Gephyrin Clusters Research Articles

Synapse formation regulated by protein tyrosine phosphatase receptor T through interaction with cell adhesion molecules and Fyn

The receptor-type protein tyrosine phosphatases (RPTPs) have been linked to signal transduction, cell adhesion, and neurite extension. PTPRT/RPTPrho is exclusively expressed in the central nervous system and regulates synapse formation by interacting with cell adhesion molecules and Fyn protein tyrosine kinase. Overexpression of PTPRT in cultured neurons increased the number of excitatory and inhibitory synapses by recruiting neuroligins that interact with PTPRT through their ecto-domains. In contrast, knockdown of PTPRT inhibited synapse formation and withered dendrites. Incubation of cultured neurons with recombinant proteins containing the extracellular region of PTPRT reduced the number of synapses by inhibiting the interaction between ecto-domains. Synapse formation by PTPRT was inhibited by phosphorylation of tyrosine 912 within the membrane-proximal catalytic domain of PTPRT by Fyn. This tyrosine phosphorylation reduced phosphatase activity of PTPRT and reinforced homophilic interactions of PTPRT, thereby preventing the heterophilic interaction between PTPRT and neuroligins. These results suggest that brain-specific PTPRT regulates synapse formation through interaction with cell adhesion molecules, and this function and the phosphatase activity are attenuated through tyrosine phosphorylation by the synaptic tyrosine kinase Fyn.

Gephyrin Oligomerization Controls GlyR Mobility and Synaptic Clustering

High local concentrations of glycine receptors (GlyRs) at inhibitory postsynaptic sites are achieved through their binding to the scaffold protein gephyrin. The N- and C-terminal domains of gephyrin are believed to trimerize and dimerize, respectively, thus contributing to the formation of submembranous gephyrin clusters at synapses. GlyRs are associated with gephyrin also at extrasynaptic locations. We have investigated how gephyrin oligomerization influences GlyR dynamics and clustering in COS-7 cells and in cultured spinal cord neurons. To this aim, we have expressed isolated N- and C-terminal domains of gephyrin that interfere with the oligomerization of the full-length protein. We also studied the effect of an endogenous splice variant, ge(2,4,5), with a decreased propensity to trimerize. A reduction of the size and number of gephyrin-GlyR clusters was found in cells expressing the various interfering gephyrin constructs. Using fluorescence recovery after photobleaching, we studied the exchange kinetics of synaptic gephyrin clusters. Real-time single-particle tracking was used to analyze the mobility of GlyRs. We found that all the tested constructs displayed faster rates of recovery than wild-type gephyrin and increased the mobility of extrasynaptic receptors, showing that gephyrin-gephyrin interactions modulate the lateral diffusion of GlyRs. Furthermore, we observed an inverse correlation between GlyR diffusion properties and gephyrin cluster size that depended on the number of binding sites blocked by the different constructs. Since alterations in the oligomerization properties of gephyrin are related to the dynamics of GlyRs, the gephyrin splice variant ge(2,4,5) may be implicated in the modulation of synaptic strength.

A balanced chromosomal translocation disruptingARHGEF9is associated with epilepsy, anxiety, aggression, and mental retardation

Clustering of inhibitory gamma-aminobutyric acid(A) (GABA(A)) and glycine receptors at synapses is thought to involve key interactions between the receptors, a "scaffolding" protein known as gephyrin and the RhoGEF collybistin. We report the identification of a balanced chromosomal translocation in a female patient presenting with a disturbed sleep-wake cycle, late-onset epileptic seizures, increased anxiety, aggressive behavior, and mental retardation, but not hyperekplexia. Fine mapping of the breakpoint indicates disruption of the collybistin gene (ARHGEF9) on chromosome Xq11, while the other breakpoint lies in a region of 18q11 that lacks any known or predicted genes. We show that defective collybistin transcripts are synthesized and exons 7-10 are replaced by cryptic exons from chromosomes X and 18. These mRNAs no longer encode the pleckstrin homology (PH) domain of collybistin, which we now show binds phosphatidylinositol-3-phosphate (PI3P/PtdIns-3-P), a phosphoinositide with an emerging role in membrane trafficking and signal transduction, rather than phosphatidylinositol 3,4,5-trisphosphate (PIP3/PtdIns-3,4,5-P) as previously suggested in the "membrane activation model" of gephyrin clustering. Consistent with this finding, expression of truncated collybistin proteins in cultured neurons interferes with synaptic localization of endogenous gephyrin and GABA(A) receptors. These results suggest that collybistin has a key role in membrane trafficking of gephyrin and selected GABA(A) receptor subtypes involved in epilepsy, anxiety, aggression, insomnia, and learning and memory.

