colocalization of multiple gabaa receptor subtypes with gephyrin at postsynaptic sites

Colocalization of Multiple GABAA

Receptor Subtypes With Gephyrin atPostsynaptic Sites

MARCO SASSOE-POGNETTO,1 PATRIZIA PANZANELLI,1 WERNER SIEGHART,2AND

JEAN-MARC FRITSCHY3*1Department of Anatomy, University of Turin, I-10126 Torino, Italy

2Department of Biochemical Psychiatry, University Clinic for Psychiatry, A-1090 Vienna,Austria

3Institute of Pharmacology, University of Zurich, CH-8057 Zurich, Switzerland

ABSTRACTClustering of gamma aminobutyric acid (GABA)A receptors to postsynaptic sites requires

the presence of both the g2 subunit and gephyrin. Here, we analyzed by double-immunofluorescence staining the colocalization of gephyrin and major GABAA-receptor sub-types distinguished by the subunits a1, a2, a3, or g2 in adult rat brain. By using confocallaser scanning microscopy, GABAA-receptor subunit staining revealed brightly stained clus-ters that were colocalized with gephyrin-positive clusters of similar size and distribution inseveral brain regions, including cerebellum, hippocampus, thalamus, and olfactory bulb. Inaddition, a diffuse staining was observed for GABAA-receptor subunits in the neuropil,presumably representing extrasynaptic receptors. Overall, only few gephyrin-positive clus-ters were not colocalized with GABAA-receptor subunit clusters. Electron microscopic anal-ysis in cerebellar cortex confirmed the selective postsynaptic localization of gephyrin. High-resolution images (voxel size, 50 3 50 3 150 nm) were restored with an iterative imagedeconvolution procedure based on a measured point-spread function to analyze the colocal-ization between GABAA-receptor subunits and gephyrin in individual clusters. This analysisrevealed a considerable heterogeneity in the micro-organization of these presumptiveGABAergic postsynaptic sites. For instance, whereas gephyrin- and g2 subunit-positiveclusters largely overlapped in the cerebellar molecular layer, the colocalization was onlypartial in glomeruli of the granule cell layer, where small gephyrin clusters typically were“embedded” in larger GABAA-receptor clusters. These findings show that gephyrin is asso-ciated with a majority of GABAA-receptor subtypes in brain, and document the usefulness ofimage deconvolution for analyzing the structural organization of the postsynaptic apparatus byfluorescence microscopy. J. Comp. Neurol. 420:481–498, 2000. © 2000 Wiley-Liss, Inc.

Indexing terms: GABAergic transmission; inhibitory synapses; synaptic clustering; confocal laser

scanning microscopy; image deconvolution

Gamma aminobutyric acid (GABA) is the main inhibi-tory neurotransmitter of the central nervous system(CNS). Its fast synaptic action is mediated by GABAA-receptors, which form hetero-pentameric chloride chan-nels assembled from a large family of homologous sub-units encoded by distinct genes (a1–6, b1–3, g1–3, e, p, d,u; for review, see Mohler et al., 1996; Hevers and Luddens,1998; Whiting et al., 1999). Heterogeneity of GABAA-receptors is thought to arise from the differential assem-bly of these subunits into receptor subtypes with distinctpharmacological and functional properties. The majorityof functional GABAA-receptors comprise variants of a and

b subunits, along with the g2 subunit. The analysis ofmutant mice lacking a particular GABAA-receptor subunitrevealed that the a and b subunits are required for as-

Grant sponsor: Swiss National Foundation for Scientific Research; Grantnumber: 31-52869-97; Grant sponsor: Italian M.U.R.S.T.

*Correspondence to: Dr. Jean-Marc Fritschy, Institute of Pharmacology,University of Zurich, Winterthurerstr. 190, CH - 8057 Zurich, Switzerland.E-mail: [email protected]

Received 18 October 1999; Revised 10 January 2000; Accepted 11 Jan-uary 2000

THE JOURNAL OF COMPARATIVE NEUROLOGY 420:481–498 (2000)

© 2000 WILEY-LISS, INC.

sembly and cell-surface targeting of the correspondingGABAA-receptor subtypes (Fritschy et al., 1997, 1998a;Homanics et al., 1997; Jones et al., 1997; Krasowski et al.,1998). In contrast, the g2 subunit, although being dispens-able for the formation of GABA-gated chloride channels,was essential for synaptic localization and clustering ofmajor GABAA-receptor subtypes (Essrich et al., 1998). Inaddition, gephyrin, a peripheral membrane protein firstidentified as a component of the glycine-receptor (GlyR)complex (Pfeiffer et al., 1984), is also required for cluster-ing of postsynaptic GABAA-receptors (Essrich et al., 1998;Kneussel et al., 1999). However, because a direct protein-protein interaction between GABAA-receptors and gephy-rin has never been observed conclusively, the role ofgephyrin in the clustering process remains elusive.

Although GABAA-receptors are ubiquitous and ex-pressed by virtually all CNS neurons, comparatively littleis known about their subcellular distribution and postsyn-aptic localization. This is due in part to methodologicallimitations, because immunohistochemical labeling ofsynaptic GABAA-receptors is highly sensitive to fixation inboth light and electron microscopic (EM) procedures(Greferath et al., 1995; Nusser and Somogyi, 1997;Fritschy et al., 1998b). Studies using postembedding EMhave demonstrated that GABAA-receptors are highly en-riched at postsynaptic sites and are differentially localizeddepending on their subunit composition (Nusser et al.,1995a, 1996b, 1998; Somogyi et al., 1996). For instance, inhippocampal pyramidal cells, a2-GABAA-receptors wereselectively localized in the axon initial segment, whereasa1-GABAA-receptors were distributed in synapses on thesoma and dendrites (Nusser et al., 1996a). However,postembedding EM is very labor-intensive and only a fewbrain regions have been investigated so far. Likewise,studies of the distribution of gephyrin have been limitedmostly to regions enriched in glycinergic synapses (Trilleret al., 1985, 1987; Altschuler et al., 1986; Wenthold et al.,1988; Cabot et al., 1995; Todd et al., 1995, 1996; Alvarez etal., 1997), and only a few data are available regarding itspresence in central GABAergic synapses (Sassoe-Pognettoet al., 1995; Giustetto et al., 1998).

