The distribution of glycine receptors and interactions with the cytoskeleton

seminars in

CELL & DEVELOPMENTAL BIOLOGY, Vol 7, 1996: pp 717–724

The distribution of glycine receptors and interactions with the cytoskeleton Catherine B´echade and Antoine Triller

classic model.1 At this peripheral synapse, the restricted distribution of the AChRs depends on both extracellular and intracellular molecules. Agrin and ARIA are two nerve-derived extracellular matrix factors which are secreted by the nerve ending: agrin induces AChR clustering and ARIA stimulates the transcription of AChR subunit genes in subsynaptic nuclei.6 The intracellular components include a peripheral protein rapsyn (also named the 43K protein) that colocalizes with AChRs and anchors the receptor to the structurally specialized subsynaptic cytoskeleton.7,8 The mechanisms of receptor clustering at the NMJ are not necessarily applicable to central synapses, where a single neuron often receives synaptic inputs from thousands of presynaptic axons which may use different neurotransmitters. Inhibitory glycinergic synapses are the central nervous system synapses for which the mechanisms of neurotransmitter receptors clustering are best characterized.

In the central nervous system, mechanisms of postsynaptic clustering of neurotransmitter receptors are best understood at glycinergic inhibitory synapses. The glycine receptor is a pentameric protein composed of three α and two β transmembrane proteins: the ligand-binding α subunit and a structural β subunit. At glycinergic synapses, these receptors form clusters at the postsynaptic membrane directly opposite to the presynaptic release sites. Anchoring of glycine receptors in the neuronal membrane depends on a peripheral membrane protein named gephyrin. Gephyrin binds to the glycine receptor via the β subunit and links the receptor to the subsynaptic cytoskeleton. Gephyrin may be involved in the anchoring of other neurotransmitter receptors such as the γ-aminobutyric acid (GABAA) receptor. Key words: glycine receptor / cytoskeleton / gephyrin / synapse ©1996 Academic Press Ltd

RAPID AND SPECIFIC synaptic transmission requires the precise apposition of the presynaptic nerve terminal to postsynaptic specialization with high densities of appropriate neurotransmitter receptors. The best known example of postsynaptic differentiation is that at the neuromuscular junction (NMJ) where there are extremely high concentrations of acetylcholine receptors (AChRs) at the motor endplate opposite the presynaptic nerve terminal.1 There is increasing evidence that some neurotransmitter receptors are clustered in the central nervous system (CNS) in front of the presynaptic releasing apparatus. Thus, the glycine receptor (GlyR), some subunits of the GABAA and of the glutamate ionotropic receptors have been found directly opposite the presynaptic release sites of the corresponding neurotransmitter.2-5 The mechanisms of the postsynaptic receptor clustering are still poorly understood. Most of our knowledge is from studies of the neuromuscular junction which is the

The glycine receptor Glycine is one of the main inhibitory neurotransmitters in the central nervous system particularly in the spinal cord and in the brain stem.9 Gamma-aminobutyric acid (GABA), the other inhibitory neurotransmitter, is more abundant in rostral parts of the CNS. The binding of the two transmitters to their corresponding receptors activates chloride conductances.10 The action of glycine is selectively antagonized by strychnine, a convulsant alkaloid.11 The postsynaptic glycine receptor (GlyR) was initially purified from rat spinal cord by affinity chromatography on strychnine columns.12 The GlyR contains three major polypeptides of 48 kDa (α) 58 kDa (β) and 93 kDa.12 The two transmembrane subunits α and β are assembled in a pentameric structure with an α3β2 stoichiometry (Figure 1A).13 The α polypeptide harbours the binding sites for glycine and strychnine.14 Expression of the α subunit in a heterologous system leads to the formation of homomeric chloride channels which display the pharmacological characteristics of native GlyRs.15 In contrast, the β subunit alone does not

From the Laboratoire de Biologie Cellulaire de la Synapse (INSERM, CJF 94-10), Ecole Normale Sup´erieure, 46 Rue d’Ulm, 75005 Paris, France ©1996 Academic Press Ltd 1084-9521/96/050717 + 08 $25.00/0

