A balance between excitatory and inhibitory synapses
is controlled by PSD-95 and neuroligin
Oliver Prange*, Tak Pan Wong†‡, Kimberly Gerrow*‡, Yu Tian Wang†, and Alaa El-Husseini*§
Departments of *Psychiatry and †Medicine, Brain Research Centre, University of British Columbia, Vancouver, BC, Canada V6T 1Z3
Communicated by Roger A. Nicoll, University of California, San Francisco, CA, August 12, 2004 (received for review June 10, 2004)
Factors that control differentiation of presynaptic and postsynaptic
elements into excitatory or inhibitory synapses are poorly defined.
Here we show that the postsynaptic density (PSD) proteins PSD-95
and neuroligin-1 (NLG) are critical for dictating the ratio of exci-
tatory-to-inhibitory synaptic contacts. Exogenous NLG increased
both excitatory and inhibitory presynaptic contacts and the fre-
quency of miniature excitatory and inhibitory synaptic currents. In
contrast, PSD-95 overexpression enhanced excitatory synapse size
and miniature frequency, but reduced the number of inhibitory
synaptic contacts. Introduction of PSD-95 with NLG augmented
synaptic clustering of NLG and abolished NLG effects on inhibitory
synapses. Interfering with endogenous PSD-95 expression alone
was sufficient to reduce the ratio of excitatory-to-inhibitory syn-
apses. These findings elucidate a mechanism by which the amounts
of specific elements critical for synapse formation control the ratio
of excitatory-to-inhibitory synaptic input.
S ynapse formation involves stabilization of initial sites of contact
between axons and dendrites, followed by recruitment of spe-
cific protein complexes to newly formed presynaptic and postsyn-
aptic structures (1–4). Neuronal contact formation is spatially and
temporally controlled by changes in protein content and shape at
areas of contact (5, 6). The total number of synapses formed and
ratio of excitatory-to-inhibitory synaptic inputs a neuron receives
are factors critical for determining neuronal excitability. Appropri-
ate synthesis and recruitment of specific factors important for
building synaptic contacts are thought to power this process.
However, the identity of molecules that dictate the balance between
excitatory and inhibitory synaptic contacts remains elusive. Several
lines of evidence indicate that the scaffolding postsynaptic density
(PSD) protein, PSD-95, is involved in orchestrating excitatory
synapse maturation and specificity (7). PSD-95 is exclusively local-
ized to glutamatergic synapses (8). Moreover, PSD-95 expression
correlates with the period of excitatory synapse maturation (7,
9–11). Augmentation of excitatory synapse activity and ion channel
clustering is driven by PSD-95 but not by related proteins, including
synapse-associated protein (SAP)-102 and SAP-97 (12–14), and
PSD-95 regulates clustering and activity of the ␣-amino-3-hydroxy-
5-methyl-4-isoxazolepropionic acid-type glutamate receptors
through a direct interaction with stargazin (7, 13–20). However, it
is unknown how PSD-95 effects are translated into changes in
apposing presynaptic terminals. A candidate molecule for mediat-
ing PSD-95 effects on presynaptic maturation is the cell adhesion
molecule neuroligin (NLG). NLG is present at excitatory postsyn-
aptic sites and associates with the third PSD-95兾Dlg兾ZO-1 homol-
ogy (PDZ) domain of PSD-95 through its C-terminal PDZ-binding
site (2, 21, 22).
The postsynaptic PDZ protein S-SCAM, another known binding
partner of NLG, is also possibly involved in modulating NLG effects
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on synapse formation (23). Transsynaptically, NLG binds with high
affinity to ␤-neurexin, and this interaction is thought to induce
synapse formation (2, 21, 24–26). Indeed, presynaptic contact
formation is induced in axons contacting nonneuronal cells express-
ing NLG, and these effects are abolished by disruption of NLG–
neurexin protein interaction (26, 27). Moreover, coexpression of
PSD-95 with NLG in heterologous cells potentiated NLG effects on
N-methyl-D-aspartic acid currents (28). These results support a role
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0405939101
for PSD-95 in the regulation of NLG-mediated effects in estab-
lishing synaptic contacts. However, it remains unclear whether
coordinated actions of NLG and PSD-95 dictate synapse specificity.