Collybistin is required for both the formation and maintenance of GABAergic postsynapses in the hippocampus

Collybistin (Cb) is a brain-specific guanine nucleotide exchange factor, which interacts with the inhibitory receptor anchoring protein gephyrin. In the hippocampus of constitutively Cb-deficient adult mice, gephyrin and gephyrin-dependent GABA A receptors (GABA ARs) are lost from postsynaptic sites. Here, we used a Cre–loxP system to inactivate the Cb gene in the forebrain at different developmental stages. Deletion of Cb during embryonic development prevented gephyrin clustering during synaptogenesis and caused an accumulation of gephyrin aggregates in the cell body of CA1 pyramidal neurons. Inactivation of the Cb gene during the third postnatal week resulted in a protracted loss of postsynaptic gephyrin clusters and the appearance of cytoplasmic gephyrin aggregates. These changes in gephyrin distribution were accompanied by a similar reduction in synaptically localized GABA AR γ2-subunit immunoreactivity. Our data show that Cb is required for both the initial localization and maintenance of gephyrin and gephyrin-dependent GABA ARs at inhibitory postsynaptic membrane specializations in the hippocampus.

Normal sleep homeostasis and lack of epilepsy phenotype in GABAA receptor α3 subunit-knockout mice

Thalamo-cortical networks generate specific patterns of oscillations during distinct vigilance states and epilepsy, well characterized by electroencephalography (EEG). Oscillations depend on recurrent synaptic loops, which are controlled by GABAergic transmission. In particular, GABA A receptors containing the alpha3 subunit are expressed predominantly in cortical layer VI and thalamic reticular nucleus (nRT) and regulate the activity and firing pattern of neurons in relay nuclei. Therefore, ablation of these receptors by gene targeting might profoundly affect thalamo-cortical oscillations. Here, we investigated the role of alpha3-GABA A receptors in regulating vigilance states and seizure activity by analyzing chronic EEG recordings in alpha3 subunit-knockout (alpha3-KO) mice. The presence of postsynaptic alpha3-GABA A receptors/gephyrin clusters in the nRT and GABA A-mediated synaptic currents in acute thalamic slices was also examined. EEG spectral analysis showed no difference between genotypes during non rapid-eye movement (NREM) sleep or at waking-NREM sleep transitions. EEG power in the spindle frequency range (10-15 Hz) was significantly lower at NREM-REM sleep transitions in mutant compared with wild-type mice. Enhancement of sleep pressure by 6 h sleep deprivation did not reveal any differences in the regulation of EEG activities between genotypes. Finally, the waking EEG showed a slightly larger power in the 11-13-Hz band in alpha3-KO mice. However, neither behavior nor the waking EEG showed alterations suggestive of absence seizures. Furthermore, alpha3-KO mice did not differ in seizure susceptibility in a model of temporal lobe epilepsy. Strikingly, despite the disruption of postsynaptic gephyrin clusters, whole-cell patch clamp recordings revealed intact inhibitory synaptic transmission in the nRT of alpha3-KO mice. These findings show that the lack of alpha3-GABA(A) receptors is extensively compensated for to preserve the integrity of thalamo-cortical function in physiological and pathophysiological situations.