In this study, we have investigated the association be-tween GABAA-receptors and gephyrin at postsynapticsites in adult rat brain to determine whether it is re-stricted to particular receptor subtypes or to certain typesof synapses (axosomatic, axodendritic, axo-axonic).Double-immunofluorescence staining was analyzed withconfocal laser scanning microscopy (CLSM) to determinethe colocalization of gephyrin with major GABAA-receptorsubtypes. These experiments were performed in lightlyfixed tissue optimized for the detection of synapticGABAA-receptors and gephyrin in situ. In such material,both markers appear as brightly stained puncta, whichrepresent presumptive clusters of GABAA-receptor sub-units or gephyrin at synaptic sites. The analysis firstfocused on the cerebellar cortex, in which an extensiveheterogeneity of GABAA-receptors has been described.The selective postsynaptic distribution of gephyrin in cer-ebellum was confirmed by pre-embedding EM. The anal-ysis was then extended to other brain regions, to includeadditional receptor subtypes and types of synapses. Thesestudies revealed an extensive coexistence of gephyrin clus-ters with major subtypes of GABAA-receptors throughoutthe brain. Finally, an iterative image deconvolution pro-cedure was applied to high-resolution images from confo-

cal laser scanning microscopy (voxel size 50 3 50 3 150nm) to compare the subcellular distribution of GABAA-receptors and gephyrin in individual clusters. This anal-ysis revealed a considerable heterogeneity in the micro-organization of clusters containing both GABAA-receptorsand gephyrin, notably in cerebellar glomeruli. These re-sults validate the use of image deconvolution for analyzingthe synaptic organization of receptor proteins by immuno-fluorescence microscopy.

MATERIALS AND METHODS

Animals

All experiments were performed on adult Wistar rats(n 5 6 for immunofluorescence and n 5 3 for EM) raisedunder standard laboratory conditions with unrestrictedaccess to food and water. The procedures used were ap-proved by the animal care committee of the University ofTurin.

Immunofluorescence staining

Parasagittal cryostat sections (12–14 mm) cut fromfresh-frozen brains were mounted onto gelatinized slidesand stored at 220°C. They were then thawed, fixed byimmersion in 2% formaldehyde (freshly depolymerizedfrom paraformaldehyde in 0.1 M phosphate buffer, pH 7.4)for 90 seconds, and rinsed two times in phosphate-buffered saline (PBS, pH 7.4). The sections were thenincubated for 2 hours in primary antibodies diluted inPBS containing 2–5% normal serum and 0.05% TritonX-100. The following antibodies were used: guinea pigantisera against the GABAA-receptor subunits a1(1:10,000), a2 (1:1,000), a3 (1:700), and g2 (1:2,000; forcharacterization, see Fritschy and Mohler, 1995; Fritschyet al., 1998b), rabbit antisera against the d subunit (1:1,000), and the a6 subunit (1:200; Nusser et al., 1996b;Jones et al., 1997). For double-immunofluorescence stain-ing, these antisera were combined with either monoclonalantibody (mAb) 7a (1:200) recognizing gephyrin or mAb2b(1:50; kindly provided by Dr. H. Betz, Frankfurt, Ger-many) recognizing the a1 subunit of the GlyR (Pfeiffer etal., 1984). The sections were rinsed with PBS and incu-bated for 30 minutes with secondary antibodies (goat anti-guinea pig or anti-rabbit coupled to indocarbocyanine[Cy3] and goat anti-mouse coupled to fluorescein isothio-cyanate [FITC]) diluted 1:1,000 and 1:50, respectively, inPBS (Jackson Immunoresearch, West Grove, PA). Theywere rinsed again and coverslipped with Mowiol or withbuffered glycerol (pH 9.2).

Data analysis

Sections were analyzed by CLSM (Leica TCS 4D, Hei-delberg, Germany), by using simultaneous dual channelrecording of double-labeled sections. The intensity of theemission lines (488 nm and 568 nm) was adjusted individ-ually to eliminate possible “bleed-through” between therecorded channels. Typically, stacks of 18–36 images(512 3 512 pixels) spaced by 250 nm were recorded at amagnification of 200 nm/pixel, with the photomultipliersettings adjusted for sampling over their full dynamicrange. The digitized images were processed with the soft-ware Imaris (Bitplane, Zurich, Switzerland). Noise waseliminated by baseline subtraction (5–10%). For display,projections of 5–8 consecutive confocal layers were calcu-

482 M. SASSOE-POGNETTO ET AL.

lated (maximal pixel intensity) and contrast-optimized.Alternatively, a three-dimensional rendering effect wasachieved from stacks of 18–36 confocal layers by a “sim-ulated fluorescence” algorithm. In these images, spatialrelationships can be inferred from shadows projected froma virtual light source.

A semiquantitative analysis of the number of GABAA-receptor and/or gephyrin clusters was performed on singleconfocal images (100 mm 3 100 mm, resolution 200 nm/pixel), using a threshold segmentation algorithm for theautomatic detection of clusters. The minimal size of clus-ters was arbitrarily set to 0.1 mm2, corresponding to threeadjacent pixels at the magnification used, and the thresh-old of intensity was set at twice the background intensitymeasured in the baseline-subtracted images (typically at10–20% of the maximal intensity). The results are derivedfrom a total of 10 measurements in five sections fromthree different animals and are expressed as mean 6 S.D.

Clusters double-labeled for gephyrin and a givenGABAA-receptor subunit were identified with “colocaliza-tion” algorithm that selected and marked all voxels la-beled with both markers above a user-defined threshold ofintensity. The threshold typically was set at an intensitygreater than twice the background measured in thebaseline-subtracted images (see Fig. 7C for an example).The selected voxels were color-coded and the correspond-ing clusters quantified as described above.

For high-resolution analysis, an image deconvolutionprocedure (Huygens System 2, Scientific Volume Imaging,Hilversum, The Netherlands) was applied to series of im-ages (voxel size 50 3 50 3 150 nm) captured with a 1003lens with a numerical aperture of 1.4 (zoom factor 4). Aniterative “maximal likelihood estimate” was calculatedfrom the raw data by using a measured point-spread func-tion. The latter was derived from stacks of 48–60 imagesof fluorescent spherical microspheres (diameter, 170 nm;Molecular Probes, Eugene, OR) embedded in bufferedglycerol (refraction index 1.442) with calibrated coverslips(0.17 6 0.01 mm; Karl Hecht Assistant GmbH; Sondheim,Germany) and recorded under identical conditions to thesections to be analyzed. Green and red fluorescent micro-spheres (maximal emission at 515 and 565 nm, respec-tively) were recorded separately to generate the point-spread function corresponding to these emissionwavelengths. The accuracy of the point-spread functionswas checked by deconvolution of microsphere images (seeFig. 8).