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C. B´echade and A. Triller form functional GlyRs.16 However, its presence together with α subunits in recombinant receptors modulates the pharmacological and conductance properties of the GlyR.17,18 The sequences of α and β subunit cDNAs have been determined. The two polypeptides share a strong sequence homology and a similar transmembrane topology.16,19 Both proteins possess a putative cleavable signal sequence, a large hydrophilic extracellular N-terminal domain and four membrane spanning segments (M1–M4) with an intracytoplasmic loop between the third and the fourth transmembrane regions (Figure 1B). This transmembrane arrangement is similar to that of subunits of nicotinic acetylcholine, GABAA and of serotonin type-3 receptors.20,21 These various polypeptides share a significant degree of amino acid similarity and are classified into a superfamily of ligand-gated ion channels.20 There are several isoforms of the GlyR α subunit. Three isoforms (α1–α3) have been cloned from rat and human cDNA libraries19,22-24 and a fourth isoform (α4) has been isolated from a mouse genomic library.25 These isoforms have very homologous peptidic sequences but different pharmacological and functional properties. They are also

expressed at different developmental stages.26 In-situ hybridization studies revealed that the α1 isoform transcripts are detected mainly at postnatal stages of development, in spinal cord and brain stem.27 In contrast, α2 mRNA is the majority form in rat brain and spinal cord at early embryonic stages and around birth. α2 mRNA is progressively replaced in the spinal cord by α1 mRNA during the first two weeks following birth but persists in higher brain regions.27 α3 transcripts are detected only postnatally and in small amounts in spinal cord, olfactory bulb and cerebellum.27 GlyR heterogeneity is further increased by alternative splicing of both the α1 and the α2 mRNAs so that there are two variants for each of the proteins.28,29 No variants of the β subunit have been detected. Contrasting with the regionalized expression pattern of the various α subunits, β subunit transcripts are widely distributed in the CNS throughout development.27 In particular, the β mRNA was detected in brain structures which contain little or no α transcripts such as the thalamus and striatum. This pattern suggests that the β subunit may also co-assemble with other unidentified α subunits and/or with other neurotransmitter receptors such as the closely related

Figure 1. Schematic model of the postsynaptic glycine receptor. (A) Model of GlyR subunit assembly: 3α and 2β transmembrane GlyR subunits, gephyrin being associated with the β subunit. (B) Predicted transmembrane topology of the α subunit showing the four transmembrane (1, 2, 3, 4) domains of the molecule. The arrow at position 38 indicates a possible glycosylation site. The putative disulfide bridges are indicated by S–S. The β subunit has the same transmembrane topology. Gephyrin associates with the M3–M4 domain (crossed arrow). (Figure modified with kind permission of Elsevier Science Ltd and the Author, from Betz H, Glycine receptors: heterogenous and widespread in mammalian brain, Trends in Neurosciences, Vol. 14, pp. 458-461, 1991.)

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Glycine receptors and the cytoskeleton system for example the cerebellum,4 cochlear nucleus31 and olfactory bulb32 of the rat.

GABAAR. However, nothing of that sort has been demonstrated. Monoclonal antibodies against the purified GlyR have been determined. They have been used to analyse the sub-cellular distribution of the receptor in relation to the presynaptic release sites of neurotransmitters.30 These antibodies recognize both α1 and α2 subunits and the β subunit (mAb4a), only the α1 subunit (mAb2b) or gephyrin (mAb7a and 5a). Immunocytochemistry in the ventral horn of the spinal cord showed that GlyR and gephyrin was unevenly distributed on the somata and dendrites, giving a patchy appearance to the neuronal surface (Figure 2A).3 It was demonstrated by immunoelectron microscopy that both GlyR and gephyrin are concentrated at the postsynaptic membrane in front of the presynaptic release apparatus (Figure 2B, C, D).3,4 Thus, they form functional postsynaptic microdomains with high concentrations of glycine activatable chloride channels in front of the presynaptic release sites. Similar patchy distributions of GlyRs were found in many other areas of the central nervous

Gephyrin, cytoskeleton and GlyR clusters The 93 kDa protein is named gephyrin. It is a nonglycosylated peripheral membrane protein.33 Gephyrin has been localized by immunoelectron microscopy at the cytoplasmic face of glycinergic postsynaptic membranes.3,4,31 This led to the suggestion that gephyrin functions as a link between the GlyR and the subsynaptic cytoskeleton. Indeed, its name derives from the Greek ‘γεφνρα’ from bridge, here between the receptor and the underlying cytoskeleton. Work with primary cultures of rat embryonic spinal neurons demonstrated the involvement of gephyrin in the formation of GlyR aggregates. In comparable cultures, neurons establish functional glycinergic synapses after one week in vitro.34 Immunocytochemical studies at this stage showed that GlyR, in the in-situ

