Gephyrin clustering is required for the stability of GABAergic synapses

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Mol. Cell. Neurosci. 36 (2007) 484–500
Gephyrin clustering is required for the stability ofGABAergic synapses

Wendou Yu,a Min Jiang,b Celia P. Miralles,a Rong-wen Li,a

Gong Chen,b and Angel L. de Blasa,⁎

aDepartment of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269, USAbDepartment of Biology, Huck Institutes of Life Sciences, The Pennsylvania State University, University Park, Pennsylvania 16802, USA

Received 2 May 2007; revised 10 August 2007; accepted 16 August 2007Available online 23 August 2007

Although gephyrin is an important postsynaptic scaffolding protein atGABAergic synapses, the role of gephyrin for GABAergic synapseformation and/or maintenance is still under debate. We report here thatknocking down gephyrin expression with small hairpin RNAs (shRNAs) incultured hippocampal pyramidal cells decreased both the number ofgephyrin andGABA(A) receptor clusters. Similar results were obtained bydisrupting the clustering of endogenous gephyrin by overexpressing agephyrin-EGFP fusion protein that formed aggregates with the endogenousgephyrin. Disrupting postsynaptic gephyrin clusters also had transsynapticeffects leading to a significant reduction of GABAergic presynaptic boutonscontacting the transfected pyramidal cells. Consistent with the morpholo-gical decrease of GABAergic synapses, electrophysiological analysis re-vealed a significant reduction in both the amplitude and frequency of thespontaneous inhibitory postsynaptic currents (sIPSCs). However, nochange in the whole-cell GABA currents was detected, suggesting a selec-tive effect of gephyrin on GABA(A) receptor clustering at postsynapticsites. It is concluded that gephyrin plays a critical role for the stability ofGABAergic synapses.© 2007 Elsevier Inc. All rights reserved.

Introduction

Gephyrin is a cytoplasmic protein that accumulates at the post-synaptic complex of GABAergic and glycinergic synapses where itforms submembranous lattices associated with postsynaptic clustersof GABAA receptors (GABAARs) and glycine receptors (GlyRs),respectively (Kneussel and Betz, 2000). Studies with a gephyrin-deficient mousemutant (geph−/−) have shown that while gephyrin isessential for the synaptic clustering of glycine receptors (Essrichet al., 1998; Feng et al., 1998; Levi et al., 2004), gephyrin is only

⁎ Corresponding author. Fax: +1 860 486 5439.E-mail address: [email protected] (A.L. de Blas).Available online on ScienceDirect (www.sciencedirect.com).

1044-7431/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.mcn.2007.08.008

essential for the clustering of some GABAARs (Kneussel et al.,1999, 2001; Levi et al., 2004).

The geph−/−mouse mutant dies soon after birth. Thus, the studyof GABAAR clusters in these mutants is normally done inembryonic tissue or neuronal cultures derived from embryonictissue. In the gephyrin knockout mouse or in the correspondingneuronal cultures, some of the observed phenotypes (i.e. decreasednumber of GABAAR clusters) might result from developmentaldefects, while the absence of a phenotypic change might be due tocompensatory mechanisms. Therefore, some of the conclusionsreached with the geph−/− mouse need to be tested with otherindependent approaches. The RNA interference (RNAi, Dykxhoornet al., 2003; Zeringue and Constantine-Paton, 2004) is an alternativeto the gene knockout technology. With the RNA interferenceapproach, there is a knockdown (not a knockout from the day ofgestation) of gephyrin, which is still expressed during the treatment.The knockdown by RNA interference is done during a short time-window (i.e. between 10 and 15 days in culture of E18 neurons). Insuch a short time and with gephyrin being present, it is considerablyless likely that compensatory and/or silencing mechanisms occur. Inthe present study, we have used gephyrin RNAi to knockdown theexpression of gephyrin in cultured hippocampal pyramidal cells.We have also used the overexpression of a gephyrin-EGFP fusionprotein construct, which forms aggregates and interferes with thenormal clustering of endogenous gephyrin. The gephyrin RNAi andgephyrin-EGFP overexpression experiments indicate that gephyrinis essential for the postsynaptic clustering of many GABAARs.

Our approaches have also led to an observation that has not beenuncovered by studying the geph−/− mouse mutant, namely thatpostsynaptic clustering of gephyrin is essential for the maintenanceof the GABAergic synapses. We have previously shown thatknocking down the γ2 GABAAR subunit in pyramidal cells leads todecreased density of both γ2 subunit-containing GABAAR (γ2-GABAAR) clusters and gephyrin clusters and to reduced GABAer-gic innervation on pyramidal cells (Li et al., 2005b). Thus, thepostsynaptic clustering of γ2-GABAARs and gephyrin is tightlylinked to each other and is essential for the stability of presynapticGABAergic contacts.

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485W. Yu et al. / Mol. Cell. Neurosci. 36 (2007) 484–500

Results

Knocking down gephyrin with gephyrin shRNAs decreases theclustering of both gephyrin and GABAARs

In cultured hippocampal neurons, gephyrin forms clustersthat highly colocalize with γ2 subunit-containing GABAAR(γ2-GABAAR) clusters (Craig et al., 1996; Christie et al., 2002b). Inthese cultures, about 90% of gephyrin clusters colocalized with γ2-GABAAR clusters and 95% of all γ2-GABAAR clusters colocalizedwith gephyrin (Christie et al., 2002b). We have investigatedwhether in these cultures the clustering of gephyrin is essential forthe clustering of GABAARs.

Transfection of cultured hippocampal pyramidal cells withshRNAs (Fig. 1A) that target either the coding region of gephyrin(Geph CR) or the non-coding 3′ UTR region of gephyrin (GephUTR) led to a large reduction of both gephyrin and γ2-GABAARclusters in the transfected cells (Figs. 1B and D), when compared tocells transfected with the corresponding mutated shRNAs GephCR3m and Geph UTR3m (Figs. 1C and E, respectively), as revealedby immunofluorescence with antibodies to gephyrin and to the γ2GABAAR subunit, respectively. Transfected pyramidal cells wereidentified by EGFP fluorescence signal that was cotransfected withshRNAs (green color, Figs. 1B–E).

Quantitative immunofluorescence analysis showed that inpyramidal cells transfected with Geph CR shRNA, gephyrin clusterdensity was significantly reduced to 22.3±2.2% when compared tothe gephyrin cluster density in non-transfected sister pyramidal cellsfrom the same culture (Fig. 1B, red fluorescence, and Fig. 2G). Thearrow in Fig. 1B shows dendrites from a non-transfected neuronwithnormal density of gephyrin clusters. Note that the neighbortransfected neuron (green) shows a high reduction in the numberof gephyrin clusters. The pyramidal neurons transfected with GephCR shRNA also showed a significant reduction in γ2-GABAARcluster density (to 27.4±1.7%), as shown in Fig. 1B blue fluo-rescence, and Fig. 2G. The remaining gephyrin and γ2-GABAARclusters showed a high level of colocalization in dendrites (Fig. 1B,arrowheads), where 98.0±3.5% of the remaining gephyrin clusterscolocalized with γ2-GABAAR clusters and 80.0±3.9% of theremaining γ2-GABAAR clusters colocalized with gephyrin. Neu-rons transfected with a control shRNA containing three point mu-tations (Geph CR3m) showed no significant reduction in gephy-rin cluster density (93.9±3.8%) or γ2-GABAAR cluster density(91.6±3.3%) compared with sister non-transfected neurons(Figs. 1C and 2G).

Neurons transfected with a shRNA targeting the 3′ untranslatedregion of gephyrin mRNA (Geph UTR) also showed a largeand significant reduction in gephyrin cluster density (to 36.3±3.0%)and γ2-GABAAR density (to 40.1±2.3%) as shown in Figs. 1D and2H. Pyramidal cells transfected with the corresponding controlshRNA containing three point mutations (Geph UTR3m) showed nosignificant reduction in gephyrin clusters (94.9±3.7%) or γ2-GABAAR clusters (91.5±4.0%) when compared to non-transfectedsister pyramidal cells (Figs. 1E and 2H) from the same culture. Thearrow in Fig. 1D shows dendrites from a non-transfected neuronwith normal density of gephyrin clusters.