Our data provide evidence that assembly of specific postsynaptic
elements may not simply serve to form contacts between presyn-
aptic and postsynaptic compartments but can determine synapse
specificity. We find that the amounts of PSD-95 control proper
localization and兾or retention of NLG at the synapse, and that
coordinated actions of NLG and PSD-95 regulate type (excitatory
vs. inhibitory), number, and morphology of newly formed synaptic
contacts. These effects are manifested in an overall change in the
ratio of excitatory兾inhibitory synaptic contact number and activity.
NEUROSCIENCE
Methods
cDNA Cloning and Mutagenesis. Hemagglutinin (HA)-tagged WT
NLG (1ab splice variant) amplified from mouse cerebellum was a
gift from Peter Scheiffele (Columbia University, New York).
HA-NLG was subcloned into pNice vector (Clontech), and the HA
tag was inserted between amino acids 45 and 46 as described by
Scheiffele et al. (27). Generation of GW1 PSD-95 fused to GFP and
PSD-95 mutants has been described (15, 29). A NLG construct with
a deletion of the last 4 aa (NLG⌬PDZb) and the other truncated
forms of NLG lacking the C-terminal cytosolic domain (containing
amino acids 1–730; NLG⌬CT GFP) or N-terminal extracellular
sequences (containing amino acids 671–843; GFP NLG⌬NT) were
constructed by PCR and subcloned into an enhanced GFP vector
(Clontech). NLG⌬CT and NLG⌬NT constructs were tagged with
GFP to allow detection of these proteins. The signal peptide of
NLG (amino acids 1–46) was added to the N terminus of NLG⌬NT
to maintain proper protein sorting.
Neuronal Cell Culture and Transfections. Dissociated primary neu-
ronal cultures were prepared from hippocampi of embryonic day
(E)18兾E19 Wistar rats as described (30). Cultures were maintained
in Neurobasal media (GIBCO-Invitrogen) supplemented with B27,
penicillin, streptomycin, and L-glutamine. Hippocampal cultures
were transfected by lipid-mediated gene transfer 3 days before
immunostaining by using the transfection agent Effectene
(GIBCO-Invitrogen) as described (31) and according to the man-
ufacturer’s protocol.
Immunocytochemistry. Coverslips were removed from culture wells,
fixed in ⫺20°C methanol, and immunolabeled for synaptic protein
as described (15, 29). The following primary antibodies were used:
PSD-95 (mouse, 1:500; Affinity BioReagents, Golden, CO), GFP
(mouse, 1:1,000; Clontech), NLG (mouse, 1:1,000; Synaptic Sys-
tems, Goettingen, Germany), synaptophysin (rabbit, 1:1,000;
Pharmingen), vesicular ␥-aminobutyric acid (GABA) transporter
Abbreviations: PSD, postsynaptic density; PDZ, PSD-95兾Dlg兾ZO-1 homology; NLG, neuroli-
gin; GABA, ␥-aminobutyric acid; VGAT, vesicular GABA transporter; VGLUT, vesicular
glutamate transporter; HA, hemagglutinin; PSC, postsynaptic current; mEPSC, miniature
excitatory PSC; mIPSC, miniature inhibitory PSC; siRNA, small interference RNA; DIV, day
in vitro.
‡T.P.W. and K.G. contributed equally to this work.
§To whom correspondence should be addressed. E-mail: alaa@interchange.ubc.ca.
© 2004 by The National Academy of Sciences of the USA
PNAS 兩 September 21, 2004 兩 vol. 101 兩 no. 38 兩 13915–13920
 (VGAT) (rabbit, 1:1,000; Synaptic Systems), vesicular glutamate
transporter (VGLUT)-1 (rabbit, 1:1,000; Synaptic Systems), GluR1
(rabbit, 1:500; Upstate Biotechnology, Lake Placid, NY), and the
N-methyl-D-aspartic acid receptor subunit 1 (mouse; 1:200; Synap-
tic Systems). The GFP antibody (guinea pig, 1:300) was generously
provided by David Bredt (University of California, San Francisco).
All antibody reactions were performed in blocking solution (2%
normal goat serum) for 1 h at room temperature.
Imaging and Analysis. Images were acquired on a Zeiss Axiovert
M200 motorized microscope by using a monochrome 14-bit Zeiss
Axiocam HR charge-coupled device camera at 1,300 ⫻ 1,030 pixels.