Figure 2. Cellular and subcellular organization of the glycine receptor and associated gephyrin. (A) Look-through projection of a stack of confocal sections showing the uneven distribution of the immunofluorescent GlyR (mAb GlyR 4a) at the surface of a large goldfish brainstem neuron. Nonlabeled patches (arrow heads) of the neuronal surface are contacted by non-glycinergic axons. Calibration bar: 10 µm. (B,C) Accumulation at postsynaptic densities of peroxidase electron dense product (B) and gold particles (C) associated with gephyrin immunoreactivity (mAb GlyR 7a). Two synaptic complexes are established by single flat vesicle-containing boutons (arrows). (D) Accumulation of the glycine receptor-associated gold particles in the synaptic cleft (arrow) of an asymmetrical synapse (mAb GlyR 4a). Calibration bar for B,C,D: 0.25 µm. (A) Goldfish brain stem; (B, C and D) Rat ventral horn.

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C. B´echade and A. Triller testing the effects of drugs affecting the organization of neuronal microtubules and microfilaments.40 Disruption of microtubules with demecolcine or nocodazole resulted in a substantial reduction in both the number of cells containing gephyrin clusters and the mean number of clusters per cell. As measured by confocal microscopy, the mean size of the clusters was increased, but their fluorescence intensity was reduced, suggesting lateral diffusion in the plasma membrane. In contrast, depolymerization of microfilaments by cytochalasin D did not affect the number of clusters but they were significantly smaller. Moreover, the packing density of gephyrin in individual clusters, as assessed by fluorescence intensity, was increased. Therefore, the size of peripheral gephyrin patches is regulated not only by microtubules but also by microfilaments. However, the two cytoskeleton elements appear to have antagonistic effects on the packing density of the postsynaptic geophyrin clusters. A model of the interactions of gephyrin with the GlyR and the cytoskeleton has been proposed40 (Figure 3): microtubules tend to condense receptor clusters and to restrict their mobility whereas microfilaments tend to disperse them. However, the structure of the subsynaptic cytoskeleton is complex and not all the elements involved in the stabilization of GlyR have been identified. cDNA cloning disclosed the existence of several gephyrin variants generated by alternative splicing of four cassettes.41 These cassettes encode parts of the

form, aggregates at the neuronal surface while synaptic contacts are established.35 Gephyrin also forms clusters which, as shown by double-labeling experiments, colocalize with GlyR aggregates.36 However, quantified confocal analysis revealed that gephyrin aggregates are present at the neuronal surface at earlier stages (3–5 days in vitro) than GlyR aggregates.36,37 It is only later that they associate with GlyRs. The first gephyrin patches to be detected are associated with the plasma membrane at points of contact between neurites or between neurites and the substrate.38 This led to a model in which gephyrin first gathers at points of cellular contact, thus defining ‘hot spots’. The transmembrane components of the GlyR, initially distributed evenly over the somato-dendritic membrane, are then trapped at these loci by lateral diffusion in the membrane.26 Many steps of this model still remain to be elucidated. Evidence for a role of gephyrin in anchoring and/ or stabilizing the GlyR was obtained using antisense oligonucleotides in cultured spinal cord neurons: inhibition of gephyrin synthesis by antisense oligonucleotides prevented the formation of GlyR clusters at the neuronal surface.36 Earlier overlay assays showed that gephyrin binds to polymerized tubulin with high affinity (KD = 2.5 nm)39 leading to the notion that microtubules could be directly involved in the stabilization of the GlyR-rich postsynaptic microdomains. Interactions between gephyrin and the cytoskeleton were then verified in cultured spinal neurons by

Figure 3. Schematic model of the interactions of the glycine receptor with the subsynaptic cytoskeleton. Gephyrin (Ge) links the β subunits of the glycine receptor to the subsynaptic microtubular (mt) web. The association of the microfilaments to gephyrin could be either direct or through an actin-binding protein (modified from Kirsch and Betz, 1995, ref 40).