For quantification of the number, diameter, and appar-ent surface area (in the x-y plane) of gephyrin- andGABAA-receptor subunit-positive clusters in deconvolutedimages, individual clusters were first reconstructed inthree dimensions using an “isosurface” algorithm. Withthis method, virtual surfaces were generated that inter-connected all voxels with an intensity superior to a setthreshold (10–20% of maximal intensity; see Fig. 10). Thenumber and size of these clusters were then measuredwith the threshold segmentation algorithm, as describedabove, with the minimal size of a cluster being set at 0.01mm2 (corresponding to 100 nm 3 100 nm).

Electron microscopy

For preembedding immunohistochemistry, the ratswere deeply anesthetized with Ketamine (100 mg/kg) andXylazine (5 mg/kg) and perfused through the left ventriclewith PBS, followed by 400 ml of 4% paraformaldehyde

(freshly dissolved in phosphate buffer). The cerebellarhemispheres were dissected and cut into 70-mm tangentialsections on a Vibratome. The sections were cryoprotectedin 30% sucrose and repeatedly frozen and thawed to en-hance antibody penetration. They were then collected inPBS and processed free-floating as described in detailelsewhere (Sassoe-Pognetto et al., 1994). After incubationin the primary (mAb7a, 1:1,000) and secondary (goat anti-mouse conjugated to biotin, 1:250, Vector, Burlingame,CA) antibodies, the sections were treated with 3,39-diaminobenzidine (DAB), and the reaction product wassilver-intensified and gold-toned. Finally, the sectionswere postfixed with 1% osmium tetroxide (in 0.1 M caco-dylate buffer), dehydrated in acetone and flat-embeddedin Epon 812. Serial ultrathin sections were taken perpen-dicular to the plane of the Vibratome sections, stainedwith uranyl acetate and lead citrate and observed in aPhilips EM 410 electron microscope.

RESULTS

Distribution of gephyrin in adult rat brain

In lightly fixed cryostat sections cut from fresh-frozenbrains, gephyrin-immunoreactivity (IR) appeared exclu-sively as brightly stained puncta distributed throughoutthe brain, presumably reflecting the clustered postsynap-tic distribution of this protein. The density of gephyrinclusters varied across brain areas, largely mirroring thedistribution of GABAergic terminals as reported withstaining for glutamic acid decarboxylase (Mugnaini andOertel, 1985). In the cerebellum, gephyrin clusters wereuniformly distributed across the molecular layer, were fewon Purkinje cell somata, and were very dense in glomerulithroughout the granule cell layer (Fig. 1). The latter clus-ters were distinctly smaller than those in the molecularlayer. In addition, a few, rather large clusters ofgephyrin-IR were distributed in the granule cell layer, notassociated with glomeruli. An intense, punctate gephyrinstaining was also evident in the deep cerebellar nuclei,outlining the soma and dendritic arbors of individual neu-rons (not shown).

Ultrastructural localization of gephyrinin cerebellum

The ultrastructural localization of gephyrin was inves-tigated in the cerebellar cortex with the preembeddingmethod. Gephyrin-IR was found exclusively at synapticjunctions, where it was concentrated at the cytoplasmicside of the postsynaptic membrane (Figs. 2, 3). This sup-ports the idea that the puncta detected with immunoflu-orescence correspond to synaptic clusters of gephyrin. Thegephyrin-positive synapses were invariably of the sym-metric type. Because the immunoreaction product tendedto mask the postsynaptic specialization, the criterion usedfor the characterization of labeled synapses was mainlythe shape of the vesicles in the presynaptic profiles. Thus,labeled synapses were characterized by the presence ofpleomorphic vesicles, whereas junctions with round vesi-cles and a well- defined postsynaptic specialization werealways unlabeled (Figs. 2C, 3A).

In the molecular layer, the gephyrin-positive synapseswere usually large, with a diameter that often exceeded400 nm. They were frequently located on the dendriticshafts of Purkinje cells, as defined by the presence of

483GABAA-RECEPTOR SUBTYPES AND GEPHYRIN

cisternal stacks (Fig. 2A,B). In some cases, the labeledsynapses involved small dendrites, which could not beidentified unambiguously (Fig. 2C). However, it is likely

that at least some of them belonged to interneurons.Gephyrin-positive synapses between presumed basket cellaxons and Purkinje cell soma were also observed (Fig. 2D).

In the granule cell layer, the labeled synapses wereusually much smaller than in the molecular layer andrarely exceeded 200 nm in diameter. Thus, the size of thegephyrin-positive synapses determined in the electron mi-croscope is consistent with the presence of immunofluo-rescent puncta of different sizes in the molecular layer andin the granule cell layer, respectively (Fig. 1). By examin-ing serial sections through distinct cerebellar glomeruli,we found that gephyrin-IR was located at synapses madeby the axon terminals of Golgi cells with the dendrites ofgranule cells (Fig. 3A–C). Conversely, the asymmetricalsynapses made by mossy fibers were not labeled (Fig. 3A).