Exposure times were individually adjusted to yield an optimum
brightness of immunofluorescent clusters without saturation. To
correct for potential out-of-focus clusters within the field of view,
focal plane (z-) stacks were acquired and a maximum intensity
projection was performed offline. Images were scaled to 16 bits and
analyzed in NORTHERN ECLIPSE (Empix Imaging, Missasauga, Can-
ada), by using custom-written software routines. Briefly, images
were processed at a constant threshold level (of 32,000 pixel values)
to create a binary (‘‘mask’’) image, which was multiplied with the
original image by using Boolean image arithmetics. The resulting
image contained a discrete number of clusters with pixel values of
the original image. Dendrites of the cell of interest and untrans-
fected control cells were outlined by using a combination of the
GFP fluorescence signal and differential interference contrast
bright-field images. Only clusters with average pixel values 2 times
greater than corresponding background pixel values were used for
analysis. Unless otherwise indicated, the size of stained clusters was
measured, which represents the total (integral) pixel value of each
object (see Supporting Text, which is published as supporting
information on the PNAS web site, for more details).
For colocalization analysis, a number of equally sized mea-
surement boxes was placed over stained clusters on the PSD-95
GFP (green) channel, and background-subtracted immunoflu-
orescence values for all imaging channels (red, green, and blue)
were correlated within each box. Colocalization was scored if
clusters in two color channels were overlapping by at least 1 pixel,
and the average staining intensity was ⬎2 background staining.
HA-NLG overexpression levels were calculated by calculating
the ratio of NLG immunofluorescence over the soma of HA-
NLG transfected with untransfected cells within the same field
of view. The two-tailed nonparametric Mann–Whitney U test
was used to compare clusters between experimental groups. For
correlation analysis all data were rank-ordered and the Spear-
man correlation coefficient was calculated.
Electrophysiology. Day in vitro (DIV) 9 neurons were transfected
with different cDNA combinations by using Lipofectamine 2000
(Invitrogen) according to the manufacturer’s protocol and ex-
pressed for 48 h before recording. For recording of miniature
postsynaptic currents (PSCs) neurons were continuously perfused
with extracellular solution [pH 7.4; 320–330 milliosmolar (mosM)]
containing 140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1.3 mM
CaCl2, 25 mM Hepes, 33 mM glucose, and 0.0005 mM tetrodotoxin
(Alomone, Jerusalem) to block voltage-gated sodium channels and
isolate action potential-independent miniature PSCs. Transfected
cells with GFP signal were identified under a fluorescent upright
microscope. Intracellular solution (pH 7.2; 300–310 mOsm) was
composed of 115 mM Cs gluconate, 17.5 mM CsCl, 10 mM Hepes,
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2 mM MgCl2, 10 mM EGTA, 4 mM ATP, 0.4 mM GTP, and 0.1%
Lucifer yellow (Sigma-Aldrich). A MultiClamp 700A amplifier
(Axon Instruments, Foster City, CA) was used for recording.
Access resistance was monitored, and recordings where series
resistance varied by ⬎10% were rejected. No electronic compen-
sation for series resistance was used. Whole-cell patch-clamp re-
cordings were performed in voltage-clamp mode while maintaining
the membrane potential either at the reversal potential for GABA
13916 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0405939101
receptor-mediated miniature PSCs (⫺60 mV) to isolate miniature
excitatory PSCs (mEPSCs) or at the reversal potential for iono-
tropic glutamate receptor-mediated miniature PSCs (⫹10 mV) to
isolate miniature inhibitory PSCs (mIPSCs). Recorded mEPSCs
and mIPSCs were antagonized completely by the ionotropic glu-
tamate receptor antagonist cyano-7-nitroquinoxaline-2,3-dione
(Sigma-Aldrich) and the GABA type A receptor antagonist bicu-
culline (Sigma-Aldrich), respectively (data not shown). Recordings
were low-pass-filtered at 2 kHz, sampled at 10 kHz, and stored in
a computer by using CLAMPEX 8.0 (Axon Instruments).