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Glycine receptors and the cytoskeleton been demonstrated in vivo in coexpression experiments.48 When the β subunit of GlyR or the β3 subunit of GABAAR were cotransfected with gephyrin in a mammalian fibroblast line, they were re-routed to intracellular gephyrin aggregates instead of being targeted to the plasma membrane. A comparable result was obtained with GlyR α1/β chimerea, but not with the GlyR α1 subunit alone. Interestingly, the sequence which binds gephyrin is lacking in the β subunit of the mouse spastic mutant.49 This mutant suffers from muscle rigidity, tremor, myoclonus jerk and exaggerated startle reactions all due to an altered glycinergic inhibition. This mutation leads to a large decrease in the levels of the adult GlyR isoform.50 In the spastic mutant, an L1 transposable element is inserted into the intron 5 of the β subunit gene which causes the formation of aberrant spliced mRNAs.51,52 In these forms, exons 4 and 5 are skipped, resulting in the synthesis of truncated β subunits which lack the cytoplasmic loop between M3 and M4. Therefore, one of the consequences of this mutation might be that the GlyR cannot bind gephyrin and, therefore, is not stabilized in the postsynaptic membrane.

amino-terminal half of the protein thus generating a variable region. This variable region may associate with different membrane receptors depending on the cassettes used. Gephyrin has no homology with the AChR-associated protein, rapsyn, and shares only similarity with molybdopterin, a cofactor required for the activity of enzymes such as xanthine- and sulphite oxidase.42 The significance of this homology is unclear. There is a potential myristilation site at the amino-terminus of the protein. Myristilation may contribute to the interaction of gephyrin with the plasma membrane. Gephyrin also possess nine consensus sequences for protein kinase C and two potential sites for cyclic nucleotide-dependent phosphorylation in its central part. Gephyrin is phosphorylated by an endogenous protein kinase.43 Phosphorylation state appears to be important for the regulation of interactions between microtubule-associated proteins such as MAP-2 and microtubules.44 This could also be the case for gephyrin. Gephyrin transcripts have also been found in nonneural rat adult tissues such as kidney, lung and liver by Northern blotting.41 This broad distribution suggests a general role for gephyrin in anchoring membrane proteins. This distribution of gephyrin in the central nervous system has been further refined by in-situ hybridization studies.45 Transcripts containing the cassette C2 can be found in the spinal cord and in most parts of the adult rat brain whereas the C3 and C4 containing mRNAs are limited to the cerebellum and the hippocampus. During development, C2 and C3 mRNAs are found in most brain regions. This ubiquitous presence of gephyrin in embryonic and adult rat brain was confirmed by immunocytochemical studies.46 Thus, the distribution of gephyrin mRNA is different from that of any of the known GlyR α subunit mRNAs, but coincides with that of the β subunit mRNA.27 The co-distribution of gephyrin and the β subunit suggests interactions between the two molecules. Overlay assay experiments using affinitypurified native GlyR and recombinant subunit fragments demonstrated that gephyrin binds to the cytoplasmic loop between transmembrane segments M3 and M4 of the β subunit.47 In contrast, gephyrin did not bind to the GABAA R nor to the β 1 or the β 3 subunits of GABAAR, despite their being the most similar to the M3–M4 loop of the GlyR β subunit. Deletion constructs of the M3–M4 loop defined a 49 amino-acid stretch as being sufficient for the binding of gephyrin. Thus, the β subunit may be involved in the formation of postsynaptic receptor clusters. Interactions between gephyrin and the β subunit have

Gephyrin and GABA receptors Although there is no biochemical evidence of the binding of gephyrin with the GABAAR,47 there is increasing evidence that gephyrin might also be involved in the postsynaptic positioning of GABAAR. First, gephyrin is present in many regions of the mammalian brain that do not contain any of the known GlyR α subunits but where GABAAR is abundant.45,46 In addition, gephyrin is found in front of GABA-containing terminals and colocalizes with GABAA receptor subunits in the rat spinal cord, cerebellum and retina.4,53-56 The presence of gephyrin postsynaptic to GABA-containing terminals can be explained in the spinal cord by the fact that many nerve terminals contain both glycine and GABA57 and, therefore, GABAAR and GlyR constitute a mosaic of receptors at postsynaptic sites. However, in other regions such as in the inner plexiform layer of the retina, GlyRs and GABAAR are notcolocalized to any substantial extent whereas gephyrin and different subunits of the GABAAR are present at the same synapses.54 Sympathetic preganglionic neurons do not receive a glycinergic innervation but contain postsynaptic gephyrin-like immunoreactivity associated to GABAergic synaptic inputs.56 Finally, in cultured rat 721