The Geph CR and the Geph UTR shRNAs reduced the levels ofgephyrin expression in the culture as shown in immunoblots afternucleofection (Fig. 1F). Quantification of the density of the gephyrinband in immunoblots after normalization with the density of the α-tubulin band, showed that Geph CR and Geph UTR shRNAs

significantly reduced gephyrin expression in hippocampal cultures(to 60.3±3.1% and 67.1±2.1%) when compared with culturestransfected with Geph CR3m and Geph UTR3m shRNAs,respectively (Fig. 1G). The reduction of gephyrin expression inthe whole culture was attenuated when compared to individualtransfected pyramidal cells because only about 40% of the cells inthe culture became transfected after nucleofection, and even the cellstransfected with Geph CR or Geph UTR shRNAs did not showcomplete disappearance of the gephyrin clusters. About 20% of thegephyrin clusters remained in the transfected cells. Thus, in thewhole culture, gephyrin protein is expected to decrease by approx-imately 32% (or 0.4×0.8), which is similar to the gephyrin reductionobserved in the immunoblots after transfecting the cultures withGeph CR and Geph UTR shRNAs (Figs. 1F and G).

Transfection of pyramidal cells with Geph CR or Geph UTRshRNAs also led to large and significant reductions in α2-GABAARcluster density (to 32.2±4.1% and 47.6±3.7%, respectively) and β2/3-GABAAR cluster density (to 47.8±3.1% and 56.6±2.5%, respec-tively), but to little but significant reduction of α1-GABAAR clusterdensity (to 85.2±2.4% and 88.9±2.5%, respectively) compared tonon-transfected sister pyramidal cells (Fig. 2). Similar to theremaining γ2-GABAAR clusters, the remaining α2-GABAAR andβ2/3-GABAAR clusters frequently colocalized with the remaininggephyrin clusters (Figs. 2C and E, arrowheads). In contrast, most ofthe remaining α1-GABAAR clusters did not colocalize with gephy-rin (Fig. 2A, arrowheads). Control experiments showed that trans-fection with mutated Geph CR3m and Geph UTR3m shRNAs hadno significant effect on the cluster density of α2-GABAARs (91.9±3.0% and 93.9±4.1%, respectively), β2/3-GABAARs (98.0±4.1%and 91.9±3.1%, respectively) or α1-GABAARs (97.2±2.5% and96.2±2.2%, respectively) as shown in Fig. 2.

These results showed that knocking down gephyrin led to a highreduction in gephyrin cluster density which was accompanied by asimilar reduction in the density of γ2- and α2-GABAAR clusters. Incontrast, only a small reduction in α1-GABAAR cluster density wasobserved. Moreover, most of the remaining γ2-GABAAR and α2-GABAAR clusters colocalized with the remaining gephyrin clusters,while most of the α1-GABAAR clusters did not. These resultsindicate that gephyrin or gephyrin clustering is essential for theclustering (and/or cluster maintenance) of most of the γ2-GABAARand α2-GABAAR, but not for the clustering of the majority of α1-GABAARs. Our results agree with those reported by Jacob et al.(2005), which showed a 50% reduction of α2-GABAAR clustersafter knocking down gephyrin. Moreover, we have expanded thestudy to additional GABAAR subunits, which has given us insightson the differential effects of knocking down gephyrin on α2-γ2- andα1-GABAARs as well as on the relationship between the remaininggephyrin and GABAAR clusters.

Overexpression of gephyrin-EGFP, a fusion protein that formsfilamentous cytoplasmic aggregates, interferes with the clusteringof endogenous gephyrin and GABAA receptors

Overexpression of gephyrin-EGFP in transfected pyramidal cellsled to the formation of large intracellular filamentous gephyrin-EGFP aggregates in the perikaryon (Fig. 3A, crossed arrows) and inthe proximal segment of some dendrites (Figs. 3B and C, crossedarrows), as identified by EGFP fluorescence (Figs. 3A–C, green) orby an anti-EGFP antibody (not shown) or an anti-gephyrin mAb(Figs. 3A–C, red). The anti-gephyrin mAb recognized not only theendogenous gephyrin but also gephyrin-EGFP as revealed by

Fig. 1. Gephyrin knockdown by shRNAs reduces the density of both gephyrin and γ2-GABAAR clusters. (A) The gephyrin shRNAs used in this study. The three pointmutations introduced in the control shRNAs are shown in red (see Experimentalmethods); (B–E) cultured hippocampal neuronswere cotransfectedwith pEGFP-N1 andGeph CR (B), Geph CR3m (C), Geph UTR (D) or Geph UTR3m (E) shRNAs. Triple-label immunofluorescence was done usingmAb to gephyrin (red color) and rabbitanti-γ2 GABAAR antibodies (blue color). EGFP fluorescence of transfected neurons is shown in green color. The smaller panels at the right side of each figure show athigher magnification the corresponding boxed area. Arrows in panels B and D show dendrites of a non-transfected neuron which has much higher density of gephyrinclusters andγ2-GABAARclusters than the dendrites of the sister neurons (green color) transfectedwithGephCRorGephUTR shRNAs, respectively. Arrowheads showgephyrin clusters that colocalize withγ2-GABAAR clusters. Scale bar: 10μm for large panels; 5 μm for the small panels. (F) Immunoblots with anti-gephyrin and anti-αtubulin antibodies of hippocampal cultures transfected by nucleofection with Geph CR, Geph CR3m, Geph UTR or Geph UTR3m shRNAs. (G) Quantification of theimmunoblots shown in panel F. Values for Geph CR and Geph UTR represent percentages of the normalized intensity of the gephyrin protein bands (normalized to thedensity of α-tubulin) when compared to that of the corresponding 100% control value (pyramidal cells transfected with Geph CR3m or Geph UTR3m shRNAs).

486 W. Yu et al. / Mol. Cell. Neurosci. 36 (2007) 484–500

immunofluorescence of HEK293 cells transfected with gephyrin-EGFP (not shown). The clusters recognized by the anti-gephyrinantibody that showed no EGFP fluorescence (or anti-EGFP

fluorescence) corresponded to clusters of endogenous gephyrin.Immunoblot of hippocampal cultures nucleofected with thegephyrin-EGFP fusion protein (with EGFP at the gephyrin C-

Fig. 2. Gephyrin knockdown reduces the clustering of various GABAAR subunits to different extent. (A–F) Cultured hippocampal neurons were cotransfected withpEGFP-N1 and Geph CR (A, C and E) or Geph CR3m (B, D and F) shRNAs. Triple label immunofluorescence was done by using combinations of the mouse mAb togephyrin and rabbit anti-α1, rabbit anti-α2 GABAAR subunit antibodies or the combination of rabbit anti-γ2 and the mouse mAb to β2/3. Note that the neuronstransfected (EGFP fluorescence) with Geph CR (C and E) show a high reduction in the cluster density of gephyrin and α2, β2/3 and γ2-GABAARs when compared toneurons transfected with the control Geph CR3m (D and F). However, the reduction ofα1 subunit containing GABAAR clusters (A) is small when comparedwith GephCR3m (B). (G andH)Quantification of the effect of the gephyrin shRNAs on gephyrin and the GABAAR cluster density. Values (mean±SEM) represent the percentagescompared to the corresponding internal control (non-transfected sister cells in the same cultures). Significant differenceswith the corresponding internal control values areindicated by asterisks (⁎⁎pb0.01; ⁎⁎⁎pb0.001 in Student's t-test). Comparisons between groups using one-way ANOVATukey test when compared at pb0.05 showedthat the mutated shRNAs (Geph CR3m or Geph UTR3m) had no effect on the density of gephyrin and GABAAR clusters. The cluster densities (number of clusters/100 μm2;mean±SEM) in sister non-transfected cells in the Geph CR shRNA transfection experiments were 21.1±0.9 (632) for gephyrin, 20.9±0.9 (628) for γ2, 23.9±0.8 (717) forα1, 15.7±0.8 (472) forα2 and 17.7±0.8 forβ2/3 (531) (the number of counted clusters is shown in parenthesis). Similar values for the non-transfected cellswere obtained in the other transfection experiments. Arrowheads show colocalizing gephyrin and GABAAR clusters. Scale bar: 5 μm.


terminal) confirmed that the transfected neurons expressed a 118-kDa protein, corresponding to the gephyrin-EGFP fusion protein, asrevealed by immunoblots with a rabbit anti-gephyrin antibody (notshown). The 118-kDa gephyrin-EGFP protein bandwas not detectedin immunoblots of non-nucleofected hippocampal cultures. Inaddition, immunoblots showed that nucleofected and non-nucleo-fected cultures had similar expression levels of the endogenous93 kDa gephyrin protein. The pyramidal neurons that overexpressedgephyrin-EGFP showed reduced density of the endogenousgephyrin clusters (51.5±3.2% Figs. 3A and E, red) 1 day aftertransfection when compared with non-transfected sister neuronsfrom the same culture (100%). Further time-dependent reduction ofgephyrin cluster density was observed 3 days (27.6±2.7%, Figs. 3Band E) and 5 days after transfection (14.6±1.9%, Figs. 3C and E).The results from Fig. 3E also indicate that normal gephyrin clustershave a half-time turnover rate of about 1.5 days.

