D.-T.L., M.-G.K., and H.A. performed research;J.-C.B.,
D.-T.L., and K.T. contributed new reagents�analytic tools; J.-C.B.,
D.-T.L., and M.-G.K.analyzed data; and J.-C.B. and R.L.H. wrote the
paper.
Conflict of interest statement: Under a licensing agreement
between Upstate Group, Inc.and The Johns Hopkins University, R.L.H.
is entitled to a share of royalties received by theUniversity on
sales of products described in this article. R.L.H. is a paid
consultant to UpstateGroup, Inc. The terms of this arrangement are
being managed by The Johns HopkinsUniversity in accordance with its
conflict-of-interest policies.
Abbreviations: 2P, two-photon; AMPA,
�-amino-3-hydroxy-5-methylisoxazole-4-propionicacid; AMPAR, AMPA
receptor; DL-AP5, D(�)-2-amino-5-phosphonovaleric acid; EPSC,
ex-citatory postsynaptic current; eEPSC, evoked EPSC; LTP,
long-term potentiation; mEPSC,miniature EPSC; MNI-GLU,
MNI-Glutamate, 4-methoxy-7-nitroindolinyl-caged-glutamate;NMDA,
N-methyl-D-aspartate; NMDAR, NMDA receptor; P(n), postnatal day
(n); PSD, postsynaptic density.
*To whom correspondence should be addressed. E-mail:
[email protected].
© 2006 by The National Academy of Sciences of the USA
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2006 � vol. 103 � no. 51 � 19535–19540
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the ratio of AMPA to NMDA receptor components of
evokedexcitatory postsynaptic currents (eEPSCs) in pyramidal
neuronsof the CA1 region of the hippocampus. We recorded
eEPSCswhile clamping the cell at �40 mV. In these conditions,
bothAMPA and NMDARs are activated by synaptically
releasedglutamate, and their respective contribution to the eEPSC
wasdetermined pharmacologically by administration of theNMDAR
antagonist DL-AP5 (100 �M; Fig. 1A1; and seeSupporting Methods,
which is published as supporting informa-tion on the PNAS web site)
(9, 12, 13). The AMPAR�NMDARratio was significantly reduced in KO
compared with age-matched littermate PSD-95�/�mice (WT; Fig. 1
A1).
Because AMPAR- and NMDAR-mediated synaptic responsesare
kinetically distinguishable, we could also approximate AMPARand
NMDAR components by measuring the amplitude of thesynaptic current
at different time points of the eEPSCs at �40 mV(see Fig. 1A2 and
Supporting Methods). Although less precise, thismethod allowed
sampling over a much broader population ofrecordings and allowed us
to estimate the developmental profile ofthe defect in synaptic
transmission induced by genetic deletionof PSD-95 (Fig. 1B). In
young animals [postnatal day (P)9–P12], theAMPA�NMDA ratio was not
altered in the KO compared withWT mice. However, this ratio was
reduced by �30% in KO slicesin a slightly older age group
(P14–P20), and this reduction persistedin the older animals tested
(Fig. 1B).
In principle, a change in rectification properties of
AMPARscould account for the decrease in AMPAR�NMDAR ratio ob-served
at �40 mV even though spermine was not included in ourpipette
solution. To control for this possibility, we obtained full
I-Vcurves of AMPAR-mediated EPSCs and found no changes
inrectification of AMPAR currents between WT and KO mice (Fig.6,
which is published as supporting information on the PNAS website).
Together, these results outline a general defect in glutama-tergic
transmission in mice lacking PSD-95. In addition, the
devel-opmental profile of this defect is broadly consistent with
the knownexpression profile of PSD-95, which begins to be highly
expressedin the hippocampus at around P10 (14).