Small Interference RNA (siRNA) Experiments. We used annealed and
HPLC-purified predesigned siRNA (Ambion, Austin, TX) to spe-
cifically interfere with PSD-95 expression levels in hippocampal
cells. Sense and antisense sequences of siRNA were GGAGAU-
CACAUUGGAAAGGtt and CCUUUCCAAUGUGAUCUC-
Ctc, respectively. DIV 7 hippocampal neurons were transfected by
using Lipofectamine 2000 (Invitrogen) and immunostained 3 days
later as described. For each well of a 24-well plate, siRNA (0.2 ␮g)
and a transfection reporter (0.5 ␮g eGFP-C1) (Clontech) were
cotransfected with 1 ␮l of Lipofectamine 2000.
Results and Discussion
NLG Drives the Formation of Excitatory and Inhibitory Presynaptic
Contacts. To evaluate the role of NLG in synapse development, we
assessed changes in synaptic contacts with expression of an HA-
tagged NLG isoform-1 (HA-NLG) in hippocampal neurons. Both
endogenous and overexpressed NLG were inefficiently clustered in
developing neurons (Fig. 7, which is published as supporting
information on the PNAS web site). Despite the lack of discrete
NLG clusters, total number and size of presynaptic terminals
contacting dendrites from cells expressing HA-NLG were signifi-
cantly increased at DIV 12 (4.7- ⫾ 0.3-fold and 2.4- ⫾ 0.3-fold,
respectively) and DIV 15 (4.1- ⫾ 0.3-fold and 2.5- ⫾ 0.3-fold,
respectively) when compared to terminals on dendrites from cells
transfected with GFP (Fig. 1 A, B, and E). In contrast, HA-NLG
expression did not significantly change the average size of PSD-95
puncta and only resulted in a modest increase (1.5- ⫾ 0.1-fold) in
the number of PSD-95 clusters (Fig. 1 C, D, and F). Similarly, no
significant change in size of either GluR1 or N-methyl-D-aspartic
acid receptor subunit 1 clusters was detected (data not shown).
These results demonstrate that NLG exerts differential effects on
presynaptic and postsynaptic compartments. Moreover, unlike
PSD-95 (7), NLG does not potentiate the size of the PSD and
assembly of postsynaptic receptor complexes.
A small increase in the number of PSD-95 clusters was observed
when compared to the total number of synapses induced by
HA-NLG overexpression. This finding suggests that HA-NLG may
have induced a heterogeneous population of synaptic contacts. To
evaluate the identity of synapses formed, we immunolabeled HA-
NLG-transfected cells with the inhibitory presynaptic marker
VGAT and the excitatory presynaptic marker VGLUT. We found
that HA-NLG expression enhanced the size (1.9- ⫾ 0.1-fold) and
number (3.4- ⫾ 0.2-fold) of both VGAT-positive presynaptic con-
tacts and the size (2.2- ⫾ 0.2-fold) and number (1.4- ⫾ 0.1-fold) of
VGLUT-positive contacts (Fig. 1 G–J). These results demonstrate
that NLG can drive the formation of excitatory and inhibitory
synapses.
Regulation of NLG Clustering by a PDZ-Dependent Pathway. The lack
of distinct clusters of HA-NLG may have resulted from insuf-
ficient amounts of endogenous PSD-95. To test this, we first
examined whether PSD-95 GFP could drive clustering of en-
dogenous NLG. Using antibodies specific to NLG subtypes 1 and
3, we found that the size of NLG clusters was significantly
enhanced (2.6- ⫾ 0.2-fold) at sites containing PSD-95 GFP (Fig.
2 A and B). The size of presynaptic contacts (labeled with
synaptophysin) was also enhanced (2.6- ⫾ 0.2-fold). Moreover,
Prange et al.

Fig. 1. NLG induces excitatory and inhibitory presynaptic contact formation.
Hippocampal neurons were transfected with HA-tagged NLG (HA-NLG) or GFP
and stained for synaptophysin (Syn), PSD-95, VGAT, or VGLUT. (A) Size and
number of Syn-positive terminals (white arrowheads; n ⫽ 10 and 11 cells) were
enhanced on dendrites from neurons expressing HA-NLG when compared
with GFP-expressing cells (B) (n ⫽ 12 and 9 cells). (C and D) A modest increase
in the number of PSD-95 puncta was observed. (E) Similar changes in Syn
cluster size and number were seen at both DIV 12 and 15. (F) Summary of
changes in PSD-95 cluster number and size in cells expressing HA-NLG. (G–I)
Size and number of VGAT-positive (n ⫽ 11 cells) and VGLUT-positive (n ⫽ 8
cells) contacts (white arrowheads) increased on dendrites from HA-NLG-
expressing cells when compared with GFP-expressing cells (n ⫽ 12 cells, 242
terminals). (J) Summary of changes in VGAT and VGLUT cluster size and
number in HA-NLG-expressing cells. In A–D and G–I, black arrowheads depict
synaptic puncta from neighboring untransfected cells. *, P ⬍ 0.05; ***, P ⬍
0.001 (Mann–Whitney U test). (Scale bars: 10 ␮m, A, C, and G; 1 ␮m, A Inset,
B Inset, D, H, and I.)