Colocalization of gephyrin and GABAA-receptor subunits

Sections processed for double-immunofluorescencestaining with mAb7a and an antiserum against one of theGABAA-receptor subunit a1, a2, a3, a6, d, or g2 wereanalyzed by CLSM to assess the colocalization of gephyrinwith GABAA-receptors. In cerebellar sections, staining forthe a1 and g2 subunits revealed numerous brightlystained puncta, corresponding presumably to postsynapticclusters. In addition, a weak diffuse staining of the neu-ropil, possibly representing extrasynaptic receptors, wasobserved. These subunits were extensively colocalizedwith gephyrin in individual clusters (Fig. 4A,C,D). Thus,in the molecular layer, the a1 and g2 subunit-IR appearedcolocalized with most gephyrin-positive clusters, whereason Purkinje cell somata most GABAA-receptor subunitclusters were single labeled (Fig. 4A,C). In the granule celllayer, the a1 and g2 subunit-IR in glomeruli was lessclearly punctate than gephyrin-IR. The pattern of colocal-ization was assessed by high resolution CLSM as de-scribed in the next section. In addition, the g2 subunit, butnot the a1 subunit, was colocalized with the large, isolatedgephyrin clusters present outside of glomeruli (Fig. 4A,C).In contrast to the a1 subunit, the a3 subunit-IR was of lowabundance in the cerebellum. It was detected primarily inthe granule cell layer, outlining the soma and dendrites ofisolated neurons probably corresponding to Golgi type IIcells. Glomeruli were devoid of a3 subunit-IR. Some a3subunit-positive clusters were also seen in the molecularlayer, mostly in the inner half. All a3 subunit-positiveclusters were colocalized with gephyrin (Fig. 4B). Finally,

Fig. 2. Electron photomicrographs showing the synaptic localiza-tion of gephyrin in the molecular layer of the cerebellar cortex. A andB show gephyrin-positive synapses on the shaft of Purkinje cell den-drites (PC), which are identified by cisternal stacks (arrowheads).Gephyrin-immunoreactivity (IR; small arrows) is concentrated at thecytoplasmic face of the postsynaptic membrane. Note the presence ofpleomorphic vesicles in the presynaptic profiles. Asymmetric syn-apses made by parallel fibers with the dendritic spines (sp) of Purkinjecells are not labeled (hollow arrows). C: Two adjacent axon terminalsare in contact with an unidentified dendritic profile. One terminal(Ax1) contains pleomorphic vesicles and makes a gephyrin-positivesynapse (arrow). The other terminal (Ax2) contains round vesiclesand makes an unlabeled, asymmetric synapse (hollow arrow).D: Gephyrin-positive synapses (arrows) between a presumed basketcell axon (Ax) and the perikaryon of a Purkinje cell (PC). Scale bar 50.4 mm.

Fig. 1. Distribution of gephyrin-immunoreactivity (IR) in cerebel-lar cortex as visualized by immunofluorescence staining of fresh-frozen tissue. Note the uniform distribution of clusters in the molec-ular layer (ML) and their paucity in the Purkinje cell layer (PCL). Inthe granule cell layer (GCL), gephyrin-IR was most abundant inglomeruli, forming densely packed small clusters at the border ofthese structures (inset). Scale bar 5 11 mm.


Figure 2


Fig. 3. Synaptic localization of gephyrin in cerebellar glomeruli. A–C: Synapses (arrows) betweenpresumed Golgi cell terminals (Go) and granule cell dendrites. Gephyrin-immunoreactivity (IR) isclustered close to the postsynaptic membrane. Mossy fibers (MF) make asymmetric synapses (hollowarrow in A) which are always unlabeled. Scale bar 5 0.4 mm.

Fig. 4. Colocalization of gephyrin and GABAA-receptor subunits incerebellar cortex visualized by double-immunofluorescence stainingand confocal laser scanning microscopy (CLSM). Each panel repre-sents the superposition of gephyrin-immunoreactivity (IR; green) andGABAA-receptor subunit-IR (red), with sites of colocalization appear-ing yellow. A: a1 subunit. Gephyrin and the a1 subunit are exten-sively colocalized. Only a few clusters in the molecular layer (ML) andin the granule cell layer (GCL) are positive for gephyrin only (arrows).

Clusters that are positive only for the a1 subunit are visible on thesomata of Purkinje cells (PC; arrowhead). B: a3 subunit. The few a3subunit-positive clusters are all colocalized with gephyrin (arrows).C,D: g2 subunit. Most g2 subunit-positive clusters are colocalizedwith gephyrin, except some around PC somata (arrowhead in C). Inthe ML (D), very few clusters are labeled only for gephyrin (arrow).Scale bar 5 10 mm.

double staining for the a1 and a3 subunit revealed thatthese two subunits were not colocalized, indicating thatthey are present in different synapses or perhaps even indifferent cell types.

The extent of colocalization between the g2 subunit andgephyrin was analyzed semiquantitatively in the molecu-lar layer. On average, 705 6 76 gephyrin clusters weredetected per 10,000 mm2 in images from CLSM with aresolution of 200 nm/pixel (n 5 10; mean 6 S.D.). In thesame sections, there were 769 6 76 clusters positive forthe g2 subunit. Of these, 649 6 76 were found to bedouble-labeled, using a “colocalization” algorithm to detectall pixels labeled with both markers, with the intensitythreshold being set at twice the intensity of backgroundstaining of the g2 subunit-IR. In other terms, 84% of g2subunit-positive clusters were colocalized with gephyrin,whereas 92% of gephyrin clusters were colocalized withthe g2 subunit. Because gephyrin was located exclusivelyin symmetric, presumably GABAergic synapses in themolecular layer (Fig. 2), these data confirm that the vastmajority of GABAA-receptor clusters represent postsynap-tic receptor aggregates associated with gephyrin.

Because glycine is known as neurotransmitter in thecerebellum (Dieudonne, 1995; Kaneda et al., 1995), andbecause gephyrin is associated with GlyRs (reviewed byKirsch et al., 1996), we checked whether the GABAA-receptor g2 subunit was colocalized with the GlyR a1subunit, by using double-staining with the mAb2b(Pfeiffer et al., 1984). In the cerebellar cortex, only a few,isolated clusters positive for mAb2b were detected, mostlylocated in the granule cell layer (Fig. 5A). These clusterswere never colocalized with the g2 subunit (Fig. 5B), in-dicating that GlyRs and GABAA-receptors are present atdistinct sites. In contrast, an extensive colocalization be-tween these two markers was detected in the deep cere-bellar nuclei, where almost all g2 subunit-positive clusterswere labeled with mAb2b (Fig. 5C,D). The g2 subunit-positive clusters were also colocalized with gephyrin (notshown), suggesting that in neurons of the deep cerebellarnuclei, GABAA-receptors, GlyRs, and gephyrin arepresent in the same synapses, as they are in spinal cord(Bohlhalter et al., 1994; Todd et al., 1996).