C. B´echade and A. Triller ments in QT6 fibroblasts have demonstrated that rapsyn can cluster dystroglycan, the agrin binding component of the dystrophin–glycoprotein complex (DGC)68,69 even in the absence of AChRs.8 Therefore, rapsyn may link the AChR to the DGC. In the QT-6 cell line, rapsyn also clusters the synapse-specific receptor tyrosine kinase MuSK and activates its kinase activity.70 Coexpression of rapsyn with MuSK and the AChR results in the specific phosphorylation of the β subunit of the AchR, similar to the phosphorylation observed in agrin treated myotubes, indicating that AChR is a substrate for MuSK. Interestingly, the phenotype of mice lacking MuSK is comparable to that of mice lacking rapsyn: AChRs and the sub membrane proteins such as utrophin or dystroglycan do not form clusters.71 Furthermore, MuSK is a component of the receptor complex that mediates agrin signalling.72 Thus, rapsyn appears to play a pivotal role in the agrin transducing pathway by interacting with AChR, the cytoskeleton via dystroglycan and the tyrosine kinase MuSK. Thus, the biology of the motor neuron to muscle synapses indicates that the presynaptic element contributes to the structure of the postsynaptic membrane. This is possibly the case at central synapses.

hippocampal neurons, a system that lacks glycinergic transmission, gephyrin clusters were also found to be associated with GABAergic synapses.58 Thus, gephyrin may be involved in the localization of some subunits of the GABAAR as well.

The neuromuscular junction as a model for central synapses Rapsyn is a protein associated with the postsynaptic membrane at the neuromuscular junction. It has a molecular mass of 43 kDa and appears to be functionally related to gephyrin.1 The importance of rapsyn in the formation and in the maintenance of AChRs clusters was first suggested by the observation that its removal from the membrane by alkaline extraction resulted in an increase in the mobility of AChR.59 Rapsyn precisely colocalizes with AChR at neuromuscular synapses and with AChR clusters in cultured myotubes.60,61 This colocalization is observed from the earliest times of neuromuscular synaptogenesis.62 Coexpression studies in heterologous systems show the involvement of rapsyn in AChRs clustering.63,64 Recombinant AChR subunits expressed in Xenopus oocytes or in quail fibroblasts QT-6 were diffusely distributed over the cell surface. Following coexpression of the rapsyn protein, AChRs became organized into clusters. Interestingly, in fibroblasts, rapsyn can form clusters even in the absence of the AChRs.63 This is reminiscent of that observed for gephyrin at the early stages of the formation of glycinergic synapses. Direct proof that rapsyn is essential for AChR clustering in vivo came from studies of mice mutants in which rapsyn expression was eliminated.65 In these mutants AChRs was undetectable at neuromuscular junctions. Moreover, several components of the AChR-associated cytoskeleton such as utrophin, syntrophin and dystroglycan which belong to the dystrophin–glycoprotein complex (DGC),66,67 failed to concentrate at synaptic sites, suggesting that rapsyn is essential for the organization of the subsynaptic cytoskeleton. Surprisingly, analysis of these mutants revealed that rapsyn is also involved in the differentiation of presynaptic elements: nerve endings formed long branches without the distinct arbours characteristic of normal terminals. How does rapsyn mediate the AChR clustering? Recent data suggest that rapsyn is part of the agrin transduction signalling pathway: myotubes from rapsyn-deficient mutants do not form AChRs clusters in response to agrin.65 Moreover, co-transfection experi-

Concluding comments Both the glycinergic central synapse and the neuromuscular junction contain a peripheral membrane protein which links neurotransmitter receptors to the subsynaptic cytoskeleton. We are still at an early stage in understanding neuro-neuronal interactions and the mechanisms of GlyR clustering at postsynaptic membranes. In particular, it is not known how glycinergic postsynaptic specializations are regulated by presynaptic factor(s)such as agrin or agrin-like molecules at the NMJ. Interestingly, another protein, the function of which could be similar to rapsyn or gephyrin, has recently been identified at glutamatergic excitatory synapses. The sequence of this protein named PSD-95/SAP90 does not exhibit any homology to those of gephyrin and rapsyn.73,74 It interacts with NMDA receptors75 and Shaker-type potassium channel subunits.76 However, the relationships between PSD-95/SAP90 and the cytoskeleton are unclear. Peripheral nicotinic acetylcholine receptors, and central glycine and glutamate NMDA receptors, interact with submembraneous molecules which have no peptide sequence in common. Understanding how the expression and subcellular localization of 722