Frequency, but Not Amplitude, of AMPAR-Mediated Miniature
EPSCs(mESPCs) Is Reduced in PSD-95�/� Mice. A reduction in the
AMPA�NMDA ratio could reflect a reduction in the number (or
function)
of AMPARs, an increase in the number (or function) of NMDAR,or a
combination of both. To distinguish between these possibilities,we
next recorded AMPAR-mediated mEPSCs. During the devel-opmental
period studied here (P8–P25), we observed in WT micean increase in
the frequency of mEPSCs with increasing age (Fig.1C). This
developmentally regulated increase in mEPSC frequencywas largely
abolished in KO mice. Thus, by the 3rd week of life, thefrequency
of mEPSCs was reduced by �50% in KO mice (Fig. 1C).We observed no
differences in the amplitude of mEPSCs betweenWT and KO mice at all
ages tested (Fig. 1C).
A decrease in the frequency of mEPSCs can reflect either
adecrease in the probability of neurotransmitter release or
adecrease in the number of AMPAR-containing synapses. Todistinguish
between these possibilities, we carried out paired-pulse ratio
analysis, a measure of presynaptic neurotransmitterrelease
probability, and found no difference between WT andKO mice (Fig. 7,
which is published as supporting information onthe PNAS web site).
The reduction in frequency of mEPSCs inKO mice may thus reflect a
reduction in the number of AMPAR-containing synapses. Furthermore,
because this reduction inmEPSC frequency can quantitatively account
for the reductionin AMPAR�NMDAR ratio observed in KO mice, these
resultssuggest that synaptic NMDAR function is largely unaffected
inthese mice. Together, these observations suggest the
intriguingpossibility that a greater proportion of synapses in KO
micewould be functionally silent [i.e., containing NMDARs but
noAMPARs (15, 16)].
2P Uncaging of MNI-GLU Reveals Silent Synapses in Developing
CA1Pyramidal Neurons. The existence of silent synapses was
originallydemonstrated by minimal stimulation experiments, which
arebelieved to monitor synaptic responses elicited by activation
ofsingle synapses (16, 17). These experiments are laborious and
donot lend themselves with ease to quantitative comparison be-tween
groups. We therefore systematically probed the functionof
individual synapses (i.e., determined whether they are func-tional
or silent) by locally and transiently ‘‘uncaging’’ glutamateby
laser photolysis of a glutamate analog, MNI-caged-L-glutamate
(MNI-GLU). Neurons (filled for �20 min with AlexaFluor 594) were
first imaged by confocal microscopy to identifydendritic spines
(Fig. 2A). In agreement with a report using
10 pA100 ms
Wild type PSD-95 -/-
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B
PSD-95 -/-Wild type
Fig. 1. AMPAR-function is de-creased in PSD-95�/� mice. (A) In
A1,the AMPA�NMDA ratio was deter-mined by subtracting the traces
ob-tained in 100 �M D-L AP5 from thoseobtained in its absence (C �
1) andwas found to be significantly lower(P � 0.05, Student’s t
test) in KO (n �9) compared with WT (n � 5) mice.These results were
obtained in age-matched littermates (P16–P20). InA2, the AMPA�NMDA
ratio was cal-culated by estimating the respectiveAMPA (1) and
NMDAR (2) current onthe traces at �40 mV based on theirdifferent
time courses (as depicted inthe Inset). The AMPA�NMDA ratiowas
significantly lower (P � 0.01, Stu-dent’s t test)
inKO(n�19)thaninWTmice (n � 18). (B) The AMPA�NMDAratios obtained
by the method de-picted in A2 are shown binned ac-cording to the
age of the animals(P9–P12, P � 0.07, n � 5 each; P14–20, P � 0.05;
n � 7 each; P21–P24, P �0.05; n � 6 for WT and n � 7 for KO). (C)
Frequency and amplitude of mEPSCs were binned according to the age
of the animals. For the frequency of events: P8–P10,P � 0.3; n � 11
for WT and n � 4 for KO mice; P14–P20, P � 0.05; n � 10 for WT and
n � 16 for KO mice; P21–P25, P � 0.05; n � 10 for WT and n � 8 for
KO mice. Forthis and subsequent figures, an asterisk indicates
statistical significance.