The time-dependent reduction in gephyrin cluster density wasalso observed when considering actual density values (5.9±0.4;3.9±0.4 and 2.5±0.3 clusters/100 μm2 at 1, 3 and 5 days aftertransfection, respectively, Fig. 3F). It is worth noting that between11 and 15 DIV (corresponding to 1 and 5 days after transfection,respectively) non-transfected cells show an increase in the densityof gephyrin (Fig. 3F) and γ2-GABAAR clusters (Fig. 3G) as part of

the normal developmental maturation of GABAergic synapses. Allthese results show that gephyrin-EGFP expression disrupts existinggephyrin clusters and interferes with formation of new gephyrinclusters. The reductions in endogenous gephyrin clusters occurredin all dendrites of the pyramidal cells showing gephyrin-EGFPaggregates, even though the aggregates per se were often restrictedto a few dendrites. The gephyrin-EGFP filamentous aggregateswere not found in non-transfected neurons, which showed normallevels of endogenous gephyrin clusters (Figs. 3C and D, arrows).

Overexpression of gephyrin-EGFP also decreased the densityof the γ2-GABAAR clusters to 95.0±4.3%, 50.1±3.5% and 22.4±2.5% (Figs. 3A–C and E, blue) 1, 3 and 5 days after transfection,respectively, when compared with non-transfected sister neurons(100%). More importantly, these experiments also showed thatthe disappearance of γ2-GABAAR clusters was delayed about 1–2 days with respect to the disappearance of endogenous gephyrinclusters (Fig. 3E), which is consistent with the notion that gephyrinclustering is essential for the stability of many of the existing γ2-GABAAR clusters. The remaining endogenous gephyrin clustersshowed high colocalization with the remaining γ2-GABAARclusters (Figs. 3A and B, white filled arrowheads). However, therewere many γ2-GABAAR clusters, particularly at 1 day and 3 daysafter transfection, that had no colocalizing gephyrin clusters


(Figs. 3A–C, black filled arrowheads), which is consistent with thedisappearance of gephyrin clusters prior to the disappearance of γ2-GABAAR clusters. The time-dependent reduction in γ2-GABAARcluster density was also observed when considering actual densityvalues (11.9±0.5; 7.0±0.5 and 4.0±0.4 clusters/100 μm2 at 1, 3and 5 days after transfection, respectively, Fig. 3G).

Transfection of pyramidal cells with gephyrin-EGFP or gephyrinshRNAs was done at day 10 in culture, a time at which many but notall gephyrin and γ2-GABAARs clusters are formed in our cultures

(Christie et al., 2002a, and legend to Fig. 3). We have previouslyshown that gephyrin clusters and γ2-GABAAR clusters are alreadypresent in these cultures after 3.5 days and that a large number ofthese clusters are present after 8.5 days in culture (Christie et al.,2002a). Figs. 3F and G show that although the density of bothgephyrin and γ2-GABAAR clusters increased in non-transfectedneurons from day 11 to 15, both densities decreased substantiallyfrom 1 day to 5 days after gephyrin-EGFP overexpression, sug-gesting that the formation of new gephyrin and γ2-GABAAR

Fig. 4. Overexpression of non-tagged gephyrin slightly increases the density of both gephyrin and GABAAR-γ2 clusters. Cultured hippocampal neurons werecotransfected with EGFP and non-tagged gephyrin. (A) Triple-label immunofluorescence was done at 1, 3 and 5 days after transfection, using a mAb to gephyrinand a rabbit anti-γ2 antibody. (B and C) The density of gephyrin and γ2-GABAAR clusters is increased in the gephyrin transfected cells when compared withnon-transfected sister cells at 1, 3 or 5 days after transfection, respectively. (⁎pb0.05, in Student's t-test). Scale bar: 5 μm.


clusters is seriously compromised in addition to the reduction ofexisting clusters. These results support the notion that gephyrinclustering is critically important for both the formation and themaintenance of many γ2 subunit containing GABAAR clusters.

It has been proposed that normal gephyrin clustering occurs byhomo-oligomerization of gephyrin molecules forming submembra-nous hexagonal lattices via the N-terminal G-domain, which canform trimers, and the C-terminal E-domain, which can form dimers(Kim et al., 2006). This clustering of gephyrin would facilitate theaccumulation of glycine receptors and GABAAR at the postsynapticmembrane (Kim et al., 2006). It has been reported that native EGFPcan also form dimers (Yang et al., 1996). Thus, the addition of EGFPto the C-terminal of gephyrin may alter the normal hexagonal clus-tering of gephyrin, leading to the formation of filamentous aggre-gates containing both gephyrin-EGFP and endogenous gephyrin.

Fig. 3. Overexpression of gephyrin-EGFP decreases endogenous gephyrin and GABAEGFP. (A–D) Triple-label immunofluorescence, at 1 day (A), 3 days (B) and 5 days (C, D(blue color). Note that the pyramidal cells overexpressing gephyrin-EGFP form large(crossed arrows). Arrows in panels C and D show that the dendrites of non-transfecttransfected neurons expressing gephyrin-EGFP (green color) show reduced cluster dcolocalizing gephyrin clusters and γ2-GABAAR clusters. Black arrowheads show γfluorescence was not detected in gephyrin clusters of normal aspect, which are presumabthe reduction of gephyrin and γ2-GABAAR clusters after transfection of pyramidal cellsdensity of the endogenous gephyrin (Geph+/EGFP−) clusters to 51.5±3.2%, 31.8±3.2%compared with control (100%) sister non-transfected neurons from the same culture (Overexpression of gephyrin-EGFP also led to a delayed reduction (with respect to gephyrand 22.4±2.5% at 1, 3 and 5 days, respectively, after transfection when compared with c0.5; 3 day 15.2±0.7; 5 day 17.7±0.6 clusters/100 μm2, panel G). Significant differenStudent's t-test). Scale bar: 10 μm for the large panels; 5 μm for the small panels.

The decreased clustering of endogenous gephyrin likely results frombeing trapped by the gephyrin-EGFP aggregates that forms at theperikaryon and the proximal dendrites, thus preventing the deliveryof both endogenous gephyrin and gephyrin-EGFP to synaptic sites.

Overexpression of non-tagged gephyrin slightly increases thedensity of both gephyrin and GABAA receptor clusters

We transfected hippocampal cultures with non-tagged gephyrin,aiming to avoid the formation of gephyrin aggregates induced by theEGFP tagging of gephyrin. Neurons transfected with gephyrin wereidentified by cotransfection with EGFP. Fig. 4A shows thatpyramidal cells transfected with non-tagged gephyrin had normalgephyrin clusters (Fig. 4, Geph column) instead of the abnormal

AR-γ2 clusters. Cultured hippocampal neurons were transfected with gephyrin-) after transfection, with amousemAb to gephyrin (red color) and rabbit anti-γ2filamentous aggregates of gephyrin-EGFP in the soma and proximal dendritesed neurons have normal density of gephyrin and γ2-GABAAR clusters, whileensity of gephyrin or γ2-GABAAR in cell dendrites. White arrowheads show2-GABAAR clusters without colocalizing gephyrin clusters. Gephyrin-EGFPly formed by endogenous gephyrin (anti-Geph+/EGFP−). (E–G) Time course ofwith gephyrin-EGFP. Overexpression of gephyrin-EGFP led to a reduction in the, 14.6±1.9% (mean±SEM) at 1, 3, 5 days, respectively, after transfection when1 day 11.4±0.5; 3 day 14.1±0.5; 5 day 17.3±0.7 clusters/100 μm2, panel F).in clusters) in the density of theγ2-GABAARclusters to 95.0±4.3%, 50.1±4.3%ontrol (100%) sister non-transfected neurons from the same culture (1 day 12.6±ces with the corresponding control are indicated by asterisks (⁎⁎⁎pb0.001 in


aggregates that occurred when cells were transfected with gephyrin-EGFP. Pyramidal cells transfected with non-tagged gephyrin alsoshowed γ2-GABAAR clusters of normal appearance (Fig. 4A, γ2column), similar to those of non-transfected sister cells. Quantifica-