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a linear correlation (P ⬍ 0.0001) between the size of PSD-95
GFP, NLG, and Syn clusters was seen at single synapses (n ⫽ 10
cells, 200 synapses). This analysis revealed that PSD-95-
mediated clustering of NLG and presynaptic enhancement are
tightly regulated at individual excitatory synapses.
Previous findings showed that NLG associates with the third
PDZ domain of PSD-95 through its C-terminal PDZ binding site
Prange et al.
NEUROSCIENCE
Fig. 2. PSD-95 regulates NLG clustering and presynaptic maturation. Hip-
pocampal neurons were transfected with either PSD-95 GFP or a mutant form
of PSD-95 containing PDZ domains 1 and 2 (PSD-95 GFP 1-PDZ2). (A–C) Cells
were stained with antibodies specific to NLG1 and NLG3 and the presynaptic
marker synaptophysin (Syn). Dendritic clusters of endogenous NLG were
compared between cells expressing PSD-95 GFP (A) (white arrowheads; n ⫽ 11
cells) and neighboring untransfected cells (B) or cells expressing PSD-95 GFP
1-PDZ2 (C). (D) Summary of changes of NLG and Syn in these neurons. PSD-95
GFP expression increased NLG and Syn cluster size. No significant change (n.s.)
in NLG and Syn cluster size was observed in neurons expressing PSD-95 GFP
1-PDZ2. (E–H) Expression of PSD-95 GFP with HA-NLG enhanced the size of NLG
clusters but reduced Syn-positive contact number when compared with neu-
rons expressing HA-NLG alone. (E and F Right) Higher magnification micro-
graphs of boxed regions overview images are shown. **, P ⬍ 0.01; ***, P ⬍
0.001 (Mann–Whitney U test). (Scale bars: 10 ␮m, A Left, C Left, E Left, and F
Left; 1 ␮m, A Right, B, C Right, E Right, and F Right.)
(22, 32). To examine whether a PDZ-dependent interaction is
involved in PSD-95-mediated clustering of NLG, we expressed a
truncated form of PSD-95 that contains only the first two PDZ
domains (PSD-95 1-PDZ2). This mutant form was previously
shown to enhance GluR1 clustering (14). However, we found
expression of PSD-95 1-PDZ2 did not significantly enhance either
NLG clustering or the size of opposed presynaptic sites (Fig. 2 C and
D). These results indicate that PSD-95-mediated clustering of
GluR1 and NLG involves two independent processes.
Similar to the enhanced clustering of endogenous NLG, we also
PNAS 兩 September 21, 2004 兩 vol. 101 兩 no. 38 兩 13917
 Fig. 3. PSD-95 restricts NLG localization to excitatory synapses. Cells trans-
fected with HA-NLG and PSD-95 GFP and stained with VGAT or VGLUT. (A
Right) Magnifications of Inset Left. (A and B) PSD-95 GFP recruited HA-NLG to
sites positive for VGLUT (n ⫽ 7 cells) but not VGAT (n ⫽ 10 cells). (C) Graph
summarizing these results. (D and E) GluR1 cluster size was enhanced in
dendrites from cells expressing PSD-95 GFP alone (n ⫽ 9 cells) or HA-NLG (n ⫽
8 cells; white arrowheads) when compared with GluR1 clusters from untrans-
fected controls (n ⫽ 10 cells). Expression of HA-NLG alone (n ⫽ 8 cells) did not
enhance GluR1 clustering. **, P ⬍ 0.01; ***, P ⬍ 0.001 (Mann–Whitney U test).