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similar methodologies (18), bath administration of MNI-GLU (5mM)
did not activate any obvious membrane conductance at�70 mV (data
not shown). Under these conditions, brief focallaser illumination
(720 nm) of a dendritic spine elicited rapidlyactivating and
inactivating inward currents (2P-EPSC; n � 100;Fig. 2). The kinetic
properties of 2P-EPSCs were indistinguish-able from those displayed
by mEPSCs recorded from the samecell (rise, 3.4 � 0.7 ms and 2.7 �
0.5 ms; decay, 8.4 � 1.9 ms and7.8 � 1.5 ms for 2P-EPSCs and
mEPSCs, respectively; n � 6; Fig.2B). To determine the spatial
profile of the uncaging spot, wefocused the laser beam at fixed
spatial intervals (0.5 �m) alongan axis perpendicular to the length
of a spine (Fig. 2C) whilerecording the elicited current. The FWHM
values of the result-ing Gaussian function (amplitude vs. location)
was 1.39 � 0.9�m, whereas that of the fluorescence profile of the
spine per sewas 0.65 � 0.01 �m, thereby confirming the local nature
of theuncaging event within the constraints expected from the size
ofthe diffraction-limited spot in our conditions and from
diffusionof uncaged glutamate. To minimize spillover of the
uncagingevent, we carefully chose spines that were separated by ��2
�mfrom their closest neighbor. Uncaging MNI-GLU while holdingthe
voltage of the neuron at different values yielded currents
thatbehaved like those induced by endogenously released
glutamate:the currents reversed at �0 mV and displayed slower decay
atdepolarized potentials, as expected from the gradual recruit-ment
of voltage-dependent NMDARs (Fig. 2D). The currentinduced by
uncaging MNI-GLU was mediated by both AMPARsand NMDARs at �40 mV
and solely by AMPARs at �70 mV(Fig. 2E). Together, these findings
show that uncaging MNI-GLU onto spines activates ionotropic
glutamate receptors in amanner closely mimicking activation by
endogenous glutamate.
To determine whether this approach was able to detect
silentsynapses, we focused our attention on young (P7–P8)
developing rathippocampal slices where silent synapses have been
shown to beprevalent (15, 16). We initially uncaged MNI-GLU onto
charac-teristically long, thin filopodia-like spines because they
are believedto be largely devoid of AMPARs (18). Fig. 2F depicts
two suchspines from which we recorded 2P-EPSCs only when the cell
wasvoltage-clamped at �40 mV. These filopodia thus appeared
toexpress on their surface functional NMDARs but no AMPARsand, as
such, fulfilled the definition generally agreed upon for
silentsynapses. As reported in ref. 18, we also observed a
correlationbetween the amplitude of AMPAR-mediated 2P-EPSC and
thevolume of spine heads, but not spine length, at this young age
(see
Fig. 8A, which is published as supporting information on the
PNASweb site).
Altogether, these results indicate that this approach
affordsremarkably precise spatial and temporal control of
ionotropicglutamate receptor activation at individual spines while
alsoallowing correlative analysis of function and morphology.
There Are More Silent Synapses in PSD-95�/� Mice as Detected
by2P-Uncaging of MNI-GLU. At silent synapses,
NMDAR-mediatedsynaptic responses are �5–15 pA in amplitude when
recorded at�40 mV (16). To detect silent synapses in the most
physiologicallyrelevant manner, we adjusted the uncaging laser
power to obtainNMDAR-mediated 2P-EPSCs of 5–15 pA at �40 mV. We
thenmeasured the AMPAR-mediated 2P-EPSCs at �70 mV andcomputed an
AMPA�NMDA ratio for each spine (see SupportingMethods for more
details). Examples of spines and corresponding2P-EPSCs traces at
�40 and �70 mV are shown in Fig. 3A. Usingthis approach in WT
(P13–P16) mice, we found that individualspines exhibited remarkable
heterogeneity as inferred from analysisof their AMPAR�NMDAR ratios
in response to 2P-uncaging(range from 0.15 to �3; Fig. 3B). The
high ratios observed areunlikely to reflect poor voltage clamping
of the spine under study(which would lead to an underestimate of
NMDAR current at �40mV) because fast AMPAR-mediated 2P-EPSCs can
readily be seenon the traces at �40 mV (Fig. 3A). This broad range
of AMPA�NMDA ratios for individual spines was also found in neurons
fromage-matched KO mice. However, the distribution of those
ratioswas highly skewed such that a greater proportion of spines
exhibitedvery low, or zero, AMPA�NMDA ratios in KO compared with
WTmice (Fig. 3B). We thus readily detected silent synapses only in
KOmice. Consistent with our previous observations, the
averagedAMPA�NMDA ratios across all spines we analyzed was
signifi-cantly lower in the KO compared with WT mice (Fig. 3 B and
D).Thus, these results suggest that the reduction in AMPA�NMDAratio
observed in KO mice selectively stems from a greater occur-rence of
silent synapses. Importantly, the distribution of AMPA�NMDA ratios
revealed that some spines in the KO mice exhibitedratios as high as
the highest encountered in the WT mice. Theseresults indicate that
a subpopulation of synapses appeared unaf-fected by PSD-95
deletion.