Fig. 5. The knockdown of gephyrin in pyramidal cells decreases the GABAergic inncotransfected with pEGFP-N1 and Geph CR (A), Geph CR3m (B), Geph UTR (Cusing amousemAb to gephyrin and a sheep anti-GAD antibody. Green color shows tto dendrites of non-transfected neurons, having a normal density of gephyrin clusterspostsynaptic gephyrin clusters. Note that the pyramidal cells transfected with Gephand GAD+ boutons than the corresponding controls Geph CR3m (B) or Geph UTRshRNAs on the density of GAD+ boutons contacting pyramidal cells (E) and on thepercentage of control (mean±SEM). Control values were determined in non-transfecGeph CR shRNA transfection experiment had 57.5±4.5 GAD+ boutons/cell (meancells by GAD+ terminals was 89±6% (n=54 cells). There is significant decrease in thhas been knocked down, compared to non-transfected cells or cells transfected withsignificant differences in the density of GAD+ boutons contacting the pyramidal celltransfected cells (one-way ANOVATukey test when compared at pb0.05). Scale b

tion revealed that the transfected cells had slightly increased densityof gephyrin clusters compared with non-transfected sister cells(Fig. 4B). These statistically significant effects were observed asearly as 1 day after transfection (transfected 12.0±0.5 vs. non-

ervation that these cells receive. (A–D) Cultured hippocampal neurons were) or Geph UTR3m (D) shRNAs. Triple-label immunofluorescence was donehe EGFP fluorescence of transfected neurons. Arrows in panels A and C pointandGAD+ boutons. Arrowheads showpresynaptic GAD+ boutons apposed toCR (A) or Geph UTR (C) shRNAs show a lower density of gephyrin clusters3m (D), respectively. (E and F) Quantification of the effect of the gephyrinnumber of pyramidal cells showing synaptic GAD+ boutons (F) expressed asted sister cells in the same cultures. The non-transfected pyramidal cells of the±SEM, n=30 cells). The percentage of innervated non-transfected pyramidale GAD+ terminals contacting the transfected pyramidal cells whose gephyrinthe mutated shRNAs (⁎⁎⁎, pb0.001, Student's t-test). However, there are nos transfected with gephyrin CR3m or gephyrin UTR3m shRNAs and the non-ar: 10 μm for large panels; 5 μm for small panels.


transfected 10.7±0.4 clusters/100 μm2). The effect was alsoobserved 3 days after transfection (15.1±0.7 vs. 12.8±0.5 clus-ters/100 μm2) and 5 days after transfection (20.0±0.7 vs. 17.6±0.7clusters/100 μm2). Similar statistically significant but smallincreases in the density of γ2-GABAAR clusters were observed(Fig. 4C) 1 day (13.0±0.5 vs. 11.3±0.4 clusters/100 μm2), 3 days(15.7±0.7 vs. 13.9±0.5 clusters/100 μm2) and 5 days (21.1±0.7 vs.18.8±0.7 clusters/100 μm2) after transfection. As indicated above,between day 11 and 15 (1 and 5 days after transfection, respectively),non-transfected cells show an increase in the density of gephyrinand γ2-GABAAR clusters as part of their normal developmentalmaturation of these cultures. Thus, gephyrin overexpression leadsonly to a small increase in gephyrin cluster density in the transfectedpyramidal cells, indicating that the maximum density of gephyrinclusters is tightly regulated. A similar small increase in the γ2-GABAAR clusters after gephyrin overexpression indicates that theclustering of γ2-GABAAR is precisely coupled to the clustering ofgephyrin during synaptic development.

In cultured pyramidal cells, decreased gephyrin clustering resultingfrom knocking down gephyrin by shRNA is accompanied bydecreased GABAergic innervation of the targeted pyramidal cells

Pyramidal cells transfected with Geph CR or Geph UTR shRNAshowed not only reduced gephyrin cluster density, as describedabove, but also reduced GABAergic innervation, as determined bythe density of GAD+ presynaptic boutons contacting transfectedpyramidal cells, compared with that of sister non-transfectedneurons (Figs. 5A and C, respectively, green color). Similar resultswere obtained when we used an antibody to VIAAT (vesicularinhibitory amino acid transporter) to identify presynaptic GABAer-gic boutons (not shown). We have previously shown that the GAD+

boutons apposed to GABAAR clusters concentrate the GABAergicsynaptic vesicle marker VIAAT (also named vesicular GABAtransporter or vGAT) and the general synaptic vesicle marker SV2(Li et al., 2005b). We have also shown that in these cultures, theseGAD-containing boutons have actively recycling synaptic vesicles(Christie et al., 2002b).

The non-transfected pyramidal cells showed normal levels ofboth gephyrin clusters and apposed GAD+ boutons (Figs. 5A and C,arrows). In pyramidal cells transfected with Geph CR or Geph UTRshRNAs, the remaining GAD+ boutons from GABAergic axonsinnervating these cells were also apposed to remaining postsynapticgephyrin clusters (Figs. 5A and C, arrowheads). The dendrites ofpyramidal cells transfected with control shRNA Geph CR3m andGephUTR3m (Figs. 5B andD, respectively) showed normal densityof GAD+ presynaptic boutons, which were apposed to postsynapticgephyrin clusters (Figs. 5B and D, arrowheads).

Quantification (Fig. 5E) shows that the density of GAD+

boutons contacting pyramidal cells transfected with Geph CR orGeph UTR shRNAs was reduced to 49.2±4.2% and 51.9±4.0%,respectively of the non-transfected sister neurons (100%). Incontrast, pyramidal cells transfected with Geph CR3m or GephUTR3m shRNAs did not show significant reduction in the densityof GAD+ boutons (91.5±4.7% for Geph CR3m and 95.1±3.1% forGeph UTR3m). Similar results were obtained when we quantifiedthe percentage of pyramidal cells that received GABAergic inner-vation (Fig. 5F). In the pyramidal cells transfected with Geph CRor Geph UTR, the percentage of these neurons that receivedGABAergic innervation was reduced (to 52.8±5.1% and 57.9±5.3%, respectively) compared to 83.9±11.9% and 91.1±9.4%

for pyramidal cells transfected with Geph CR3m and GephUTR3m.

We also performed control rescue experiments by cotransfectingneurons with Geph UTR shRNA and gephyrin mRNA thatcontained the coding region but not the 3′-UTR. In this way, theGeph UTR shRNA targeted the endogenous gephyrin mRNA butnot the exogenous gephyrin mRNA. Supplementary Fig. 1 showsthat the exogenous gephyrin mRNA rescued gephyrin clustering,γ2-GABAAR clustering and GABAergic innervation by GAD+

boutons to control levels.We have previously shown that knocking down the GABAAR γ2

subunit in pyramidal cells with γ2 shRNAs led not only to reducedclustering of both postsynaptic GABAARs and gephyrin but also to areduced presynaptic GABAergic innervation of these neurons byGABAergic interneurons (Li et al., 2005b). We are now showingthat knocking down gephyrin with gephyrin shRNA also leads toreduced gephyrin and GABAAR clustering and to decreasedGABAergic innervation of the pyramidal cells. What is commonin both experimental conditions is the reduced clustering of bothgephyrin and γ2-GABAARs. Therefore, the postsynaptic clusteringof gephyrin and γ2-GABAARs is mutually dependent on each other,and the presynaptic GABAergic innervations rely upon intactpostsynaptic gephyrin and γ2-GABAAR clusters.

The notion that the postsynaptic clustering of gephyrin isessential for maintaining the normal level of GABAergic innerva-tion of the pyramidal cells is also supported by the gephyrin-EGFPoverexpression experiments shown next.

In cultured pyramidal cells, decreased clustering of endogenousgephyrin resulting from overexpression and aggregation ofgephyrin-EGFP is also accompanied by decreased GABAergicinnervation of the targeted pyramidal cells

As shown in above, overexpression of gephyrin-EGFP intransfected pyramidal cells led to the formation of filamentousaggregates of gephyrin-EGFP (anti-gephyrin+/EGFP+, crossedarrows, Fig. 6A) and to decreased density of endogenous gephyrinclusters (anti-gephyrin+/EGFP−, arrowheads, Fig. 6A), whencompared with sister non-transfected cells (Fig. 6B). Now we areshowing that the transfected pyramidal cells also have reducedGABAergic innervation from interneurons as shown by decreaseddensity of GAD+ presynaptic boutons contacting these cells(arrowheads, Fig. 6A, blue) when compared to non-transfectedcells (arrowheads, Fig. 6B, blue). In both transfected and non-transfected cells, the GABAergic terminals were apposed toendogenous gephyrin clusters (arrowheads, Fig. 6A, red) thatshowed no EGFP fluorescence. However, the GAD+ terminalswere not apposed to the gephyrin-EGFP aggregates (crossedarrows, Fig. 6A), indicating that the presynaptic GABAergicterminal did not form synaptic contacts with the dendritic areaswhere gephyrin-EGFP aggregates were localized. Quantificationshowed that pyramidal cells overexpressing gephyrin-EGFP had43.6±2.5 GAD+ boutons/cell (n=30 cells) compared with 97.8±3.0 GAD+ boutons/cell in sister non-transfected pyramidal cells(Fig. 6C). The proportion of transfected neurons that receivedGABAergic innervations was 54.8±3.1% (n=62 cells) comparedwith 88.6±3.6% for non-transfected neurons (n=70 cells, Fig. 6D).These experiments further support the notion that the normalpostsynaptic clustering of gephyrin, rather than the level ofexpression, is essential for maintaining the normal levels of pre-synaptic GABAergic innervation. As indicated above, immunoblots

