Development Of GABAergic Synapses Research Articles

Structural and functional characterization of the IgSF21-neurexin2α complex and its related signaling pathways in the regulation of inhibitory synapse organization.

The prevailing model behind synapse development and specificity is that a multitude of adhesion molecules engage in transsynaptic interactions to induce pre- and postsynaptic assembly. How these extracellular interactions translate into intracellular signal transduction for synaptic assembly remains unclear. Here, we focus on a synapse organizing complex formed by immunoglobulin superfamily member 21 (IgSF21) and neurexin2α (Nrxn2α) that regulates GABAergic synapse development in the mouse brain. We reveal that the interaction between presynaptic Nrxn2α and postsynaptic IgSF21 is a high-affinity receptor-ligand interaction and identify a binding interface in the IgSF21-Nrxn2α complex. Despite being expressed in both dendritic and somatic regions, IgSF21 preferentially regulates dendritic GABAergic presynaptic differentiation whereas another canonical Nrxn ligand, neuroligin2 (Nlgn2), primarily regulates perisomatic presynaptic differentiation. To explore mechanisms that could underlie this compartment specificity, we targeted multiple signaling pathways pharmacologically while monitoring the synaptogenic activity of IgSF21 and Nlgn2. Interestingly, both IgSF21 and Nlgn2 require c-jun N-terminal kinase (JNK)-mediated signaling, whereas Nlgn2, but not IgSF21, additionally requires CaMKII and Src kinase activity. JNK inhibition diminished de novo presynaptic differentiation without affecting the maintenance of formed synapses. We further found that Nrxn2α knockout brains exhibit altered synaptic JNK activity in a sex-specific fashion, suggesting functional linkage between Nrxns and JNK. Thus, our study elucidates the structural and functional relationship of IgSF21 with Nrxn2α and distinct signaling pathways for IgSF21-Nrxn2α and Nlgn2-Nrxn synaptic organizing complexes in vitro. We therefore propose a revised hypothesis that Nrxns act as molecular hubs to specify synaptic properties not only through their multiple extracellular ligands but also through distinct intracellular signaling pathways of these ligands.

Human mutations in SLITRK3 implicated in GABAergic synapse development in mice.

This study reports on biallelic homozygous and monoallelic de novo variants in SLITRK3 in three unrelated families presenting with epileptic encephalopathy associated with a broad neurological involvement characterized by microcephaly, intellectual disability, seizures, and global developmental delay. SLITRK3 encodes for a transmembrane protein that is involved in controlling neurite outgrowth and inhibitory synapse development and that has an important role in brain function and neurological diseases. Using primary cultures of hippocampal neurons carrying patients' SLITRK3 variants and in combination with electrophysiology, we demonstrate that recessive variants are loss-of-function alleles. Immunostaining experiments in HEK-293 cells showed that human variants C566R and E606X change SLITRK3 protein expression patterns on the cell surface, resulting in highly accumulating defective proteins in the Golgi apparatus. By analyzing the development and phenotype of SLITRK3 KO (SLITRK3-/-) mice, the study shows evidence of enhanced susceptibility to pentylenetetrazole-induced seizure with the appearance of spontaneous epileptiform EEG as well as developmental deficits such as higher motor activities and reduced parvalbumin interneurons. Taken together, the results exhibit impaired development of the peripheral and central nervous system and support a conserved role of this transmembrane protein in neurological function. The study delineates an emerging spectrum of human core synaptopathies caused by variants in genes that encode SLITRK proteins and essential regulatory components of the synaptic machinery. The hallmark of these disorders is impaired postsynaptic neurotransmission at nerve terminals; an impaired neurotransmission resulting in a wide array of (often overlapping) clinical features, including neurodevelopmental impairment, weakness, seizures, and abnormal movements. The genetic synaptopathy caused by SLITRK3 mutations highlights the key roles of this gene in human brain development and function.

DSCAM gene triplication causes excessive GABAergic synapses in the neocortex in Down syndrome mouse models.

Down syndrome (DS) is caused by the trisomy of human chromosome 21 (HSA21). A major challenge in DS research is to identify the HSA21 genes that cause specific symptoms. Down syndrome cell adhesion molecule (DSCAM) is encoded by a HSA21 gene. Previous studies have shown that the protein level of the Drosophila homolog of DSCAM determines the size of presynaptic terminals. However, whether the triplication of DSCAM contributes to presynaptic development in DS remains unknown. Here, we show that DSCAM levels regulate GABAergic synapses formed on neocortical pyramidal neurons (PyNs). In the Ts65Dn mouse model for DS, where DSCAM is overexpressed due to DSCAM triplication, GABAergic innervation of PyNs by basket and chandelier interneurons is increased. Genetic normalization of DSCAM expression rescues the excessive GABAergic innervations and the increased inhibition of PyNs. Conversely, loss of DSCAM impairs GABAergic synapse development and function. These findings demonstrate excessive GABAergic innervation and synaptic transmission in the neocortex of DS mouse models and identify DSCAM overexpression as the cause. They also implicate dysregulated DSCAM levels as a potential pathogenic driver in related neurological disorders.

Loss of IQSEC3 Disrupts GABAergic Synapse Maintenance and Decreases Somatostatin Expression in the Hippocampus

Gephyrin interacts with various GABAergic synaptic proteins to organize GABAergic synapse development. Among the multitude of gephyrin-binding proteins is IQSEC3, a recently identified component at GABAergic synapses that acts through its ADP ribosylation factor-guanine nucleotide exchange factor (ARF-GEF) activity to orchestrate GABAergic synapse formation. Here, we show that IQSEC3 knockdown (KD) reduced GABAergic synaptic density invivo, suggesting that IQSEC3 is required for GABAergic synapse maintenance invivo. We further show that IQSEC3 KD in the dentate gyrus (DG) increases seizure susceptibility and triggers selective depletion of somatostatin (SST) peptides in the DG hilus in an ARF-GEP activity-dependent manner. Strikingly, selective introduction of SST into SST interneurons in DG-specific IQSEC3-KD mice reverses GABAergic synaptic deficits. Thus, our data suggest that IQSEC3 is required for linking gephyrin-GABAA receptor complexes with ARF-dependent pathways to prevent aberrant, runaway excitation and thereby contributes to the integrity of SST interneurons and proper GABAergic synapse maintenance.