Synaptogenesis in the cerebellar cortex: Differential regulation of gephyrin and GABAA receptors at somatic and dendritic synapses of Purkinje cells

In rodent cerebellar cortex, synaptogenesis occurs entirely postnatally, allowing study of the mechanisms of synapse formation in vivo. Here we monitored the clustering of GABA(A) receptors and the scaffolding protein gephyrin at GABAergic postsynaptic sites during rat cerebellar development. We found that GABA(A) receptors and gephyrin co-aggregate at nascent synapses in the molecular and Purkinje cell layers with a similar time course. With few exceptions, gephyrin and GABA(A) receptor subunits clustered selectively in front of presynaptic boutons expressing the vesicular inhibitory amino acid transporter VIAAT and no ectopic localization of these molecules was observed. Surprisingly, gephyrin clusters outlining the cell body of Purkinje cells were transient, and disappeared rapidly at the end of the second postnatal week. The loss of gephyrin from perisomatic synapses was coincident with a significant reduction in the size of GABA(A) receptor clusters. Furthermore, these changes were accompanied by a developmental decrease in the size of synaptic appositions, as documented by electron microscopy. These findings suggest that gephyrin takes part in the initial assembly of postsynaptic specializations and reveal an unsuspected heterogeneity in the molecular organization of the postsynaptic apparatus at somatic and dendritic synapses of mature Purkinje cells.

Developmental maturation of synaptic and extrasynaptic GABAA receptors in mouse thalamic ventrobasal neurones

Thalamic ventrobasal (VB) relay neurones express multiple GABA(A) receptor subtypes mediating phasic and tonic inhibition. During postnatal development, marked changes in subunit expression occur, presumably reflecting changes in functional properties of neuronal networks. The aims of this study were to characterize the properties of synaptic and extrasynaptic GABA(A) receptors of developing VB neurones and investigate the role of the alpha(1) subunit during maturation of GABA-ergic transmission, using electrophysiology and immunohistochemistry in wild type (WT) and alpha(1)(0/0) mice and mice engineered to express diazepam-insensitive receptors (alpha(1H101R), alpha(2H101R)). In immature brain, rapid (P8/9-P10/11) developmental change to mIPSC kinetics and increased expression of extrasynaptic receptors (P8-27) formed by the alpha(4) and delta subunit occurred independently of the alpha(1) subunit. Subsequently (> or = P15), synaptic alpha(2) subunit/gephyrin clusters of WT VB neurones were replaced by those containing the alpha(1) subunit. Surprisingly, in alpha(1)(0/0) VB neurones the frequency of mIPSCs decreased between P12 and P27, because the alpha(2) subunit also disappeared from these cells. The loss of synaptic GABA(A) receptors led to a delayed disruption of gephyrin clusters. Despite these alterations, GABA-ergic terminals were preserved, perhaps maintaining tonic inhibition. These results demonstrate that maturation of synaptic and extrasynaptic GABA(A) receptors in VB follows a developmental programme independent of the alpha(1) subunit. Changes to synaptic GABA(A) receptor function and the increased expression of extrasynaptic GABA(A) receptors represent two distinct mechanisms for fine-tuning GABA-ergic control of thalamic relay neurone activity during development.

Induction of GABAergic Postsynaptic Differentiation by α-Neurexins

Beta-neurexin and neuroligin cell adhesion molecules contribute to synapse development in the brain. The longer alpha-neurexins function at both glutamate and gamma-aminobutyric acid (GABA) synapses in coupling to presynaptic calcium channels. Binding of alpha-neurexins to neuroligins was recently reported, but the role of the alpha-neurexins in synapse development has not been well studied. Here we report that in COS cell neuron coculture assays, all three alpha-neurexins induce clustering of the GABAergic postsynaptic scaffolding protein gephyrin and neuroligin 2 but not of the glutamatergic postsynaptic scaffolding protein PSD-95 or neuroligin 1/3/4. alpha-Neurexins also induce clustering of the GABA(A) receptor gamma2 subunit. This synapse promoting activity of alpha-neurexins is mediated by the sixth LNS (laminin neurexin sex hormone-binding protein) domain and negatively modulated by upstream sequences. Although inserts at splice site 4 (S4) in beta-neurexins promote greater clustering activity for GABA than glutamate proteins in coculture assay, alpha-neurexin-specific sequences confer complete specificity for GABA proteins. We further report a developmental increase in the ratio of -S4 to +S4 forms of neurexins correlating with an increase in glutamate relative to GABA synaptogenesis and activity regulation of splicing at S4. Thus, +S4 beta-neurexins and, even more selectively, alpha-neurexins may be mediators of GABAergic synaptic protein recruitment and stabilization.