(Scale bars: 10 ␮m, A Left; 1 ␮m, A Right, B, and D.)
found that coexpression of HA-NLG with PSD-95 increased the
average size of HA-NLG clusters (1.5- ⫾ 0.1-fold), which correlated
with a reduction in the total number of presynaptic terminals (2.5-
⫾ 0-fold) (Fig. 2 E–H). Notably, 85 ⫾ 3% of HA-NLG and PSD-95
GFP coclusters were present at sites apposed to excitatory presyn-
aptic terminals (VGLUT-labeled) (Fig. 3 A–C). These sites were
also positive for the excitatory postsynaptic receptor GluR1 (Fig.
3D). Consistent with the modest effect of NLG on postsynaptic
elements, HA-NLG did not potentiate PSD-95-mediated clustering
of GluR1 (Fig. 3E). Taken together, these results demonstrate that
PSD-95 controls NLG and GluR1 accumulation at postsynaptic
sites and restricts NLG clusters to excitatory synapses.
To further characterize the relationship between NLG and
PSD-95 in regulating synapse morphology, we compared correla-
tion of the presynaptic and postsynaptic contacts in neurons trans-
fected with PSD-95 and WT NLG or a mutant form of NLG lacking
the PDZ-binding domain (HA-NLG⌬PDZb) (Fig. 4). This analysis
revealed that the shape of presynaptic and postsynaptic contacts
was highly correlated only at sites coexpressing PSD-95 GFP and
WT HA-NLG (Fig. 4C). This finding supports the notion that both
proteins influence presynaptic contact morphology.
Next, to prevent binding to extracellular partners, we replaced the
N-terminal domain (amino acids 46–694) of NLG with a sequence
coding for GFP (GFP NLG⌬NT) (Fig. 8, which is published as
supporting information on the PNAS web site). Clusters of GFP
NLG⌬NT partially colocalized (to 43 ⫾ 5%) with PSD-95. At these
sites, the number of synaptophysin-positive contacts was reduced
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(by 86 ⫾ 4%). This finding indicates that coupling of these
molecules may regulate synapse formation at sites containing
PSD-95.
PSD-95 Modulates the Specificity of NLG-Induced Synapses. To test
whether PSD-95 modulates the specificity of HA-NLG-induced
synapses, we evaluated the ratios of inhibitory and excitatory
synaptic contacts by comparing the number of VGAT- and
13918 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0405939101
Fig. 4. PDZ-dependent interactions regulate the morphology of synapses
induced by PSD-95 and NLG. (A and B) Neurons were transfected with PSD-95
GFP and either WT HA-NLG or a mutant HA-NLG lacking the PDZ-binding site
(HA-NLG⌬PDZb) and immunostained as indicated. (A) Colocalization (white
arrowheads) of HA-NLG with PSD-95 GFP and apposed synaptophysin (Syn)-
labeled presynaptic terminals. (A1 and A2) Localization of HA-NLG clusters
and apposed presynaptic terminal to PSD-95 GFP. (B) HA-NLG⌬PDZb is not
recruited to PSD-95 GFP clusters (white arrowheads). (C) The correlation
between the size of PSD-95 GFP, HA-NLG, and Syn clusters at single synaptic
sites (linear fit; red line) was stronger in cells coexpressing PSD-95 GFP and WT
HA-NLG (P ⬍ 0.0001; n ⫽ 10 cells, 219 clusters) when compared to cells
expressing PSD-95 GFP and HA-NLG⌬PDZb (P ⬍ 0.001; n ⫽ 10 cells, 225
clusters). (Scale bars: 10 ␮m, A and B; 1 ␮m, A1, A2, B1, and B2.)
VGLUT-positive puncta relative to the total number of synapses
(Fig. 5). Expression of PSD-95 GFP resulted in a significant
reduction in number (2.5- ⫾ 0.1-fold) and size (1.5- ⫾ 0.1-fold) of
HA-NLG-induced VGAT-positive contacts, to levels similar to
those of GFP-expressing controls (Fig. 5A Upper). In contrast,
PSD-95 GFP did not alter the number or size of VGLUT-positive
terminals induced by HA-NLG (Fig. 5A Lower).
We next sought to examine functional correlates of these synaptic
changes by using an electrophysiological approach (Fig. 5 B and C).
We compared changes in excitatory and inhibitory currents in
hippocampal neurons transfected with either GFP alone, HA-NLG
and GFP, PSD-95 GFP, or PSD-95 GFP and HA-NLG by using
whole-cell voltage-clamp recordings of mEPSCs and mIPSCs.