Glycine receptors and the cytoskeleton 18. Bormann J, Rundstrom ¨ N, Betz H, Langosh D (1993) Residues within transmembrane segment M2 determine chloride conductance of glycine receptor homo- and hetero-oligomers. EMBO J 12:3729-3737 19. Grenningloh G, Rienitz A, Schmitt B, Methfessel C, Zensen M, Beyreuther K, Gundelfinger ED, Betz H (1987) The strychninebinding subunit of the glycine receptor shows homology with nicotinic acetylcholine receptors. Nature 328:215-220 20. Betz H (1990) Ligand-gated ion channels in the brain: the amino acid receptor superfamily. Neuron 5:383-392 21. Maricq AV, Peterson AS, Brake AJ, Meyers RM, Julius D (1991) Primary structure and functional expression of the 5HT3 receptor, a serotonin-gated ion channel. Science 254:432-437 22. Kuhse J, Schmieden V, Betz H (1990) A single amino acid exchange alters the pharmacology of neonatal rat glycine receptor subunit. Neuron 5:867-873 23. Kuhse J, Schmieden V, Betz H (1990) Identification and functional expression of a novel ligand binding subunit of the inhibitory glycine receptor. J Biol Chem 265:22317-22320 24. Grenningloh G, Schmieden V, Schofield P, Seeburg PH, Siddique T, Mohandas T, Becker C-M, Betz H (1990) Alpha subunit variants of the human glycine receptor: primary structures, functional expression, and chromosomal localization of the corresponding genes. EMBO J 9:771-776 25. Matzenbach B, Maulet Y, Sefton L, Courtier B, Avner P, Gu´enet J-L, Betz H (1994) Structural analysis of mouse glycine receptor α subunit genes. J Biol Chem 269:2607-2612 26. B´echade C, Sur C, Triller A (1994) The inhibitory neuronal glycine receptor. BioEssays 16:735-744 27. Malosio M-L, Marqu`eze-Pouey B, Kuhse J, Betz H (1991) Widespread expression of glycine receptor subunit mRNAs in the adult and developing rat brain. EMBO J 10:2401-2409 28. Malosio M-L, Grenningloh G, Kuhse J, Schmieden V, Schmitt B, Prior P, Betz H (1991) Alternative splicing generates two variants of the α 1 subunit of the inhibitory glycine receptor. J Biol Chem 266:2048-2053 29. Kuhse J, Kuryatov A, Maulet Y, Malosio M-L, Schmieden V, Betz H (1991) Alternative splicing generates two isoforms of the α 2 subunit of the inhibitory glycine receptor. FEBS Lett 283:73-77 30. Pfeiffer F, Simler R, Grenningloh G, Betz H (1984) Monoclonal antibodies and peptide mapping reveal structural similarities between the subunits of the glycine receptor of rat spinal cord. Proc Natl Acad USA 81:7224-7227 31. Altschuler R, Betz H, Parakkal MH, Reeks KA, Wenthold RJ (1986) Identification of glycinergic synapses in the cochlear nucleus through immunocytochemical localization of the postsynaptic receptor. Brain Res 369:316-320 32. Van den Pol AN, Gorcs T (1988) Glycine and glycine receptor immunoreactivity in brain and spinal cord. J Neurosci 8:472-492 33. Schmitt B, Knaus P, Becker C-M, Betz H (1987) The Mr 93,000 polypeptide of the glycine receptor is a peripheral membrane protein. Biochemistry 26:805-811 34. Jackson MB, Lecar H, Brenneman DE, Fitzgerald S, Nelson PG (1982) Electrical development in spinal cord cell culture. J Neurosci 2:1052-1061 35. Nicola MA, Becker CM, Triller A (1992) Development of glycine receptor alpha subunit in cultivated rat spinal neurons: an immunocytochemical study. Neurosci Lett 138:173-178 36. Kirsch J, Wolters I, Triller A, Betz H (1993) Gephyrin antisense oligonucleotides prevent glycine receptor clustering in spinal cord. Nature 366:745-748 37. B´echade C, Colin I, Kirsch J, Betz H, Triller A (1996) Expression of glycine receptor α subunits and gephyrin in cultured spinal neurons. Eur J Neurosci 8:429-435 38. Colin I, Rostaing P, Triller A (1996) Gephyrin accumulates at specific plasmalemma loci during neuronal maturation in vitro. J Comp Neurol, in press

gephyrin and PSD-95/SAP90 are regulated by glycinergic or glutamatergic afferent innervations, respectively, may help in the understanding of the development of the molecular heterogeneity of the postsynaptic somato-dendritic membrane.

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