Fig. 6. Transfection of pyramidal cells with gephyrin-EGFP leads to decreased GABAergic innervation while transfection with non-tagged gephyrin leads to littleor no effect on GABAergic innervation. (A, B) Triple label immunofluorescence of cultured hippocampal pyramidal cells that were transfected with gephyrin-EGFP using a mouse mAb to gephyrin (red) and a sheep anti-GAD antibody (blue). Crossed arrows in panel A show aggregates of gephyrin-EGFP. There is asignificant reduction of gephyrin clusters and GAD+ boutons contacting the pyramidal neurons overexpressing gephyrin-EGFP (A), when compared with thesister non-transfected cells from the same culture (B). Arrowheads show presynaptic GAD+ boutons apposed to postsynaptic gephyrin clusters. There is noapposition of gephyrin-EGFP aggregates with GAD+ boutons. (C and D) Quantification shows that overexpression of gephyrin-EGFP reduces the density ofGAD+ boutons contacting the transfected pyramidal cells and the number of these cells that are contacted by GAD+ terminals (⁎⁎⁎pb0.001, in Student's t-test).(E and F) Triple-label immunofluorescence of pyramidal cells cotransfected with EGFP and non-tagged gephyrin. Arrowheads show gephyrin clusters apposedto GAD+ boutons. (G and H) Quantification shows that pyramidal cells transfected with non-tagged gephyrin had little or no increase in either the density ofGAD+ boutons (⁎p=0.041, Student's t-test) or the percentage of pyramidal cells innervated by GAD+ terminals (p=0.063). Scale bar: 10 μm for large panels;5 μm for small panels.



of hippocampal cultures transfected with gephyrin-EGFP did notshow significant changes in the expression of endogenous gephyrin.

Transfection of pyramidal cells with non-tagged gephyrin leads toa slight increase in the GABAergic innervations of these cells

We have shown in Fig. 4 that transfecting pyramidal cells withnon-tagged gephyrin leads to a small increase in gephyrin and γ2-GABAAR cluster density over non-transfected sister neurons. We

Fig. 7. The knockdown of gephyrin by gephyrin shRNA decreases inhibitory synapfrom non-transfected (left panel), Geph CR3m-transfected (middle panel) and Geph(B) Bar graphs showing that the average sIPSC amplitude was similar between npN0.9). In contrast, the sIPSC amplitude in the Geph CR group was greatly decregraphs showing that the average sIPSC frequency was similar between non-transfecthe sIPSC frequency in the Geph CR group was significantly reduced (0.16±0.04 Hrapid application of GABA (20 μM) from non-transfected (left panel), Geph CR3m-tSummarized data showing no difference in the GABA current amplitude amongpN0.6) and Geph CR transfected neurons (1823±207 pA; pN0.1).

have investigated whether this small increase in gephyrin and γ2-GABAAR clusters affects the GABAergic innervation of these cells.Pyramidal cells transfected with non-tagged gephyrin (Fig. 6E,green) showed a slight increase in GABAergic innervation (Fig. 6E,blue) when compared with sister non-transfected pyramidal cells(Fig. 6F) of the same culture measured as GAD+ boutons contactingthese cells. Quantification showed that there was small butsignificant increase in the GABAergic innervation that thetransfected cells received when compared to sister non-transfected

tic currents. (A) Representative recordings of spontaneous synaptic responsesCR-transfected neurons (right panel). Cells with typical sIPSCs are presented.on-transfected (112.5±23.2 pA) and Geph CR3m groups (109.8±19.0 pA;ased (56.0±9.0 pA; pb0.02) comparing to the Geph CR3m group. (C) Barted (0.79±0.27 Hz) and the Geph CR3m group (0.92±0.22 Hz; pN0.7), butz; pb0.002). (D) Typical recordings showing whole-cell currents induced byransfected (middle panel) and Geph CR-transfected neurons (right panel). (E)non-transfected (2045±288 pA), Geph CR3m transfected (2252±202 pA;


cells (72.3±2.5 vs. 66.0±1.8 boutons/cell, respectively, n=30 cells,p=0.041, Fig. 6G). Nevertheless, there was no significant change inthe percentage of the transfected pyramidal cells that receivedGABAergic innervation (92.9±2.3% vs. 85.7±2.5%, p=0.063,n=30, Fig. 6H). Thus, overexpression of non-tagged gephyrin inpyramidal cells leads to little or no effect on the density ofGABAergic innervation of these cells.

Fig. 8. The knockdown of gephyrin with gephyrin shRNA does not affect the dencotransfected with EGFP and Geph CR (A, C) or Geph CR3m (B, D) shRNAs. Tgephyrin (blue color) and a rabbit anti-GluR1 (A and B, red color) or a guinea pig anPyramidal cells transfected with Geph CR shRNAs show a high reduction of gephyrGeph CR3m (B, blue, black arrowheads). However, the same neurons showed no obvGlut1+ boutons (C, red, white arrowheads), compared to the neurons transfected wiGluR1, GluR2/3, NR1, PSD-95 clusters and vGlut1+boutons in pyramidal cells transcompared to the corresponding internal control (non-transfected sister cells in the sathe cluster density of various glutamatergic markers between transfected and non-trtest showed that the Geph CR, Geph UTR and the mutated shRNAs (Geph CR3m an95 clusters or vGlut1 boutons (when compared at pb0.05). The cluster densities (numshRNA transfection experiments were 16.0±0.5 (481) for GluR1, 16.5±0.7 (508)±0.5 (402) for vGlut1 (the number of clusters or vGlut1+ boutons is shown in pexperiments. Scale bar: 10 μm for large panels; 5 μm for small panels.

Knocking down gephyrin by gephyrin shRNA decreases inhibitorysynaptic currents

To further investigate whether knocking down gephyrin willcause any functional changes of GABAergic synaptic transmission,we performed electrophysiological recordings on neurons trans-fected with either Geph CR or Geph CR3m shRNA (together with

sity of glutamatergic synapses. (A–D) Cultured hippocampal neurons wereriple-label immunofluorescence was done using a combination of a mAb toti-vGlut1 (C and D, red color). Transfected neurons show green fluorescence.in clusters (A, blue, black arrowheads), compared to neurons transfected withvious difference in the density of GluR1 clusters (A, red, white arrowheads) orth Geph CR3m (B and D, respectively). (E, F) Quantification of the density offected with gephyrin shRNA. Values (mean±SEM) represent the percentagesme culture). There was no significant difference (pN0.05, Student's t-test) inansfected cells. Comparisons between groups using one-way ANOVATukeyd Geph UTR3m) had no effect on the density of GluR1, GluR2/3, NR1, PSD-ber of clusters/100 μm2; mean±SEM, 100%) in the controls of the Geph CR

for GluR2/3, 16.2±0.1 (501) for NR1, 15.7±0.7 (471) for PSD-95 and 13.4arentheses). Similar control values were obtained in the other transfection


EGFP). We examined the frequency and amplitude of spontaneousIPSCs in both transfected groups as well as non-transfectedneurons. We found that the sIPSC amplitude significantlydecreased in cells expressing Geph CR shRNA in comparisonwith either non-transfected or Geph CR3m-transfected cells(Figs. 7A and B). The significant decrease of the sIPSC amplitudeafter knocking down gephyrin is in accordance with the reductionin γ2-GABAAR clusters observed in our immunofluorescenceexperiments and with the notion that gephyrin plays a critical rolein stabilizing postsynaptic GABAA receptors. Moreover, inaddition to the amplitude change, we also found that the sIPSCfrequency was significantly reduced in the Geph CR group but notin cells transfected with Geph CR3m (Figs. 7A and C). The largefrequency decrease observed in neurons transfected with Geph CRcannot be solely explained by a decrease in the amplitude andsuggests a potential functional deficit in the presynaptic GABArelease mechanism. This presynaptic deficit is also consistent withour immunofluorescence data, which show that the density ofpresynaptic GABAergic boutons was significantly reduced afterknocking down gephyrin. To further examine whether the knock-down of gephyrin affected the expression level of GABAARs oncell membranes, we recorded whole-cell GABA currents in cellsfrom non-transfected, Geph CR3m-transfected and Geph CR-transfected groups. No significant difference was found in thewhole-cell GABA currents among all three different groups(Figs. 7D and E). These data suggest that although gephyrin iscritical for the clustering of many GABAARs at postsynapticsites, knocking down gephyrin may not affect the total ex-pression and insertion level of GABAARs in neuronal plasmamembranes.