Miniature frequency reflects both the size and number of presyn-
aptic contacts, whereas the average amplitude of miniature currents
correlates with postsynaptic parameters (33). Ectopic expression of
HA-NLG significantly increased the frequency of mEPSCs (2.1- ⫾
0.3-fold) and IPSCs (1.9- ⫾ 0.4-fold). A similar increase in mEPSC
frequency was observed in neurons expressing PSD-95 GFP with
(1.8- ⫾ 0.3-fold) or without HA-NLG (2- ⫾ 0.4-fold). Moreover,
HA-NLG enhanced the average amplitude of mEPSCs only when
coexpressed with PSD-95. This finding is consistent with the lack of
changes in GluR1 clustering upon overexpression of NLG. In
addition, HA-NLG induced an increase in mIPSC frequency that
was abolished upon coexpression with PSD-95 GFP, a result
paralleling our immunocytochemical data. Strikingly, expression of
PSD-95 GFP alone resulted in a significant decrease (by 64 ⫾ 11%)
Prange et al.

Fig. 5. Manipulation of NLG and PSD-95 expression alters the ratio of
excitatory to inhibitory synaptic contacts. (A Upper) The number and size of
VGAT-labeled contacts significantly decreased in cells coexpressing HA-NLG
and PSD-95 GFP (n ⫽ 10 cells, 701 terminals) compared with cells expressing
HA-NLG alone (n ⫽ 11 cells, 2,145 terminals). (A Lower) No change in number
and size of VGLUT-labeled excitatory presynaptic contacts in cells coexpressing
HA-NLG and PSD-95 GFP (n ⫽ 7 cells, 572 terminals) compared with cells
expressing HA-NLG alone (n ⫽ 8 cells, 705 terminals). (B and C) Electrophysi-
ological recordings from hippocampal neurons transfected with HA-NLG and
GFP (to visualize cells) (n ⫽ 26), PSD-95 GFP (n ⫽ 8), HA-NLG and PSD-95 GFP
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(n ⫽ 9), or GFP alone (controls, n ⫽ 16). Spontaneous mEPSCs (B) and mIPSCs
(C) were measured at holding potentials of ⫺60 mV and ⫹10mV, respectively.
(B) Enhanced mEPSC frequency and amplitude in cells expressing PSD-95 GFP
and HA-NLG with PSD-95 GFP. Enhancement of mEPSC frequency, but not of
amplitude, in cells expressing HA-NLG with GFP is shown. (C) mIPSC frequency
was enhanced in cells expressing HA-NLG with GFP, reduced in cells expressing
PSD-95 GFP, and unchanged in cells expressing HA-NLG with PSD-95 GFP. No
difference in the average mIPSC amplitude between cell groups was found.
*, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001 (Mann–Whitney U test).
Prange et al.
NEUROSCIENCE
Fig. 6. Altered expression of PSD-95 influences the ratio of excitatory-to-
inhibitory presynaptic contacts. (A) Hippocampal cells were transfected with
either GFP (controls, Left) or PSD-95 GFP (Center) and immunostained at DIV
12 for GFP, synaptophysin (Syn), and the GABA inhibitory presynaptic marker
VGAT. PSD-95 GFP expression resulted in a significant decrease in the percent-
age of VGAT-positive inhibitory contacts (Upper Right) but did not alter the
number of PSD-95 clusters (Lower Right) (n ⫽ 15 cells, 288 VGAT⫹, 778
VGAT⫺) compared with controls (n ⫽ 12 cells, 242 VGAT⫹, 272 VGAT⫺). (B and
C) Introduction of GFP with either a scrambled siRNA (control siRNA; Left) or
PSD-95 siRNA (Center). (B Right) A reduction in the number of PSD-95-positive
puncta in cells cotransfected with GFP and PSD-95 siRNA (n ⫽ 11 cells, 301
puncta) compared with controls (n ⫽ 7 cells, 406 puncta) was found. (C Right)
A significant increase in the percentage of inhibitory contacts (VGAT⫹) and a
concurrent decrease in the percentage of excitatory contacts (VGAT⫺) (Upper)
was found, but there was no change in the total number of synapses in cells
expressing GFP and PSD-95 siRNA (n ⫽ 11 cells, 322 VGAT⫹, 282 VGAT⫺)
compared with controls (n ⫽ 12 cells, 230 VGAT⫹, 494 VGAT⫺) (Lower). **, P ⬍
0.01; ***, P ⬍ 0.001 (Mann–Whitney U test). (Scale bars: ⫽ 1 ␮m.)
in mIPSC frequency (Fig. 5C). However, none of the tested
constructs affected the average amplitude of mIPSCs analyzed.