The analysis of GABAA-receptor subtypes in the cere-bellar cortex was completed by the visualization of the a6and d subunits, which are exclusively expressed by gran-ule cells (Laurie et al., 1992; Gao and Fritschy, 1995). Incontrast to the a1, a3, and g2 subunit, immunofluores-cence did not reveal a punctate staining for the a6 and dsubunit (Fig. 6A,C). Rather, individual glomeruli ap-peared “filled” with a prominent, but diffuse IR that wasnot selectively colocalized with gephyrin clusters. Even athigh-resolution (50 nm/pixel), no association betweenthese GABAA-receptor subunits and gephyrin could bedetected (Fig. 6B,D).

To determine whether the colocalization betweengephyrin and GABAA-receptors was restricted to specificreceptor subtypes or to certain brain regions, we investi-gated the a2 and a3 subunits in brain areas enriched withthese two subunits. Thus, in olfactory bulb, striatum, hip-pocampus, and cerebral cortex, the vast majority of a2subunit-positive clusters were colocalized with gephyrin,as shown for olfactory bulb granule cells (Fig. 7A). Like-wise, clusters immunoreactive for the a3 subunit in olfac-tory bulb, reticular nucleus of the thalamus (Fig. 7B),cerebral cortex, brainstem reticular formation, and spinal

trigeminal complex were systematically colocalized withgephyrin. As seen for the g2 subunit in the molecular layerof the cerebellum, over 80% and 90% of a2 and a3 subunit-positive clusters were colocalized with gephyrin, respec-tively. This is shown in color-separated images for the a3subunit in the reticular nucleus of the thalamus (Fig. 7C).These data indicated that, throughout the brain, a majorfraction of GABAA-receptors, containing either the a2 ora3 subunit, are clustered selectively at sites enriched withgephyrin.

High-resolution analysis using an imagedeconvolution procedure

To gain further insight into the subcellular distributionof GABAA-receptor subunits and gephyrin within individ-ual clusters, we used an iterative image deconvolutionprocedure (maximum likelihood estimate, MLE), based ona measured point-spread function, for improving thesignal-to-noise ratio of high-resolution images from CLSM(voxel size: 50 3 50 3 150 nm). The point-spread functionwas derived from images of fluorescent microspheres (di-ameter 170 nm; Fig. 8). Unlike conventional image resto-ration procedures, which enhance signal-to-noise only atthe cost of weak specific signals, image deconvolution gen-erates a model of the object to be visualized based on allthe information available in the raw images. The accuracyof the model depends, however, on several parametersthat can be approximated but not measured exactly (seeDiscussion). Therefore, the quality of the deconvolutionwas first assessed with known objects (the microspheresused for measuring the point-spread function; Fig. 8). Theapparent diameter of such beads in deconvoluted imageswas 230 6 20 nm for the green channel and 301 6 27 nmfor the red channel. The discrepancy with the expectedvalue (170 nm) indicates a greater deviation of the modelfor longer wavelengths.

The deconvolution procedure was then applied todouble-immunofluorescence images from individual glo-meruli in the cerebellar granule cell layer, to determinewhether gephyrin-positive puncta were colocalized withthe GABAA-receptor g2 subunit in these small GABAergicsynapses. Given the size of glomeruli (typically , 5 mm indiameter), they were visualized at high resolution with a1003 oil-immersion lens. As shown in Figure 9A, thesignal-to-noise ratio of such images was too low to allowthe distinction of individual g2 subunit clusters. Likewise,gephyrin clusters could not be resolved along the z-axis.However, MLE deconvolution provided a dramatic en-hancement of signal-to-noise, resulting in a sharp delim-itation of both g2 subunit-positive and gephyrin-positiveclusters within a glomerulus (Fig. 9B). As expected, theimprovement was particularly noticeable along the z-axis,where the resolution is inherently at least three timeslower than in the x-y plane. The distribution of gephyrinand g2 subunit-positive clusters and their spatial arrange-ment around the glomeruli could then be visualized byusing a three-dimensional rendering algorithm (Fig.9C,D).

This analysis revealed that positive clusters typicallywere located at the “edges” of the glomeruli, which pre-sumably were filled with an unstained mossy fiber termi-nal. This corresponded to the expected distribution ofGABAergic synapses on granule cell dendrites. The ma-jority of g2 subunit-positive clusters were closely associ-ated with gephyrin clusters, but the two markers were not


exactly colocalized. Rather, gephyrin clusters weresmaller and more numerous than g2 subunit-positive clus-ters, and appeared to be partially “embedded” in the latter(Fig. 9C,D). In addition, the g2 subunit-IR frequently wasseen to bridge the space between gephyrin-positive punctaand to be less sharply delimited, suggesting the presenceof extrasynaptic receptors between neighboring synapseslabeled with gephyrin (Fig. 10A,C). To gain further insighton this issue, double-labeled voxels were identified andmarked using a “colocalization” algorithm (Fig. 10B,D).This approach revealed that the majority of clusters inglomeruli typically comprised a center containing only

gephyrin, surrounded by an inner “ring” of double-labeledpixels and an outer “ring” containing only the g2subunit-IR (Fig. 10B,D). These results suggest that gephy-rin and the g2 subunit have a distinct distribution in thesepostsynaptic sites, with gephyrin presumably being moreabundant in the “core” of the synapse and the g2 subunitat the edge, and perhaps extrasynaptically. This differ-ence may also reflect the fact that gephyrin is a cytoplas-mic protein, whereas the g2 subunit is recognized on anextracellular epitope.

A semiquantitative analysis of the number of gephyrinand g2 subunit clusters detectable in images restored by

Fig. 5. The GABAA-receptor g2 subunit and the glycine-receptor(GlyR) a1 subunit are markers of distinct synapses in cerebellarcortex (A,B), but not in the dentate nucleus (C,D), as visualized bydouble-immunofluorescence staining and confocal laser scanning mi-croscopy (CLSM). Pairs of images corresponding to each marker wereacquired simultaneously and are displayed in separate panels (A,B

and C,D). Note that none of the GlyR clusters in the cerebellar cortex(A) is colocalized with the g2 subunit-immunoreactivity (IR; B; arrow-heads), whereas in the dentate nucleus the colocalization is extensive(C,D). The arrows in C point to clusters positive only for the GlyR a1subunit. Scale bars 5 10 mm for A,B; 5 mm for C,D.


deconvolution (Fig. 10 E–G) was performed, with the min-imal size of a cluster being set at 0.01 mm2. It revealed amean number of 91 6 37 gephyrin clusters (range 40–170)and 67 6 23 clusters positive for the g2 subunit (range30–100) per glomerulus (n 5 11), corroborating the visualimpression that gephyrin clusters were more numerousthan g2 subunit-positive clusters. Colocalization analysisshowed, however, that only 7.3 6 3.1 gephyrin clusters perglomerulus were devoid of g2 subunit-IR, and even fewerg2 subunit-positive clusters (2.3 6 0.7 per glomerulus)were devoid of gephyrin-IR. Although the smaller numberof g2 subunit clusters confirmed that GABAA-receptorclusters may “contain” several gephyrin clusters, thesefigures match precisely the number of symmetric synapsesfound in cerebellar glomeruli by EM analysis (range 43–145; mean 5 87; n 5 4; Jakab and Hamori, 1988).