Divergence Between the Effects of PSD-95�/� Gene Deletion
onSynaptic Function and Spine Volume. As determined by 2P-uncaging
on synapses from young (P7–P8) rat, we found that
10 msec
2P-EPSCmEPSC
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Fig. 2. The electrophysiological re-sponse to 2P uncaging of
MNI-GLUclosely mimics that of endogenouslyreleased glutamate. (A)
2P image of aCA1 pyramidal neuron filled with 40�M Alexa Fluor 594.
(B) On this imageof a spine, the yellow dot (Upper)represents the
spot of laser illumina-tion. (Lower) An averaged currenttrace
depicting a 2P-EPSC induced bylaser illumination is
superimposedwith an averaged mEPSC trace col-lected from the same
neuron. (C) Thelaser spot was delivered at fixed spa-tial intervals
(0.5 �m) around thespine and the amplitude (F; redtrace) and rise
time (E) of the 2P-EPSCs are plotted as a function ofdistance from
the center of the spine.The intensity profile of the spine
isdepicted by the blue trace. (D) Traces depicting 2P-EPSCs while
holding the cell at different voltages are shown. The peak
amplitude of the 2P-EPSC is plottedas a function of the holding
voltage. (E) Traces depicting 2P-EPSCs at �70 mV and �40 mV,
elicited from the spine shown, are shown during baseline, after
bathadministration of NBQX (20 �M) and after the subsequent
administration of DL-APV (100 �M). (F) On these two neighboring
spines, displaying a characteristicthin profile, derived from a
young (P8) rat, 2P uncaging elicited responses at �40 mV but not at
�70 mV.
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small, filopodia-like spines appeared to be largely devoid
ofAMPARs (Fig. 2F and ref. 18), although they
expressedNMDAR-mediated dependent current (Fig. 2F). We thus
hy-pothesized that the silent synapses observed in KO mice
wouldpredominantly be located on smaller, less developed,
filopodia-like spines. To address this possibility, we plotted the
AMPA�NMDA ratio obtained by 2P-uncaging for each spine againsttheir
respective volume (see Supporting Methods). In WT mice,we found
that spine volume was not correlated with AMPA�NMDA ratios (Fig.
3C; R2 � 0.04, n � 20). This lack ofcorrelation was expected
because these recordings were carriedout in mice at an age
(P13–P16) where AMPAR current was notcorrelated to spine volume
(data not shown) and where silentsynapses were not readily detected
under our conditions (Fig.3B). Interestingly, we also found that
spine volume was notcorrelated with AMPA�NMDA ratio in the KO mice
(Fig. 3C;R2 � 0.08; n � 25). Closer examination revealed that the
volumeof spines that were found to be silent in KO mice spanned
closeto the entire range of spine volumes of the population.
Thus,these results show that the silent synapses observed in KO
miceare not restricted to smaller, less ‘‘developed’’ spines.