Treatments that lead to decreased density of gephyrin clusters,γ2-GABAAR clusters and GABAergic innervation do not affect thedensity of glutamatergic synapses

As shown above, pyramidal cells transfected with Geph CRshRNA showed significant decreased density in gephyrin clusters(Figs. 8A and C, blue panels, black arrowheads) when comparedwith cells transfected with Geph CR3m (Figs. 8B and D, res-pectively, blue panels, black arrowheads). However, the same trans-fected cell (Geph CR shRNA) showed no apparent changes in thedensity of GluR1 AMPA receptor clusters (Figs. 8A vs. B, redpanels, white arrowheads) or vGlut1-contaning presynaptic gluta-matergic boutons (Figs. 8 C vs. D, red panels, white arrowheads).Quantification (Figs. 8E and F) showed that Geph CR or Geph UTRshRNA had no significant effect on the density of AMPA receptorGluR1 and GluR2/3 subunit clusters, NMDA receptor NR1 subunitclusters, PSD-95 clusters or vGlut1-containing presynaptic gluta-matergic boutons, when compared with sister non-transfected cellsfrom the same culture (100%) or with cells transfected with GephCR3m or Geph UTR3m, respectively.

We have shown above that pyramidal cells transfected withgephyrin-EGFP showed gephyrin-EGFP aggregates and reduceddensity of gephyrin clusters, γ2-GABAAR clusters and GABAer-gic innervation. However, the gephyrin-EGFP transfected cellsalso showed no changes in the density of PSD-95 or vGlut1presynaptic terminals (not shown). These experiments furtherindicate that the disruption of gephyrin clusters by either gephyrinshRNAs or gephyrin-EGFP leads to a specific disruption of thestability of GABAergic synapses but not of glutamatergicsynapses.

Discussion

We have addressed the function of gephyrin at GABAergicsynapses by using experimental approaches independent of thegephyrin knockout mouse which had generated somewhat con-troversial results previously (Kneussel et al., 1999, 2001; Kneusseland Betz, 2000; Levi et al., 2004). With the use of both gephyrinRNAi and gephyrin-EGFP aggregation to disrupt gephyrin cluster-ing, we have found that not only the number of gephyrin clusters aregreatly reduced in transfected hippocampal pyramidal cells, but alsothe number of γ2, α2 and β2/3 GABAAR subunit clusters are allsignificantly reduced correspondingly in these cells. In contrast,there was little reduction in the number of α1 GABAAR subunitclusters. Our approach has also allowed us to reveal some functionalroles of gephyrin that had not been previous observed by studyingthe gephyrin knockout mouse. Thus, the time course following thedisruption of gephyrin clusters has shown that gephyrin clustering isessential for the maintenance of existing postsynaptic GABAARclusters. More interestingly, we have also shown that disruptingnormal postsynaptic gephyrin clustering in these cells leads to asignificant reduction in the total number of GABAergic synapses.Thus, our results support the notion that gephyrin clustering isessential for the stability of GABAergic synapses.

By studying a gephyrin knockout mouse (geph−/−), others haveconcluded that gephyrin is essential for the clustering of GABAARs(Kneussel et al., 1999; Kneussel and Betz, 2000). Nevertheless,although using the same mouse mutant line, others have reported theexistence of many GABAAR synaptic clusters in the geph−/−mousemutant (Levi et al., 2004). Thus, it has been reported that the intactspinal cord or cultured hippocampal neurons from the gephyrin-deficient (geph−/−) mouse show a large reduction in the number ofclusters of the γ2, α2 and β2/3 GABAAR subunits while theclustering of the α1 GABAAR subunit was not affected (Kneusselet al., 2001; Levi et al., 2004) when compared with littermate controlmice (geph+/+ or geph+/−), which agrees with our results using adifferent experimental approach.

It has been proposed that the interaction with synaptic gephyrinreduces the lateral diffusion of GABAARs and glycine receptors,thus facilitating their accumulation at GABAergic synapses andglycinergic synapses, respectively (Meier et al., 2001; Jacob et al.,2005; Charrier et al., 2006). This type of mechanism would explainwhy disrupting gephyrin clusters by shRNAs or gephyrin-EGFPaggregation leads to the disruption of many GABAAR clusters. Thedisruption of the GABAAR clusters by gephyrin-EGFP over-expression was delayed by approximately 1–2 days following thedisruption of the gephyrin clusters. Our results support the notionthat gephyrin clustering is essential for maintenance of manyGABAAR clusters at GABAergic synapses.

An important observation derived from our studies is thatinterfering with the normal postsynaptic clustering of gephyrinand/or disrupting existing gephyrin clusters in pyramidal neurons(by either gephyrin shRNAs or overexpression and aggregation ofthe gephyrin-EGFP) also leads to reduced presynaptic GABAergicinnervation that these pyramidal neurons received, as shown by thedecreased number of GAD-containing (or VIAAT-containing)presynaptic terminals contacting these pyramidal cells. We havepreviously shown that knocking down the GABAAR γ2 subunit inpyramidal cells with γ2 shRNAs not only disrupted GABAARclusters and gephyrin clusters in pyramidal cells but also decreasedthe GABAergic innervation that these cells received (Li et al.,2005b). These results and the results presented in this commu-


nication indicate that (i) the disruption of the postsynaptic γ2-GABAARs clusters is accompanied by disruption of gephyrinclusters; (ii) the disruption of gephyrin clusters is accompanied bythe disruption of many GABAARs clusters; and (iii) the co-ordinated disruption of the postsynaptic clustering of GABAARsand gephyrin has transsynaptic effects that lead to reducedinnervation of pyramidal cells by GABAergic interneurons. Theresults obtained by immunofluorescence were confirmed bymeasuring inhibitory synaptic currents in hippocampal culturestransfected with gephyrin shRNAs. Moreover, the disruption of theGABAARs and gephyrin clustering and GABAergic innervation bygephyrin shRNAs, or γ2 GABAAR subunit shRNAs or gephyrin-EGFP overexpression does not affect the density of AMPAreceptor clusters, PSD-95 clusters or presynaptic vGlut1-contain-ing glutamatergic boutons (Fig. 8 and Li et al., 2005b).

It has been reported that in the geph−/− knockout mouse therewas no difference in the number of VIAAT containing terminals inthe spinal cord (Kneussel et al., 2001) or in GAD+-containingterminals in hippocampal cultures (Levi et al., 2004) when comparedto the geph+/+ or the geph+/− littermates. The apparent discrepancyin the results between the knockout mouse and the RNAi expe-riments could be explained because in the geph−/− mouse none ofthe neurons express gephyrin at any stages of development while inthe RNAi experiments some neurons have gephyrin knocked down,while other sister neurons in the same culture express normal levelsof gephyrin and clustering. In the latter configuration, GABAergicsynapses on pyramidal cells that have low levels of postsynapticgephyrin and GABAAR clusters might be at a competitive dis-advantage with the synapses that the same interneurons form withneighboring pyramidal cells that have normal levels of gephyrin andGABAAR clusters (for a more extensive discussion and other pos-sible explanations for the different results obtained with the mouseknockout and RNAi technologies, see Li et al., 2005b). Anotherpossible explanation is that, although the density of presynapticGABAergic terminals in the geph−/− mutant is similar to that ofthe geph+/+ mouse, in the mutant mouse some GABAergicterminals could be mislocalized. Another possible explanation isthe off-target effects of the gephyrin shRNAs (Alvarez et al., 2006).However, this is unlikely since (i) the mutated shRNAs show noeffect on GABAergic synapses and (ii) the effects of Geph UTR onGABAergic synapses could be reversed by exogenous gephyrinmRNA that did not contain the 3′-UTR (rescue experiment). More-over, the same neurons that showed decreased density of severalGABAergic pre- and postsynaptic markers showed little effect onthe density of α1-GABAAR clusters and no effect on the density ofglutamatergic synaptic markers. Moreover, overexpression ofgephyrin-EGFP interfered with endogenous gephyrin clusteringand had effects similar to those of gephyrin shRNAs, namely de-creased density of both postsynaptic GABAAR clusters and pre-synaptic GABAergic terminals.