The size of gephyrin clusters in glomeruli and in themolecular layer was assessed in deconvoluted images forcomparison with the size of GABAergic synapses identi-fied ultrastructurally. In glomeruli, the average diameterof gephyrin clusters was 0.27 6 0.013 mm and their ap-parent surface area 0.0652 6 0.0064 mm2 (n 5 505 clus-ters in 5 glomeruli; the S.D. refers to the glomeruli). Inthis data set, only 18 “clusters” were eliminated with thelower limit for detection set at 0.01 mm2. In the molecularlayer, the average diameter was 0.385 6 0.018 mm andthe apparent surface area, 0.1335 6 0.07 mm2 (n 5 272clusters in 5 samples). These data show that the valuesderived from the deconvoluted images are within therange of those measured in electron photomicrographs.

The only partial colocalization of gephyrin and the g2subunit within individual clusters in cerebellar glomeruli

Fig. 6. Lack of colocalization between gephyrin and the GABAA-receptor subunits a6 (A,B) and d(C,D), as visualized in the cerebellar granule cell layer by double-immunofluorescence staining andconfocal laser scanning microscopy (CLSM). Staining for both subunits (red) is diffuse in glomeruli(arrowheads) and not associated with gephyrin clusters (green). Scale bars 5 5 mm for A,C; 2 mm for B,D.


Fig. 7. Colocalization of gephyrin with the GABAA-receptor a2subunit in the olfactory bulb granule cell layer (A) and the a3 subunitin the reticular nucleus of the thalamus (B,C) visualized by double-immunofluorescence staining and confocal laser scanning microscopy(CLSM). The majority of GABAA-receptor subunit clusters (red) inboth areas are double-labeled with gephyrin (green) and appear yel-low in A and B. In C, the colocalization algorithm used to identifydouble-labeled clusters is illustrated for the boxed area of B. The

histogram shows the distribution of red and green pixels in this area(18 confocal images spaced by 250 nm are superimposed), with theselected pixels being located in the shaded area. The distribution ofthese pixels representing colocalization of the a3 subunit (red) andgephyrin (green) is shown in yellow, and matches precisely the clus-ters formed by both markers. Only one gephyrin cluster was foundsingle labeled (arrowhead). Scale bars 5 5 mm.

suggested an unexpected degree of heterogeneity in thesubsynaptic organization of GABAergic synapses. To de-termine whether this partial colocalization is a commonfeature of GABAergic synapses, we investigated with thesame approach the colocalization between gephyrin andthe g2 subunit in three additional, well-defined GABAer-gic synapses (Fig. 11). First, in the molecular layer of thecerebellum, where gephyrin and the g2 subunit-positiveclusters are markedly larger than those in glomeruli, andmostly located on dendrites. Second, in the dentate nu-cleus, where the clusters are of medium size, also containGlyRs (Fig. 5), and are located on the soma. Third, on theaxon initial segment of CA3 pyramidal cells, where a highdensity of GABAA-receptors has been reported (Nusser etal., 1996a). In all three regions, images from double-immunofluorescence staining were recorded at high mag-nification (voxel size 50 3 50 3 150 nm) and deconvolutedwith the MLE algorithm, by using the same point-spreadfunction as for images of glomeruli. Despite the differen-tial localization of GABAergic synapses in these threeregions (dendrites, soma, axon initial segment), the colo-calization between the g2 subunit and gephyrin was foundto “fill” the double-labeled clusters (Fig. 11A–C), and noevidence for a differential localization of gephyrin and theg2 subunit within a cluster could be found. Thus, theparticular arrangement detected in cerebellar glomeruliappears to be unique for these types of synapses, whichare otherwise characterized by their small size.

DISCUSSION

The results demonstrate the ubiquitous presence ofgephyrin in GABAA-receptor clusters containing either

the a1, a2, a3, or g2 subunit, independently of their local-ization in distinct types of synapses (axosomatic, axoden-dritic, and axo-axonic). Because the g2 subunit is the mostubiquitous GABAA-receptor subunit, these findings are inline with the corequirement of the g2 subunit and gephy-rin for postsynaptic clustering of major GABAA-receptorsubtypes (Essrich et al., 1998; Kneussel et al., 1999). Fur-thermore, because gephyrin apparently is present only inpostsynaptic sites, these results suggest that it representsa marker of a large subset of GABAergic synapses inbrain, in addition to glycinergic synapses. Finally, theexcellent correspondence between the results of preem-bedding EM and deconvolution of CLSM images validatesthe latter approach for investigating the subcellular dis-tribution of synaptic proteins in situ.

Association of gephyrin with multipleGABAA-receptor subtypes

The association of gephyrin with GABAA-receptors inpostsynaptic densities was first demonstrated for the a3subunit in the retina (Sassoe-Pognetto et al., 1995). In thistissue, however, GABAA-receptors containing the a1 sub-unit were not colocalized with gephyrin, suggesting ini-tially that gephyrin was associated only with certain re-ceptor subtypes. The present results indicate that in braingephyrin is colocalized with GABAA-receptors containingthe three most predominant a subunit variants, as well asthe g2 subunit, suggesting its association with a majorityof postsynaptic GABAA-receptors.