Although the results outlined above show that AMPA�NMDAratios
were not correlated to spine volume, it remains possible thatthere
was a generalized reduction in spine volume in KO mice. Forthe
spines from which we computed 2P-AMPA�NMDA ratios, wedid not
observe any significant difference in spine volume betweenWT and KO
mice (Fig. 3 C and D). To avoid a possible selectionbias toward a
morphologically homogenous population, we alsodetermined spine
volume from a much broader population ofspines and also found that
spine volumes were unchanged in KOmice (Fig. 3E). These results
suggest that the general deficit insynaptic function observed after
PSD-95 gene deletion is notaccompanied by any significant changes
in the average spinevolume. However, we observed a small but
significant increase inspine length in the KO mice (see Fig. 9,
which is published assupporting information on the PNAS web
site).
Decay Kinetics of NMDAR-Mediated Synaptic Currents Are Slower
inthe PSD-95�/� Mice. During the course of the experiments aimedat
determining AMPA�NMDA ratio of eEPSCs at �40 mV (Fig.
1 B and C), we noticed that the late portion of synaptic
currentsrecorded at �40 mV (which predominantly reflects activation
ofNMDARs) tended to exhibit slower decay in the KO mice
(timeconstants: WT, 78.2 � 8.9 ms, n � 17; KO, 106.8 � 7.1 ms, n
�15; P � 0.05; current decay fitted with a simple
monoexponen-tial). To address this issue more rigorously, we
recorded isolatedNMDAR-mediated synaptic currents (in 0.1 mM
Mg2��10 mMglycine�10 �M NBQX; Fig. 4A). The isolated NMDAR
synapticcurrents in both WT and KO mice were best fitted by a
doubleexponential decay (Fig. 10A, which is published as
supportinginformation on the PNAS web site). As shown in Fig. 4B,
theweighed time constant for NMDAR decay kinetics were
signif-icantly longer in KO compared with WT mice.
There are several mechanisms that could account for thelonger
NMDAR decay kinetics observed in KO mice. Studies inheterologous
cells have shown that NMDA receptors containingNR2B-D subunits
exhibit slower deactivation than those con-taining NR2A (19),
raising the possibility that the longer decaykinetics observed in
KO mice may reflect synaptic NMDAR ofdifferent subunit composition.
The specific changes in decaykinetics of NMDAR-mediated currents
(Fig. 10) are fully con-sistent with this idea (19). To further
test this possibility, wedetermined the effects of the preferential
NR2B-containingNMDAR antagonist ifenprodil on isolated
NMDAR-mediatedcurrents. We found that the inhibitory effect of
ifenprodil (3�M) on NMDAR-mediated currents was significantly
greater inthe KO mice, thereby suggesting that there is a greater
contri-bution of NR2B subunit-containing NMDARs to the
NMDAR-mediated synaptic current after PSD-95 gene deletion (Fig.
4C).
Although our 2P-uncaging experiments were initially designed
todetermine AMPA�NMDA ratio at individual spines, we could getan
estimate of NMDAR kinetics at single spines by fitting to
amonoexponential decay of the NMDA portion of the 2P-EPSC at�40 mV.
Consistent with our previous observations, we also foundthat the
decay kinetics of 2P-NMDAR currents were significantlylonger in the
KO compared with WT mice (Fig. 4 D and E).
There Was No Correlation Between NMDAR Kinetics and AMPARContent
at Individual Synapses. It has been well described thatthere is a
gradual shift from mainly NR2B- to NR2A-containing
B1 B2
C
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Spine volume (µm3)
Fig. 3. Analysis of glutamatergic synapses by 2P uncaging of
MNI-GLU and analysis of spine morphology in PSD-95�/� mice. (A)
Images of individual spines fromWT and KO mice are shown with their
respective 2P-EPSCs elicited at �70 and �40 mV. (B1) Distributions
of 2P-AMPA�NMDA ratio onto individual spines areplotted for both WT
and KO mice. (B2) Cumulative distribution plots of the ratios shown
in B1. (C) The 2P-AMPAR�NMDAR ratio obtained from uncaging
ontoindividual spines is plotted against the volume of those
spines. (D) The average AMPAR�NMDAR ratio (WT, 1.13 � 0.14, n � 20;
KO, 0.63 � 0.14, n � 26; P � 0.05)obtained for individual spines by
2P-uncaging is plotted on the y axis against the average volume of
the spines (WT, 0.16 � 0.02 �m3, n � 20; KO, 0.17 � 0.02�m3, n �
25; P � 0.7) onto which the AMPAR�NMDAR were determined. (E) The
volumes of spines were determined from a much broader population
than inD; WT, 0.17 � 0.01 �m3, n � 271 spines, 7 cells, 4 mice; KO,
0.17 � 0.01 �m3, n � 509 spines, 12 neurons, 5 mice).