Gephyrin expression and clustering affects the size of glutamatergic synaptic contacts

We have recently shown that disrupting the expression and post-synaptic clustering of gephyrin in cultured hippocampal pyramidal cells, by either gephyrin RNAi (RNA interference) or over-expression of a dominant negative gephyrin-enhanced green fluorescent protein (EGFP) fusion protein, leads to decreased number of post-synaptic gephyrin and GABA(A) receptor clusters and to reduced GABAergic innervation of these cells. On the other hand, increasing gephyrin expression led to a small increase in the number of gephyrin and GABA(A) receptor clusters and to little or no effect on GABAergic innervation. We are now reporting that altering gephyrin expression and clustering affects the size but not the density of glutamatergic synaptic contacts. Knocking down gephyrin with gephyrin RNAi, or preventing gephyrin clustering by over-expression of the dominant negative gephyrin-enhanced green fluorescent protein fusion protein, leads to larger post-synaptic PSD-95 clusters and larger pre-synaptic glutamatergic terminals. On the other hand, over-expression of gephyrin leads to slightly smaller PSD-95 clusters and pre-synaptic glutamatergic terminals. The change in size of PSD-95 clusters were accompanied by a parallel change in the size of NR2-NMDA receptor clusters. It is concluded that the levels of expression and clustering of gephyrin, a protein that concentrates at the post-synaptic complex of the inhibitory synapses, not only has homotypic effects on GABAergic synaptic contacts, but also has heterotypic effects on glutamatergic synaptic contacts. We are proposing that gephyrin is a counterpart of the post-synaptic glutamatergic scaffold protein PSD-95 in regulating the number and/or size of the excitatory and inhibitory synaptic contacts.

Gephyrin clustering is required for the stability of GABAergic synapses

Although gephyrin is an important postsynaptic scaffolding protein at GABAergic synapses, the role of gephyrin for GABAergic synapse formation and/or maintenance is still under debate. We report here that knocking down gephyrin expression with small hairpin RNAs (shRNAs) in cultured hippocampal pyramidal cells decreased both the number of gephyrin and GABA(A) receptor clusters. Similar results were obtained by disrupting the clustering of endogenous gephyrin by overexpressing a gephyrin-EGFP fusion protein that formed aggregates with the endogenous gephyrin. Disrupting postsynaptic gephyrin clusters also had transsynaptic effects leading to a significant reduction of GABAergic presynaptic boutons contacting the transfected pyramidal cells. Consistent with the morphological decrease of GABAergic synapses, electrophysiological analysis revealed a significant reduction in both the amplitude and frequency of the spontaneous inhibitory postsynaptic currents (sIPSCs). However, no change in the whole-cell GABA currents was detected, suggesting a selective effect of gephyrin on GABA(A) receptor clustering at postsynaptic sites. It is concluded that gephyrin plays a critical role for the stability of GABAergic synapses.

Interaction of the N-Terminal Domain of the AMPA Receptor GluR4 Subunit with the Neuronal Pentraxin NP1 Mediates GluR4 Synaptic Recruitment

Synaptogenesis requires recruitment of neurotransmitter receptors to developing postsynaptic specializations. We developed a coculture system reconstituting artificial synapses between neurons and nonneuronal cells to investigate the molecular components required for AMPA-receptor recruitment to synapses. With this system, we find that excitatory axons specifically express factors that recruit the AMPA receptor GluR4 subunit to sites of contact between axons and GluR4-transfected nonneuronal cells. Furthermore, the N-terminal domain (NTD) of GluR4 is necessary and sufficient for its recruitment to these artificial synapses and also for GluR4 recruitment to native synapses. Moreover, we show that axonally derived neuronal pentraxins NP1 and NPR are required for GluR4 recruitment to artificial and native synapses. RNAi knockdown and knockout of the neuronal pentraxins significantly decreases GluR4 targeting to synapses. Our results indicate that NP1 and NPR secreted from presynaptic neurons bind to the GluR4 NTD and are critical trans-synaptic factors for GluR4 recruitment to synapses.