Several differences were nevertheless observed betweenthe distribution of GABAA-receptors and gephyrin. First,a diffuse GABAA-receptor subunit-IR can be seen through-

Fig. 8. Effect of image deconvolution on the appearance of a fluo-rescent microsphere (diameter 170 nm; max. emission wavelength565 nm). A: Images in the three planes of reference obtained afterconventional restoration procedures (baseline subtraction and con-trast optimization) of a stack of 60 confocal layers; the small trianglesin the x-y plane indicate the position of the other planes). Note thatthe light emitted by the microsphere spreads over considerable dis-tances, notably along the z-axis. B: Model of the microsphere gener-

ated by iterative deconvolution from the same confocal images. Theapparent size of the bead in the x-y plane is slightly larger than itsreal size, and most of the signals dispersed below and above the beadhave been removed. The resolution is thereby improved, especiallyalong the z-axis. The ovoid shape of the microsphere in the x-z and y-zplanes, as compared to the x-y plane, reflects the lower resolutionalong the z-axis that is inherent to confocal laser scanning microscopy(CLSM). Scale bar 5 0.5 mm.


out the brain in addition to clusters, suggesting the pres-ence of a sizable pool of extrasynaptic receptors (Essrich etal., 1998; Fritschy et al., 1998b), as shown by postembed-ding EM (Nusser et al., 1995a,b; Somogyi et al., 1996).Second, our results also revealed that some GABAA-receptor subunit clusters, for instance in the molecularlayer of the cerebellum and around Purkinje cells, aredevoid of gephyrin-IR. Althoguh it will be necessary toestablish whether these clusters effectively correspond topostsynaptic aggregates, this finding provides further ev-idence that the association of GABAA-receptor subunitswith gephyrin could be regulated by additional factors.Third, certain GABAA-receptor subtypes apparently arenot aggregated in clusters, as shown here for the a6 or thed subunit in cerebellar granule cells. Although the d sub-unit is known to be selectively present in extrasynaptic

receptors in cerebellum (Nusser et al., 1998), the lack ofclustering of the a6 subunit was unexpected, because thissubunit has been located postynaptically in both GABAer-gic and glutamatergic synapses in cerebellar glomeruli(Nusser et al., 1996b). However, the lack of colocalizationwith gephyrin suggests that the a6 subunit contributes toa population of GABAA-receptors that is not associatedwith gephyrin. Furthermore, a similar subcellular distri-bution of the a6 and d subunit immunofluorescence is inline with the fact that these two subunits are coassembledin a major subset of GABAA-receptors in cerebellum (Pol-lard et al., 1993; Jones et al., 1997; Jechlinger et al., 1998).

The analysis of GABAA-receptor clusters in the deepcerebellar nuclei revealed that these receptors can be co-localized with GlyRs and with gephyrin, indicating that incertain cells gephyrin may interact with both types of

Fig. 9. Effect of image deconvolution on the visualization ofgephyrin- (green) and g2 subunit-positive (red) clusters in cerebellarglomeruli, as seen by double-immunofluorescence staining and confo-cal laser scanning microscopy (CLSM). A: Conventional restoration(baseline subtraction and contrast optimization) applied to confocalimages of a single glomerulus was insufficient to allow the distinctionof g2 subunit clusters in the three planes of reference and of gephyrinclusters along the z-axis. A single confocal section is shown in thethree planes of reference. B: Model generated by iterative deconvolu-

tion of the same raw data set. Gephyrin- and g2 subunit-positiveclusters become clearly visible owing to the enhanced signal-to-noiseratio and increased resolution. C,D: Spatial relationships betweengephyrin- and g2 subunit-positive clusters in two glomeruli visualizedby iterative deconvolution. The three-dimensional structure of theglomeruli was rendered from a stack of 40 confocal sections by asimulated fluorescence-processing algorithm that projects shadowsfrom a virtual light source. Scale bars 5 1 mm.


Fig. 10. Colocalization between gephyrin- and g2 subunit-positiveclusters in glomeruli analyzed with a colocalization algorithm (seeFig. 7C). A,B: Partial colocalization between the g2 subunit-immunoreactivity (IR; red) and gephyrin-IR (green), as seen afterimage deconvolution in a single confocal section through a glomeru-lus. The spatial relationship between the two markers is shown in Aand sites of colocalization in B. The ring-like appearance indicatesthat the two proteins have a differential distribution within individ-ual clusters. C: Three-dimensional rendering of the entire glomerulusdepicted in A (stack of 48 confocal layers). The boxed area is enlargedin D, to illustrate sites of colocalization at higher magnification. Thesix panels in D represent different levels along the z-axis through

these clusters, spaced by 0.5 mm, with the sites of colocalizationmarked in yellow. E–G: Color-separated images of a glomerulus de-picting g2 subunit-positive clusters (E), gephyrin clusters (F), anddouble-labeled clusters (G). These images were generated with analgorithm depicting the three-dimensional rendering of the surface ofthe clusters. Note that most gephyrin-positive clusters appear smallerthan the corresponding g2 subunit-clusters. Arrowheads in E and Fpoint to single-labeled clusters. This technique was used to analyzesemiquantitatively the number of g2 subunit- and gephyrin-positiveclusters in glomeruli (see text). Scale bars 5 1 mm for A–C; 0.5 mm forD; 1 mm for E–G.


Fig. 11. Colocalization patterns between gephyrin and the g2 sub-unit visualized by image deconvolution in three populations of syn-apses. A: Axodendritic synapses in the molecular layer of the cerebel-lum. B: Axosomatic synapses in the dentate nucleus. C: Axo-axonicsynapses in the axon-initial segment of hippocampal CA3 pyramidalcell. The left column depicts with three-dimensional rendering (sim-ulated fluorescence processing) the distribution of clusters recon-structed from 30–40 confocal images. The boxed areas are enlarged in

the right column and show the spatial relationship between the twomarkers in individual clusters (top) and colocalized pixels in yellow(bottom) in stacks of 4–6 superposed confocal layers. Arrows point toclusters positive for the g2 subunit only, and arrowheads to clusterspositive for gephyrin only. Note that in all three types of synapse, thetwo markers are similarly distributed within individual clusters.Scale bars 51 mm.

receptors present at the same postsynaptic site. BecauseGABAA-receptors and GlyRs are frequently colocalized inspinal cord neurons (Bohlhalter et al., 1994; Todd et al.,1996), gephyrin is likely to play a dual role in numerousinhibitory synapses in the CNS. In addition, analysis by insitu hybridization histochemistry revealed moderateamounts of GlyR a3 subunit in the granule cell layer of thecerebellum (Malosio et al., 1991). It is therefore not ex-cluded that some gephyrin clusters in glomeruli, notablythose not colocalized with GABAA-receptor subunits, cor-respond to the presence of GlyR clusters.