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NMDARs during postnatal development (20, 21). In addition,the
process of synaptic maturation is accompanied by the
gradualincorporation of AMPARs into synapses (22). These
studiesraised the possibility that the incorporation of AMPARs
insynapses would parallel the shift in NMDAR subunit composi-tion
from mainly NR2B-containing receptors toward NR2A-containing
receptors and that these processes might be causallyrelated. If
this were the case, one would expect a tight correlationbetween the
NR2 subunit composition of a given synapse and itsAMPA receptor
content. To address this issue, we plotted theAMPA�NMDA ratio of
2P-EPSCs against the decay kinetics of2P-NMDAR-mediated responses
at �40 mV for all spinesanalyzed (same as those in Fig. 3).
Surprisingly, we found nocorrelation between AMPA�NMDAR ratio of
2P-EPSCs and2P-NMDAR-mediated decay kinetics in both WT (R2 �
0.001;n � 20) and in KO mice (R2 � 0.096; n � 26). These
resultssuggest that the level of AMPAR expression and the
subunit
composition of NMDAR at individual synapses are not
causallyrelated. We also found no correlation between
2P-NMDAR-mediated decay kinetics and spine volume during this
develop-mental period in both WT and KO mice (see Fig. 10B).
The Magnitude of Long-Term Potentiation (LTP) Is Greater in
PSD-95�/� Mice. To examine the functional consequences of
thecomplete PSD-95 KO on synaptic plasticity, we examined LTPin the
CA1 region of the hippocampus using field potentialrecordings in
the stratum radiatum. The magnitude of LTP,induced by delivering a
� burst-like protocol, was much greaterin KO mice than in WT
littermates (field EPSP slope, comparedwith baseline, at 30–40 min
after LTP induction: WT, 182 �13%; KO, 331 � 41%; Fig. 11, which is
published as supportinginformation on the PNAS web site). This
finding is similar to thatreported for mice expressing the
truncated form of PSD-95 (11).
DiscussionPSD-95 and other members of the MAGUK superfamily of
pro-teins are thought to be critical for the proper formation
andmaintenance of excitatory synapses. Using a combination of
genetargeting, electrophysiological, and imaging approaches, we
reportthat AMPAR function was impaired in PSD-95 KO mice.
Specif-ically, this reduction was caused by a greater proportion of
synapsesthat lack functional AMPARs (i.e., silent synapses) rather
than bya general decrease of functional AMPAR across all synapses.
Thisfinding suggests a synapse selectivity for PSD-95’s action.
Ourresults further showed that silent synapses were found on
morpho-logically mature spines in the KO mice. These results thus
outlinea distinct role of PSD-95 in controlling glutamatergic
synapsefunction independent of spine morphology. Moreover, in
confir-mation of a previous report, we find that disruption of the
PSD-95gene results in a greater magnitude of LTP.
Complete genetic deletion of PSD-95 leads, by approximatelythe
2nd week of life, to a reduction in the synaptic expression
ofAMPARs, but not NMDARs, as was apparent mainly from
theobservation that the reduction in the frequency of
AMPAR-mediated mEPSCs in KO mice could quantitatively account
forthe reduction in AMPA�NMDA ratio. This finding obtained byusing
a loss-of-function strategy is conceptually consistent withstudies
showing that overexpression of PSD-95 increasesAMPAR function (6,
7, 9, 10). Interestingly, our 2P-uncagingexperiments directly
demonstrated a greater proportion of syn-apses that expressed
NMDARs, and not AMPARs in the KOmice. These synapses are referred
to as silent because NMDARsare nonfunctional at rest. The greater
proportion of silentsynapses in KO mice bears important
consequences for synapticplasticity because it is believed that
silent synapses represent thepreferential site of AMPAR insertion
during LTP (23) (but seerefs. 24 and 25). It is thus conceivable
that the enhancedmagnitude of LTP we observed in KO mice directly
stems fromthe greater proportion of silent synapses. The corollary
impli-cation of these findings is that, whereas PSD-95 appears to
be akey molecule in controlling synaptic AMPARs expression, itdoes
not appear to play an important role in the actual recruit-ment
process of AMPARs to synapses during LTP.