Neurofascin regulates the formation of gephyrin clusters and their subsequent translocation to the axon hillock of hippocampal neurons

Little is known about the role of cell adhesion molecules (CAMs) in inhibitory synapse development. In particular, a functional link between CAMs and the clustering of postsynaptic scaffold component gephyrin, which is a critical determinant of γ-aminobutyric acid A (GABA) receptor clustering, still needs to be elaborated. At early stages of inhibitory synapse formation, gephyrin and CAM neurofascin are diffusely expressed in the soma of hippocampal neurons. Subsequently, gephyrin clusters become localized to the axon hillock and neurofascin is observed all over the soma including the axon hillock suggesting a function for neurofascin in gephyrin clustering. Transfection of expression vectors for different isoforms and mutants of neurofascin revealed that neurofascin is required for the formation of gephyrin clusters presumably dependent on extracellular interactions. Furthermore, expression of neurofascin is necessary for the translocation of gephyrin clusters to the axon hillock of hippocampal neurons as shown by shRNA-mediated knockdown. In addition, overexpression of an embryonic neurofascin isoform is sufficient for functional rescue after knockdown of endogenous neurofascin.

Multiple Association States between Glycine Receptors and Gephyrin Identified by SPT Analysis

The scaffolding protein gephyrin is known to anchor glycine receptors (GlyR) at synapses and to participate in the dynamic equilibrium between synaptic and extrasynaptic GlyR in the neuronal membrane. Here we investigated the properties of this interaction in cells cotransfected with YFP-tagged gephyrin and GlyR subunits possessing an extracellular myc-tag. In HeLa cells and young neurons, single particle tracking was used to follow in real time individual GlyR, labeled with quantum dots, traveling into and out of gephyrin clusters. Analysis of the diffusion properties of two GlyR subunit types—able or unable to bind gephyrin—gave access to the association states of GlyR with its scaffolding protein. Our results indicated that an important portion of GlyR could be linked to a few molecules of gephyrin outside gephyrin clusters. This emphasizes the role of scaffolding proteins in the extrasynaptic membrane and supports the implication of gephyrin-gephyrin interactions in the stabilization of GlyR at synapses. The kinetic parameters controlling the equilibrium between GlyR inside and outside clusters were also characterized. Within clusters, we identified two subpopulations of GlyR with distinct degrees of stabilization between receptors and scaffolding proteins.

Vertebrate-specific sequences in the gephyrin E-domain regulate cytosolic aggregation and postsynaptic clustering

Gephyrin is a multifunctional protein contributing to molybdenum cofactor (Moco) synthesis and postsynaptic clustering of glycine and GABA(A) receptors. It contains three major functional domains (G-C-E) and forms cytosolic aggregates and postsynaptic clusters by unknown mechanisms. Here, structural determinants of gephyrin aggregation and clustering were investigated by neuronal transfection of EGFP-tagged deletion and mutant gephyrin constructs. EGFP-gephyrin formed postsynaptic clusters containing endogenous gephyrin and GABA(A)-receptors. Isolated GC- or E-domains failed to aggregate and exerted dominant-negative effects on endogenous gephyrin clustering. A construct interfering with intermolecular E-domain dimerization readily auto-aggregated but showed impaired postsynaptic clustering. Finally, two mutant constructs with substitution of vertebrate-specific E-domain sequences with homologue bacterial MoeA sequences uncovered a region crucial for gephyrin clustering. One construct failed to aggregate, but retained Moco biosynthesis capacity, demonstrating the independence of gephyrin enzymatic activity and aggregation. Reinserting two vertebrate-specific residues restored gephyrin aggregation and increased formation of postsynaptic clusters containing GABA(A) receptors at the expense of PSD-95 clusters - a marker of glutamatergic synapses. These results underscore the key role of specific E-domain regions distinct from the known dimerization interface for controlling gephyrin aggregation and postsynaptic clustering and suggest that formation of gephyrin clusters influences the homeostatic balance between inhibitory and excitatory synapses.