In another study using gephyrin shRNA, the density of VIAAT-containing terminals in hippocampal cells although reduced, it wasreported not to be significant (Jacob et al., 2005). In the latter study,neurons were nucleofected at day 0 and the synaptic markers weretested by immunofluorescence at 14 DIV. In our study, neuronswere transfected at 10 DIVand synaptic markers were assayed at 15DIV. Knocking down gephyrin from day 0 (Jacob et al., 2005)might lead to a compensatory mechanism as it could be the case inthe gephyrin knockout mouse. Another explanation is that Jacobet al. 2005 used high-density neuronal cultures, which complicatesthe determination of whether a presynaptic terminal is contacting

the dendrite of a transfected cell or the dendrite of a non-transfectedcell from the same bundle, since dendrites from several neuronstend to bundle together, particularly in the high-density cultures. Wepurposely used low-density cultures where the detection ofindividual synapses on transfected cells can be assessed moreaccurately. In experimental conditions similar to the ones we haveused in the present communication, and in agreement with ourresults, Fang et al. (2006) have reported that knocking down thepalmitoyl acyltransferase GODZ with GODZ RNAi in culturedpyramidal cells, led to reduced number of GABAAR clusters and todecreased GABAergic innervation of these cells. All of theseexperiments suggest that the postsynaptic clustering of gephyrinand GABAAR is required for the normal formation and/ormaintenance of the GABAergic presynaptic contacts and functionalsynapses.

It is conceivable that the disruption of postsynaptic clustering ofgephyrin and GABAARs also disrupts the postsynaptic clustering ofa cell recognition molecule that transsynaptically interacts with apresynaptic cell recognition partner. Candidate cell recognitionmolecules are postsynaptic neuroligins (NLs), which transsynapti-cally interact with presynaptic neurexins (NRXs) and laterally withpostsynaptic NRXs (Taniguchi et al., 2007). The NLs–NRXsinteractions play a central role in the formation and/or maintenanceof both glutamatergic and GABAergic synapses (Scheiffele et al.,2000; Dean et al., 2003; Graf et al., 2004; Prange et al., 2004;Boucard et al., 2005; Chih et al., 2005, 2006; Levinson et al., 2005;Dean and Dresbach, 2006; Graf et al., 2006; Varoqueaux et al.,2006). Of particular interest are NL-1A and NL-2A, which arepreferentially localized at GABAergic synapses while NL-1B, NL-1AB, NL-3 and NL-4 preferentially localize at glutamatergic sy-napses (Song et al., 1999; Graf et al., 2004; Varoqueaux et al., 2004;Boucard et al., 2005; Levinson et al., 2005; Chih et al., 2006).Knocking down postsynaptic NLs by RNAi in hippocampalpyramidal cells leads to preferential decreased number of GABAer-gic over glutamatergic presynaptic contacts (Chih et al., 2005).Moreover, NL-2 has been demonstrated to be critical in molecularreconstitution of GABAergic synapses in HEK293 cells (Dong etal., 2007). Transsynaptic effects leading to the reduction in thenumber of presynaptic GABAergic terminals that innervate thepostsynaptic neuron occur after (i) disruption of the postsynapticgephyrin clusters (as shown in the present communication); (ii)disruption of the postsynaptic γ2−GABAAR clusters (Li et al.,2005b); (iii) or by preventing the trafficking of postsynapticGABAARs at the synapse (Fang et al., 2006). These transsynapticeffects could result from the simultaneous disruption of theclustering of postsynaptic neuroligins at GABAergic synapses. Apreliminary meeting report suggests that accumulation of NL-2 atGABAergic synapses is strictly dependent on postsynapticGABAARs (Luscher B, unpublished observations). The uncluster-ing of postsynaptic NLs would result in a weakened interaction withpresynaptic neurexins at GABAergic synapses, followed by thewithdrawal of presynaptic GABAergic innervation.

Other postsynaptic cell recognition molecules that could beinvolved in these transsynaptic effects are EphB receptors. SomeEphB receptors interact with glutamate receptor interacting proteinsor GRIPs (Bruckner et al., 1999; Contractor et al., 2002). We haverecently found that some splice variants of GRIP1, a family ofmolecules containing up to 7-PDZ domains, are postsynapticallylocalized at GABAergic synapses (Charych et al., 2004b, 2006;Kittler et al., 2004; Li et al., 2005a). Postsynaptic EphB receptorstranssynaptically interact with presynaptic ephrinBs.


Thus, it is conceivable that the clustering of postsynapticneuroligins, EphB receptors and other transsynaptic moleculestogether with the concentration of their interacting partners at thepresynaptic GABAergic terminals lead to the strengthening of thetranssynaptic interactions and the stabilization of the GABAergicsynaptic contacts.

Experimental methods

Antibodies

The following GABAAR antibodies raised in our laboratory were used:the rabbit and guinea pig anti-rat α1 GABAAR subunit antibodies were toamino acids 1–15 (QPSQDELKDNTTVFT); the rabbit anti-rat α2 GABAARsubunit antibody was to amino acids 417–423 (PVLGVSP); the mousemonoclonal (mAb) anti β2/3 GABAAR subunit antibody (clone 62-3G1) wasraised to the affinity purified GABAA receptor (De Blas et al., 1988; Vitoricaet al., 1988), which recognizes an N-terminal epitope present in both β2 andβ3 subunits but not in β1 subunit (Ewert et al., 1992); rabbit and guinea piganti-rat γ2 GABAAR subunit antibodies were to amino acids 1–15(QKSDDDYEDYASNKT). These anti-GABAAR antibodies have beenthoroughly characterized and their specificities determined previously byELISA, immunoprecipitation of GABAARs, immunoblotting, displacementby antigenic peptide, rat brain immunochemistry at the light microscopy andelectron microscopy levels, immunofluorescence of hippocampal culturesand of HEK293 cells transfected with various GABAAR subunits and someknockout and knockdown mutant mice (De Blas et al., 1988; Vitorica et al.,1988; Homanics et al., 1999; Miralles et al., 1999; Christie et al., 2002a,b;Riquelme et al., 2002; Christie and De Blas, 2003; Charych et al., 2004a,b;Chandra et al., 2005; Li et al., 2005a,b; Christie et al., 2006; Serwanski et al.,2006). The mouse mAb to gephyrin (clone mAb 7a) used in immunocy-tochemistry was from Cedarlane (Accurate Chemical & Scientific Corp.,Westbury, NY). The rabbit anti-gephyrin antibody used in immunoblots wasfrom Dr. Ben Bahr (University of Connecticut, Storrs, CT). The mouse anti-PSD-95 (postsynaptic density protein 95) antibody was from UpstateBiotechnology (Lake Placid, NY). Guinea pig anti-vGlut1 (vesicularglutamate transporter 1) and rabbit anti-GluR1 antibodies were fromChemicon (Temecula, CA). Rabbit anti-GluR2/3 was a gift from Dr. RobertJ. Wenthold (NIDCD, Bethesda, MD). Sheep anti-GAD was a gift from Dr.Irwin J. Kopin (NINDS, Bethesda, MD). Mouse anti-α tubulin was fromSigma (Saint Louis, MO). All fluorophore-conjugated secondary antibodieswere from Jackson ImmunoResearch (West Grove, PA).

Construction of various gephyrin plasmid vectors

Two gephyrin small hairpin RNAs (shRNAs) were made (Fig. 1A), onetargeting the gephyrin coding region (Geph CR, nucleotides 609–633)corresponding to the seventh exon of the gephyrin gene (GenBankaccession number NM_022865.3; gi145553981), and the second onetargeting nucleotides 2401–2425 of the gephyrin 3′ UTR non-coding region(Geph UTR) corresponding to the twenty-first exon. Control shRNAs foreach (Geph CR3m and Geph UTR3m, respectively) were made carryingthree point mutations in the sense and antisense strands (Fig. 1A). TheseshRNA were inserted in the mU6 vector and synthesized under the controlof RNA U6 polymerase III promoter (Yu et al., 2002; Li et al., 2005a,b).Gephyrin hairpin DNA oligonucleotides and their corresponding comple-mental oligonucleotides were synthesized and purified by PAGE (IntegratedDNA Technologies Inc. Coralville, IA). The DNA oligonucleotides weredesigned to create compatible overhangs on each end after annealing. Thedouble-stranded DNA was inserted into the mU6pro vector between theBbsI and XbaI sites. The antisense strand matched the target gene perfectly.A mismatched nucleotide was introduced in the sense strand to facilitatesequencing of the hairpin DNA (Fig. 1A).

The human gephyrin cDNA clone FJ06168 (Gene named KIAA1385)was kindly provided by Dr. Nobumi Kusuhara (Kazusa DNA ResearchInstitute, Japan). This gephyrin isoform, without the c5 cassette, is involved

in clusteringGABAARs (Meier andGrantyn, 2004). The full-length gephyrincoding sequence was amplified by PCR and inserted into the HindIII andXhoI sites of pcDNA3.1(+) or NheI and SacII sites of pEGFP-N1, aiming togenerate non-tagged gephyrin or a gephyrin-EGFP fusion protein with theEGFP tag located at the C-terminal of gephyrin, respectively. The quality ofthe constructs was confirmed by DNA sequencing and protein expressionwas detected by both immunofluorescence and immunoblotting aftertransfection of HEK293 cells and hippocampal neurons.