Manipulations of the Levels of Endogenous PSD-95 Alters the Ratio of
Excitatory-to-Inhibitory Synaptic Contacts. The opposite effects ex-
erted by PSD-95 GFP on mEPSCs and mIPSCs suggests that
PSD-95 may have sequestered cell adhesion molecules involved in
excitatory and inhibitory synapse formation to specifically drive the
development of excitatory synapses. Indeed, overexpression of
PSD-95 GFP significantly decreased the percentage of inhibitory
(VGAT-positive) contacts (by 53 ⫾ 5%), without altering the total
number of puncta positive for PSD-95 (Fig. 6A). These results
mirror the physiological changes in the ratio of excitatory兾
inhibitory synaptic currents we observed (see Fig. 5).
We next tested whether blocking expression of endogenous
PSD-95 has opposite effects on the ratio of excitatory-to-inhibitory
synaptic input. Therefore, we generated an siRNA that was de-
signed to specifically decrease endogenous levels of PSD-95 (see
Methods) (Fig. 6 B and C). Cotransfection of PSD-95 siRNA
together with GFP reduced PSD-95 clusters (by 56 ⫾ 5%). In
contrast, no change in the number of PSD-95 clusters was observed
in neurons expressing a scrambled siRNA (Fig. 6B). Expression of
PSD-95 siRNA also resulted in an increase (1.5- ⫾ 0.1-fold) in
inhibitory (VGAT-positive) and a decrease (by 28 ⫾ 7%) in
excitatory (VGAT-negative) synaptic contacts. However, no
change in total number of synaptic contacts was observed (Fig. 6C).
PNAS 兩 September 21, 2004 兩 vol. 101 兩 no. 38 兩 13919
 These results demonstrate that the amounts of PSD-95 available
can dictate the balance between excitatory and inhibitory presyn-
aptic inputs.
In conclusion, our results show a striking difference between the
effects of PSD-95 and NLG in dictating synaptic identity. Whereas
NLG expression results in a dramatic increase of both excitatory
and inhibitory presynaptic contacts, PSD-95 selectively induces
maturation of excitatory presynaptic and postsynaptic elements.
PSD-95, by recruiting NLG at sites of contact, plays a decisive role
in controlling synaptic contact number, morphology, and specific-
ity. In contrast, the lack of specificity of synapses induced by NLG
suggests that NLG is strictly involved in establishing initial synaptic
contacts regardless of their phenotype. The identity of these
contacts may be determined by events that require the recruitment
of additional factors. This study reveals that PSD-95 is one of the
potential factors involved in this process. By assembling a core
postsynaptic complex containing stargazin, ␣-amino-3-hydroxy-5-
methyl-4-isoxazolepropionic acid receptor, and GKAP, PSD-95
may determine synaptic identity (34).
How does NLG communicate with excitatory and inhibitory
presynaptic elements? A known binding partner of NLG is ␤-neur-
exin, a neuron-specific cell surface protein that exists in several
alternatively spliced isoforms (22, 27, 35, 36). Similar to NLG,
neurexins also contain PDZ-binding motifs that bind to type II
PDZ domains present in CASK and syntenin but not PSD-95. The
association of ␤-neurexin to a tripartite protein complex formed of
CASK, Mint-1, and Veli has been proposed to act as a nucleation
site for coupling cell adhesion molecules to synaptic vesicle exocy-
tosis (36). Also, neurexins are directly coupled to synaptotagmins,
core molecules of the synaptic vesicle machinery that regulate
neurotransmitter exocytosis (37). Therefore, binding of NLG to
␤-neurexin may activate an array of presynaptic molecular re-
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We thank Dr. Tim Murphy and Pamela Arstikaitis for valuable sugges-
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Canadian Institutes for Health Research, the Michael Smith Foundation
for Health Research, and the EJLB Foundation (to A.E.-H.). A.E.-H. is
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