Molecular Basis of Gephyrin Clustering at Inhibitory Synapses: ROLE OF G- AND E-DOMAIN INTERACTIONS

Gephyrin is a bifunctional modular protein that, in neurons, clusters glycine receptors and gamma-aminobutyric acid, type A receptors in the postsynaptic membrane of inhibitory synapses. By x-ray crystallography and cross-linking, the N-terminal G-domain of gephyrin has been shown to form trimers and the C-terminal E-domain dimers, respectively. Gephyrin therefore has been proposed to form a hexagonal submembranous lattice onto which inhibitory receptors are anchored. Here, crystal structure-based substitutions at oligomerization interfaces revealed that both G-domain trimerization and E-domain dimerization are essential for the formation of higher order gephyrin oligomers and postsynaptic gephyrin clusters. Insertion of the alternatively spliced C5' cassette into the G-domain inhibited clustering by interfering with trimerization, and mutation of the glycine receptor beta-subunit binding region prevented the localization of the clusters at synaptic sites. Together our findings show that domain interactions mediate gephyrin scaffold formation.

Regulation of Gephyrin Assembly and Glycine Receptor Synaptic Stability

Gephyrin is required for the formation of clusters of the glycine receptor (GlyR) in the neuronal postsynaptic membrane. It can make trimers and dimers through its N- and C-terminal G and E domains, respectively. Gephyrin oligomerization could thus create a submembrane lattice providing GlyR-binding sites. We investigated the relationships between the stability of cell surface GlyR and the ability of gephyrin splice variants to form oligomers. Using truncated and full-length gephyrins we found that the 13-amino acid sequence (cassette 5) prevents G domain trimerization. Moreover, E domain dimerization is inhibited by the gephyrin central L domain. All of the gephyrin variants bind GlyR beta subunit cytoplasmic loop with high affinity regardless of their cassette composition. Coexpression experiments in COS-7 cells demonstrated that GlyR bound to gephyrin harboring cassette 5 cannot be stabilized at the cell surface. This gephyrin variant was found to deplete synapses from both GlyR and gephyrin in transfected neurons. These data suggest that the relative expression level of cellular variants influence the overall oligomerization pattern of gephyrin and thus the turnover of synaptic GlyR.

Alteration of GABAergic synapses and gephyrin clusters in the thalamic reticular nucleus of GABAA receptor α3 subunit‐null mice

Multiple GABAA-receptor subtypes are assembled from alpha, beta and gamma subunit variants. GABAA receptors containing the alpha3 subunit represent a minor population with a restricted distribution in the CNS. In addition, they predominate in monoaminergic neurons and in the nucleus reticularis thalami (nRT), suggesting a role in the regulation of cortical function and sleep. Mice with a targeted deletion of the alpha3 subunit gene (alpha3(0/0)) are viable and exhibit a subtle behavioural phenotype possibly related to dopaminergic hyperfunction. Here, we investigated immunohistochemically the consequences of the loss of alpha3 subunit for maturation of GABAA receptors and formation of GABAergic synapses in the nRT. Throughout postnatal development, the regional distribution of the alpha1, alpha2, or alpha5 subunit was unaltered in alpha3(0/0) mice and the prominent alpha3 subunit staining of nRT neurons in wildtype mice was not replaced. Subcellularly, as seen by double immunofluorescence, the alpha3 and gamma2 subunit were clustered at postsynaptic sites in the nRT of adult wildtype mice along with the scaffolding protein gephyrin. In alpha3(0/0) mice, gamma2 subunit clustering was disrupted and gephyrin formed large aggregates localized at the cell surface, but unrelated to postsynaptic sites, indicating that nRT neurons lack postsynaptic GABAA receptors in mutant mice. Furthermore, GABAergic terminals were enlarged and reduced in number, suggesting a partial deficit of GABAergic synapses. Therefore, GABAA receptors are required for gephyrin clustering and long-term synapse maintenance. The absence of GABAA-mediated transmission in the nRT may have a significant impact on the function of the thalamo-cortical loop of alpha3(0/0) mice.