In principle, the reduction in synaptic AMPAR function in KOmice
could be caused by a decrease in synaptic expression ofAMPARs
across all synapses. Our results, however, support analternate
scenario whereby the deficit in synaptic AMPAR expres-sion appears
to be restricted to only a subset of synapses. Twoindependent sets
of experiments lead to this interpretation. First,the amplitude of
mEPSCs was unaltered by PSD-95 gene deletion(there were simply
fewer events). Second, the distribution ofAMPA�NMDA ratios obtained
from 2P-uncaging onto individualspines revealed that a subset of
spines in KO mice displayed equallyhigh ratios to those obtained
from spines in WT mice. Together,these results suggest that a
population of synapses have matured (at
Fig. 4. Analysis of NMDAR function in PSD-95�/� mice. (A)
Current tracesshowing NMDAR-mediated synaptic currents recorded in
10 �M glycine, 20�M NBQX, and 0.1 mM Mg2�at �60 mV. (B) The decay
kinetics of NMDAR-mediated synaptic currents were longer in KO mice
(weighted time constants:WT, 75 � 2 ms, n � 8; KO, 113 � 7 ms, n �
8; P � 0.01). (C) The inhibition ofNMDAR-mediated synaptic currents
induced by ifenprodil (3 �M) was largerin KO mice (WT, n � 8; KO, n
� 9; P � 0.05, unpaired Student’s t test testedbetween 30 and 35
min). Stimulation frequency was 0.06 Hz. (D) Current tracesshowing
mixed AMPA and NMDA currents elicited by 2P-uncaging of MNI-GLU
recorded in normal Ringer solution while holding the cell at �40 mV
inWT and KO mice. (E) The decay kinetics of the NMDAR portion of
the mixed2P-current was approximated by fitting a single
exponential decay and waslonger in KO mice (WT, 173 � 13 ms, n �
20; KO, 250 � 16 ms; n � 25; P � 0.01).(F) The decay kinetics of
the NMDAR portion of the current induced by2P-uncaging of MNI-GLU
(as determined in E) is plotted against the 2P-AMPA�NMDA ratio
obtained from the same spines (elicited following the sameprocedure
as in Fig. 3).
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least with respect to synaptic AMPAR insertion) normally in
theabsence of PSD-95. These results therefore suggest an
intriguingselectivity in the actions of PSD-95 toward a specific
population ofsynapses. Whether this apparent specificity reflects a
functioninherent to PSD-95 or rather a compensatory mechanisms
afterPSD-95 genetic deletion is unclear. Other members of theMAGUK
superfamily of proteins might be compensating for thelack of PSD-95
in a subset of synapses.
The decay kinetics of NMDAR currents were slower in KO mice.This
effect can be accounted for, at least in part, by a greatersynaptic
expression of NR2B-containing NMDAR. In particular,our results
suggest that the normal, developmentally regulated,switch of
primarily NR2B- to NR2A-containing synaptic NMDARis hindered in KO
mice. Interestingly, the subunit composition ofNMDARs has been
suggested to decisively impact the polarity ofsynaptic plasticity,
although contradictory findings are found in theliterature (26–30).
Because LTP is greater, and NMDARs con-taining the NR2B subunit
appear to dominate in the KO mice, ourresults are broadly
consistent with the idea that NR2B-containingNMDARs preferentially
trigger LTP (27). However, we did notfind any evidence that the
silent synapses found in KO mice wereenriched in NR2B-containing
NMDARs. Because LTP is believedto be preferentially induced on
silent synapses (15, 16, 23), thesefindings do not support the idea
that NR2B-containing NMDARspreferentially link to the induction of
LTP in KO mice. However,it is possible that spines with higher
NR2B-containing NMDAR inKO mice are the ones that are more amenable
to LTP regardlessof their initial AMPAR content (i.e., regardless
of whether they aresilent or not) in KO mice. Our difficulty in
inducing reliable LTPby 2P-uncaging unfortunately precluded us from
directly testingthese possibilities at this point.