Transfection of hippocampal cultured neurons for immunofluorescence

Primary hippocampal cultures were prepared from embryonic day 18(E18) Sprague–Dawley rat brains by the method of Goslin et al. (1998) asdescribed previously (Christie et al., 2002a,b; Christie and De Blas, 2003;Charych et al., 2004a,b; Li et al., 2005a,b) and maintained in glial cellconditioned medium containing 1% N2 supplement (Invitrogen, Carlsbad,CA). Cultured hippocampal neurons (10 day old in culture) werecotransfected with 3 μg of the shRNA vector and 1 μg of the pEGFP-N1vector (molar ratio 4:1), or with 3 μg of gephyrin vector and 1 μg of thepEGFP-N1 vector (molar ratio 2:1) or 4 μg of gephyrin-EGFP vectors usingthe CalPhos Mammalian Transfection Kit (BD Biosciences, San Jose, CA),according to the instructions provided by the company. Fluorescenceimmunocytochemistry was performed 5 days after transfection with shRNAor 1–5 days after transfection with gephyrin-EGFP or gephyrin.

Nucleofection of hippocampal cultured neurons and protein expression

Nucleofection was used to determine the effect of shRNAs on proteinexpression. Primary rat hippocampal neurons, prepared by the method ofGoslin et al. (1998) as described above, were transfected with the shRNAs athigh culture density using the Rat Neuron Nucleofector Kit (Amaxa GmbH,Koln, Germany) according to the instructions provided by the manufacturer.Transfection was done at day 0 before plating. Twenty-four hours later, themedium was changed to Dulbecco's modified Eagle's medium (DMEM)supplemented with 5% horse serum, 1% N2 supplement and 1 mM sodiumpyruvate solution (Invitrogen) in a 5% CO2 atmosphere. Neurons werecollected at day 16, dissolved in sample dissociation buffer and subjected tosodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE)followed by immunoblotting with anti-gephyrin and anti-α tubulinantibodies and detected by chemiluminescence (ECL; Amersham Bios-ciences, England). Intensity of the protein bands was quantified afterscanning the X-ray film optical density with a BioRad Gel Doc 2000imaging system driven by Quantity One 4.4.0 software (BioRad, Hercules,CA).

Transfection of hippocampal cultured neurons for electrophysiologyexperiments

Hippocampal cultures were generated from E16 to E18 rat embryos asrecently described (Chen et al., 2003; Yao et al., 2006). Briefly, thehippocampal CA1–CA3 region was dissected, cut into ~1 mm3 cubes andincubated with 0.05% trypsin-EDTA in Hanks' balanced salt solution(HBSS) for 20 min at 37 °C. The tissue blocks were triturated in HBSS with10% horse serum. Dissociated cells were plated onto a monolayer ofastrocytes, in MEM, 5% fetal bovine serum, 20% B27 (Invitrogen,Carlsbad, California, USA), 0.5 mM L-glutamine and 25 unit ml−1

penicillin/streptomycin, 4 μM arabinosyl cytosine in a 5% CO2 atmosphere.Cells were transfected at 7–8 DIV by using a modified Ca2+-phosphatetransfection protocol as described elsewhere (Jiang and Chen, 2006).Electrophysiology was done 48–72 h after transfection.

Immunofluorescence of hippocampal cultures

Immunofluorescence was done as described elsewhere (Christie et al.,2002a,b; Charych et al., 2004a,b; Li et al., 2005a,b). Except wherementioned, all steps were done at room temperature. Briefly, neurons werefixed in 4% paraformaldehyde/4% sucrose/phosphate-buffered saline (PBS,


pH 7.4) for 15 min followed by washing with PBS. Aldehyde groups werequenched by incubation with 50 mM NH4Cl in PBS for 10 min followed bypermeabilization with 0.25% Triton X-100 in PBS for 10 min. Cells wereincubated with 5% normal donkey serum in PBS for 30 min followed byincubation with a mixture of primary antibodies diluted in 0.25% Triton X-100 in PBS overnight at 4 °C. Cells were washed and incubated with amixture of secondary antibodies (anti-species specific IgG all made indonkey, Jackson Immunochemicals, West Grove, PA) conjugated tofluorescein isothiocyanate (FITC), Texas Red or aminomethylcoumarin(AMCA) fluorophores (1:200 dilution in 0.25% Triton X-100 in PBS) for1 h at 37 °C. The coverslips were washed with PBS followed by washingwith PBS (pH8.5) and mounting using Prolong Gold anti-fade mountingsolution (Molecular Probes, Eugene, OG).

Image acquisition and analysis

Digital images for hippocampal cultures were collected using a 60×pan-fluor objective on a Nikon Eclipse T300 microscope with a SensysKAF 1401E CCD camera driven by IPLab 3.0 acquisition software(Scanalytics, Fairfax, VA). Images obtained from different channels wereprocessed and merged with PhotoShop 7.0 (Adobe) for analysis. Brightnessand contrast were adjusted, the image was changed from 16 bits/channel to8 bits/channel (1315×1035 pixel resolution), sharpened using the unsharpmask tool (setting: amount=125%, radius=1.5 pixel, threshold=0 level),color was added to each channel and the images were merged for colorcolocalization. Fluorescent images in figures were presented beforesubtraction of the diffuse background signal in dendrites.

Quantification of clusters

For quantification of cluster density in neurons, the backgroundfluorescence of each channel seen in the dendrites was subtracted and themaximum intensities of the fluorophore channels were normalized. Threeindependent immunofluorescent experiments were performed for eachcombination of antibodies. A total of 30 dendritic fields were analyzed from20 to 30 randomly selected pyramidal neurons. Each measurement wastaken from a 25-μm long, 4-μm-wide dendritic segment. Density valueswere calculated as number of clusters per 100 μm2 of dendritic surface. Thenumber of gephyrin clusters analyzed for each experiment was in the rangeof 374–750. Nevertheless, when clustering was highly inhibited byshRNAs, the number of clusters analyzed ranged 141–178. For quantifyingthe number of neurons that received GABAergic innervation, 31–62transfected and 54–70 non-transfected pyramidal neurons were randomlyselected from three individual experiments and the number of neurons thatwere contacted by GAD+ presynaptic terminal was recorded. The density ofthe GAD+ presynaptic boutons was calculated as the number of boutons percell.

Whole-cell patch-clamp recordings

Whole-cell recordings of sIPSCs and GABA application-inducedcurrents were made in voltage-clamp mode using a MultiClamp 700Aamplifier (Molecular Devices Corporation, Sunnyvale, CA, USA). Patchpipettes were pulled from borosilicate glass and fire polished (4–6 MΩ).The recording chamber was continuously perfused with a bath solutionconsisting of (mM): 128 NaCl, 30 glucose, 25 HEPES, 5 KCl, 2 CaCl2,1 MgCl2, pH 7.3 adjusted with NaOH. Patch pipettes were filled with thefollowing (mM): 135 KCl, 10 Tris–phosphocreatine, 2 EGTA, 10 HEPES,4MgATP, 0.5 Na2GTP, pH 7.3 adjusted with KOH. The series resistance wastypically 10–20 MΩ. The membrane potential was held at −70 mV. Datawere acquired using pClamp 9 software (Molecular Devices Corporation,Sunnyvale, CA, USA), sampled at 2–10 kHz and filtered at 1 kHz. Offlineanalysis was done with Clampfit 9 software. The spontaneous IPSC (sIPSC)events were separated from sEPSCs according to their distinguish-able decay time constants (τ=2–5 ms for sEPSCs; τ=9–18 ms for sIPSCs)and analyzed using MiniAnalysis software (Synaptosoft). The decay timeconstants were obtained from pharmacological isolation using CNQX

(10 μM) to inhibit sEPSCs or using BIC (20 μM) to inhibit sIPSCs. All datawere expressed as mean±SEM and Student's t-test was used for statisticalanalysis.

Acknowledgments

This work was supported by The National Institute ofNeurological Disorders and Stroke grants NS38752 and NS39287to Angel De Blas, and Johnson & Johnson/Pennsylvania StateUniversity Innovative Technology Research Grant to Gong Chen.We would like to thank Dr. Nobumi Kusuhara for the gephyrinconstruct. We would also like to thank Dr. Ben Bahr for the rabbitanti-gephyrin antibody, Dr. Irwin J. Kopin for the sheep anti-GADantibody and Dr. Robert J. Wenthold for the rabbit anti GluR2/3antibody.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.mcn.2007.08.008.

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Gephyrin clustering is required for the stability of GABAergic synapses

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