Although there is evidence that gephyrin is present inthe majority of glycinergic and GABAergic synapses inspinal cord and brain, some exceptions have been re-ported. In the rat retina, the axon terminals of cone bipo-lar cells have been shown to express the a1 subunit of theGlyR, but not gephyrin, at sites of glycinergic synapticinput from AII amacrine cells (Sassoe-Pognetto et al.,1994). Likewise, gephyrin was localized at axodendritic,but not at axo-axonic synapses in the dorsal horn of ratspinal cord (Mitchell et al., 1993). Thus, receptor cluster-ing in axon terminals seems to occur independently ofgephyrin. In contrast, gephyrin was colocalized withGABAA-receptor subunits in the axon initial segment ofCA3 pyramidal cells, suggesting that the mechanism thatunderlie receptor clustering may vary in different cellsubcompartments.

Visualization of postsynaptic proteins byimage deconvolution

The present results demonstrate that the resolution ofCLSM images can be markedly improved by image decon-volution, allowing the visualization of subcellular neuro-nal structures with an apparent diameter of 100–300 nm.The main advantage of image deconvolution is that thecalculated model is based on all the information present inthe raw data set, whereas “conventional” image restora-tion procedures eliminate specific signals of low intensity.The latter are not necessarily “background,” as seen in theimages of fluorescent microspheres (Fig. 8), where “specif-ic” signals could be detected several mm away from the siteof emission. Consequently, the effects of image deconvolu-tion are most striking in noisy images, as they result fromboth suppression of “background” noise and enhancementof specific signals. However, the accuracy of the modelsgenerated by image deconvolution depends on parameters(numerical aperture of the lens, sampling density, refrac-tive index, size of pinhole, signal-to-noise ratio) that canonly be estimated but not measured with high precision.Even for a geometrically simple object such as a micro-sphere, some deviations from the expected size were ob-served (Fig. 8), especially for the longer emission wave-length. To validate this approach for “unknown” objects,such as gephyrin clusters, it was therefore essential tocompare the results derived from deconvoluted imageswith those from EM. Both the number and size ofgephyrin-positive clusters in glomeruli and in the molec-ular layer were very close to EM values, indicating thatthe models generated by the MLE deconvolution algo-rithm were representative of the sampled data.

These results indicate that image deconvolution proce-dures can be used to investigate the subcellular distribu-tion of proteins concentrated at specific sites, such as thepostsynaptic specialization. In particular, this method re-veals differences in the subcellular localization of gephy-

rin and the GABAA-receptor g2 subunit within singlepostsynaptic sites. Because gephyrin is a cytoplasmic pro-tein, whereas the g2 subunit epitope is located extracel-lularly, this differential localization could explain that theg2 subunit and gephyrin do not appear perfectly colocal-ized in deconvoluted images. In other words, these resultsshow that fluorochromes emitting at different wave-lengths can be discriminated in deconvoluted images at aresolution below 100 nm.

The differential distribution of the GABAA-receptor g2subunit and gephyrin in individual clusters in cerebellarglomeruli is unlikely to be an artifact of image deconvolu-tion, because it was not observed in clusters imaged underidentical conditions in other brain areas (Fig. 11). Rather,it is likely to reflect local variations in the distribution ofthe two proteins. Similar variations have been demon-strated previously for the GABAA-receptor b3 subunit(Todd et al., 1996) and the GlyR a1 subunit (Alvarez et al.,1997). Our results suggest that gephyrin clusters in cere-bellar glomeruli are restricted to the postsynaptic special-ization, whereas the larger GABAA-receptor clusters sug-gested the existence of a sizable pool of extrasynapticreceptors. This is consistent with quantitative data on thedistribution of GABAA-receptors obtained with immuno-gold electron microscopy (Nusser et al., 1995b).

A recent study in spinal cord (Alvarez et al., 1997) hasdemonstrated that the density of gephyrin clusters andtheir topographical organization and architecture varywidely in different neurons, as well as in different den-dritic regions of individual neurons. This structural vari-ability of gephyrin clusters is likely to reflect variations inthe organization of postsynaptic receptors, and dynamicchanges in the organization of the postsynaptic apparatus.Our present results indicate that the reciprocal organiza-tion of gephyrin and GABAA-receptors may vary at differ-ent types of GABAergic synapses. This is reminiscent ofwhat has been found at glutamatergic synapses, wheresegregation of receptors and receptor-associated postsyn-aptic proteins at specific subsynaptic domains is thoughtto represent novel mechanisms by which neurons processand encode information (Ottersen and Landsend, 1997;Zhang et al., 1999).

The differential localization of gephyrin and the g2 sub-unit in glomerular synapses is in line with the hypothesisthat the interaction between these two proteins is indirectand involves additional partner(s). One potential candi-date is GABAA-receptor-associated protein (GABARAP),which has been shown to interact selectively with the g2subunit (Wang et al., 1999). It is unclear, however,whether GABARAP also interacts with gephyrin, or di-rectly with the subsynaptic cytoskeleton. Another candi-date protein localized postsynaptically in a subset ofGABAergic synapses is dystrophin (Knuesel et al., 1999).A role of dystrophin for GABAA-receptor clustering and/orstabilization has been demonstrated, based on a decreasein GABAA-receptor cluster size and numbers in mice lack-ing dystrophin (mdx mice). Interestingly, however, gephy-rin clusters were not affected in mdx mice (Knuesel et al.,1999), arguing against a role of dystrophin for clusteringof gephyrin and against a direct link between gephyrinand GABAA-receptors. In view of the present results, itwould be of major interest to analyze the distribution ofboth GABARAP and dystrophin at postsynaptic sites withimage deconvolution procedures.


ACKNOWLEDGMENTS

We thank Dr. Hanns Mohler for advice and encourage-ment, Dr. Thomas Bachi and Dr. Mathias Hochli for sup-port with CLSM, and Dr. Hans Van der Voort for advicewith image deconvolution. This study was supported bythe Swiss National Foundation for Scientific Research(grant 31-52869-97 to J.M.F.) and the Italian M.U.R.S.T.

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colocalization of multiple gabaa receptor subtypes with gephyrin at postsynaptic sites

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