Because thin, filopodia-like spines express less AMPARs on
theirsurface than larger, more mature dendritic spines (18), we
hadinitially hypothesized that the silent synapses observed in KO
micewould preferentially be located on smaller spines. However,
com-bined 2P-uncaging and spine morphological analysis revealed
notonly that spine volume were unchanged in KO mice but also
thatthe silent synapses encountered in these mice were distributed
onspines independently of their volume. These data thus suggest
thatsynapse functional maturation and spine morphological
develop-ment can be dissociated.
The number of AMPARs expressed at synaptic sites is believedto
be governed by both activity-dependent (mainly NMDAR-dependent) and
activity-independent processes (22, 26, 31, 32).Although results
from this and other studies (6, 8–10) converge inshowing that
synaptic AMPARs are controlled by PSD-95, themolecular details of
this effect are still unclear. First, the enhance-ment of AMPAR
function by transient overexpression of PSD-95
does not require synaptic activity (10), thereby suggesting that
thisrole of PSD-95 does not merely result from a general
facilitatoryaction on NMDAR-dependent signaling. The observation
that LTPwas not abolished (it was actually enhanced) in KO mice
supportsthis view while also arguing against a direct role of
PSD-95 in therecruitment process of AMPAR to synapses during
NMDAR-dependent LTP. Yet, it was also found that the transient
overex-pression of PSD-95 mimicked and occluded LTP (6, 10),
therebysuggesting that LTP and PSD-95 control synaptic AMPAR
bycommon mechanisms. Thus, it appears that PSD-95 is
sufficient,although clearly not necessary, to enhance AMPAR number
atsynapses. The data presented here demonstrate that PSD-95 playsa
critical role in controlling AMPAR expression at a subset
ofsynapses. It is difficult to infer from our data whether the lack
ofAMPAR at these synapses reflects a synaptic insertion deficit
orrather a synaptic stabilization deficit. Because the actual
process ofinsertion of AMPARs after an LTP protocol is not
compromisedin KO mice, it is possible that PSD-95 mainly plays a
role inmaintaining the stability of synaptic AMPARs.
MethodsGeneration of PSD-95-Deficient Mouse. PSD-95 gene
deletion wasobtained by standard homologous recombination
technique. Formore details, see Supporting Methods.
Electrophysiology. Whole-cell recordings or field recordingswere
obtained in the CA1 region of rat or mice hippocampus(P7–P25). For
more details, see Supporting Methods.
2P Uncaging and Imaging. Whole-cell recordings of CA1
pyramidalneurons were obtained by following standard procedures,
exceptthat intracellular solution was supplemented with 40 �M
AlexaFluor 594 to outline neuronal morphology. Slices were bathed
innormal Ringer’s solution supplemented with MNI-GLU. A sectionof a
neuron was imaged, and the tip of a spine was illuminated (0.8msec)
with a laser tuned at 720 nm while monitoring the
electro-physiological responses. For more details, see Supporting
Methods.
We thank Elise Shumsky and Dr. Zbigniew Iwinski (Carl Zeiss
Micro-Imaging group) for their continuous technical assistance, Dr.
HaruoKasai (University of Tokyo, Tokyo, Japan) for helpful
discussion onspine volume calculation, Jason Tien for invaluable
expertise in devel-oping the spine volume calculation algorithm,
Dr. Gareth Thomas forcritical reading of the manuscript, and Dr
James Trimmer (University ofCalifornia, Davis, CA) for providing a
series of anti-PSD-95 polyclonalantibodies. This work was supported
by the Howard Hughes MedicalInstitutes (R.L.H.), by grants from the
National Institutes of Health (toR.L.H.), the National Alliance for
Research on Schizophrenia andDepression (to J.-C.B.), and the
Mental Illness Research Association (toJ.-C.B.).
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