Differential expression of GABAA and glycine receptors in ALS‐resistant vs. ALS‐vulnerable motoneurons: possible implications for selective vulnerability of motoneurons

Summary Amyotrophic lateral sclerosis (ALS) is a devastating motoneuronal degenerative disease, which is inevitably fatal in adults. ALS is characterized by an extensive loss of motoneurons in the cerebrospinal axis, except for those motoneurons that control eye movements and bladder contraction. The reason for this selectivity is not known. Systematic differences have been found in the organization of excitatory synaptic transmission in ALS-resistant vs. ALS-susceptible motor nuclei. However, although motoneurons express high levels of glycine receptors (GlyR) and GABA(A) receptors (GABA(A)R), no such studies have been carried out yet for inhibitory synaptic transmission. In this study, we compared the subunit composition, patterns of expression, density and synaptic localization of inhibitory synaptic receptors in ALS-resistant (oculomotor, trochlear and abducens) and ALS-vulnerable motoneurons (trigeminal, facial and hypoglossi). Triple immunofluorescent stainings of the major GABA(A)R subunits (alpha1, alpha2, alpha3, and alpha5), the GlyR alpha1 subunit and gephyrin, were visualized by confocal microscopy and analysed quantitatively. A strong correlation was observed between the vulnerability of motoneurons and the subunit composition of GABA(A)R, the GlyR/GABA(A)R density ratios and the incidence of synaptic vs. extrasynaptic GABA(A)R. These differences contrast strikingly with the uniform gephyrin cluster density and synaptic GlyR levels recorded in all motor nuclei examined. These results suggest that the specific patterns of inhibitory receptor organization observed might reflect functional differences that are relevant to the physiopathology of ALS.

Compensatory alteration of inhibitory synaptic circuits in cerebellum and thalamus of γ‐aminobutyric acid type A receptor α1 subunit knockout mice

Targeted deletion of the alpha1 subunit gene results in a profound loss of gamma-aminobutyric acid type A (GABA(A)) receptors in adult mouse brain but has only moderate behavioral consequences. Mutant mice exhibit several adaptations in GABA(A) receptor subunit expression, as measured by Western blotting. By using immunohistochemistry, we investigated here whether these adaptations serve to replace the missing alpha1 subunit or represent compensatory changes in neurons that normally express these subunits. We focused on cerebellum and thalamus and distinguished postsynaptic GABA(A) receptor clusters by their colocalization with gephyrin. In the molecular layer of the cerebellum, alpha1 subunit clusters colocalized with gephyrin disappeared from Purkinje cell dendrites of mutant mice, whereas alpha3 subunit/gephyrin clusters, presumably located on dendrites of Golgi interneurons, increased sevenfold, suggesting profound network reorganization in the absence of the alpha1 subunit. In thalamus, a prominent increase in alpha3 and alpha4 subunit immunoreactivity was evident, but without change in regional distribution. In the ventrobasal complex, which contains primarily postsynaptic alpha1- and extrasynaptic alpha4-GABA(A) receptors, the loss of alpha1 subunit was accompanied by disruption of gamma2 subunit and gephyrin clustering, in spite of the increased alpha4 subunit expression. However, in the reticular nucleus, which lacks alpha1-GABA(A) receptors in wild-type mice, postsynaptic alpha3/gamma2/gephyrin clusters were unaffected. These results demonstrate that adaptive responses in the brain of alpha1(0/0) mice involve reorganization of GABAergic circuits and not merely replacement of the missing alpha1 subunit by another receptor subtype. In addition, clustering of gephyrin at synaptic sites in cerebellum and thalamus appears to be dependent on expression of a GABA(A) receptor subtype localized postsynaptically.

The state of the actin cytoskeleton determines its association with gephyrin: Role of ena/VASP family members

The role the cytoskeleton plays in generating and/or maintaining gephyrin-dependent receptor clusters at inhibitory synapses is poorly understood. Here, the effects of actin cytoskeleton disruption were investigated in eGFP–gephyrin-transfected cells and hippocampal neurons. While gephyrin was not associated with microfilaments in transfected cells, it colocalized with G-actin and cytochalasin-D-induced F-actin patches. The linker region between the MoeA and MogA homology domains of gephyrin was required for colocalization with F-actin patches and for the binding of gephyrin to ena/VASP, an actin anti-capping factor that, in vitro, caused gephyrin binding to polymerized actin. In hippocampal neurons, treatment with cytochalasin D resulted in the redistribution of the neuronal ena/VASP homologue Mena into actin patches and, at early stages of development, a reduction in the number of gephyrin clusters. Our data suggest that Mena binding to F-actin allows for gephyrin recruitment to the leading edge of uncapped actin filaments.