The small GTPase ARF6 regulates GABAergic synapse development

ADP ribosylation factors (ARFs) are a family of small GTPases composed of six members (ARF1–6) that control various cellular functions, including membrane trafficking and actin cytoskeletal rearrangement, in eukaryotic cells. Among them, ARF1 and ARF6 are the most studied in neurons, particularly at glutamatergic synapses, but their roles at GABAergic synapses have not been investigated. Here, we show that a subset of ARF6 protein is localized at GABAergic synapses in cultured hippocampal neurons. In addition, we found that knockdown (KD) of ARF6, but not ARF1, triggered a reduction in the number of GABAergic synaptic puncta in mature cultured neurons in an ARF activity-dependent manner. ARF6 KD also reduced GABAergic synaptic density in the mouse hippocampal dentate gyrus (DG) region. Furthermore, ARF6 KD in the DG increased seizure susceptibility in an induced epilepsy model. Viewed together, our results suggest that modulating ARF6 and its regulators could be a therapeutic strategy against brain pathologies involving hippocampal network dysfunction, such as epilepsy.

A Conserved Tyrosine Residue in Slitrk3 Carboxyl-Terminus Is Critical for GABAergic Synapse Development

Single-passing transmembrane protein, Slitrk3 (Slit and Trk-like family member 3, ST3), is a synaptic cell adhesion molecule highly expressed at inhibitory synapses. Recent studies have shown that ST3, through its extracellular domain, selectively regulates inhibitory synapse development via the trans-synaptic interaction with presynaptic cell adhesion molecule, receptor protein tyrosine phosphatase δ (PTPδ) and the cis-interaction with postsynaptic cell adhesion molecule, Neuroligin 2 (NL2). However, little is known about the physiological function of ST3 intracellular, carboxyl (C)-terminal region. Here we report that in heterologous cells, ST3 C-terminus is not required for ST3 homo-dimerization and trafficking to the cell surface. In contrast, in hippocampal neurons, ST3 C-terminus, more specifically, the conserved tyrosine Y969 (in mice), is critical for GABAergic synapse development. Indeed, overexpression of ST3 Y969A mutant markedly reduced the gephyrin puncta density and GABAergic transmission in hippocampal neurons. In addition, single-cell genetic deletion of ST3 strongly impaired GABAergic transmission. Importantly, wild-type (WT) ST3, but not the ST3 Y969A mutant, could fully rescue GABAergic transmission deficits in neurons lacking endogenous ST3, confirming a critical role of Y969 in the regulation of inhibitory synapses. Taken together, our data identify a single critical residue in ST3 C-terminus that is important for GABAergic synapse development and function.

Genetic Deletion of GABAA Receptors Reveals Distinct Requirements of Neurotransmitter Receptors for GABAergic and Glutamatergic Synapse Development.

In the adult brain GABAA receptors (GABAARs) mediate the majority of synaptic inhibition that provides inhibitory balance to excitatory drive and controls neuronal output. In the immature brain GABAAR signaling is critical for neuronal development. However, the cell-autonomous role of GABAARs in synapse development remains largely unknown. We have employed the CRISPR-CAS9 technology to genetically eliminate GABAARs in individual hippocampal neurons and examined GABAergic and glutamatergic synapses. We found that development of GABAergic synapses, but not glutamatergic synapses, critically depends on GABAARs. By combining different genetic approaches, we have also removed GABAARs and two ionotropic glutamate receptors, AMPA receptors (AMPARs) and NMDA receptors (NMDARs), in single neurons and discovered a striking dichotomy. Indeed, while development of glutamatergic synapses and spines does not require signaling mediated by these receptors, inhibitory synapse formation is crucially dependent on them. Our data reveal a critical cell-autonomous role of GABAARs in inhibitory synaptogenesis and demonstrate distinct molecular mechanisms for development of inhibitory and excitatory synapses.

Preoperative anxiety-induced glucocorticoid signaling reduces GABAergic markers in spinal cord and promotes postoperative hyperalgesia by affecting neuronal PAS domain protein 4.

Preoperative anxiety is common in patients undergoing elective surgery and is closely related to postoperative hyperalgesia. In this study, a single prolonged stress model was used to induce preoperative anxiety-like behavior in rats 24 h before the surgery. We found that single prolonged stress exacerbated the postoperative pain and elevated the level of serum corticosterone. Previous studies have shown that glucocorticoid is associated with synaptic plasticity, and decreased spinal GABAergic activity can cause hyperalgesia in rodents. Here, single prolonged stress rats’ lumbar spinal cord showed reduced glutamic acid decarboxylase-65, glutamic acid decarboxylase-67, GABA type A receptor alpha 1 subunit, and GABA type A receptor gamma 2 subunit, indicating an impairment of GABAergic system. Furthermore, neuronal PAS domain protein 4 was also reduced in rats after single prolonged stress stimulation, which has been reported to promote GABAergic synapse development. Then, intraperitoneal injection of RU486 (a glucocorticoid receptor antagonist) rather than spironolactone (a mineralocorticoid receptor antagonist) was found to relieve single prolonged stress-induced hyperalgesia and reverse neuronal PAS domain protein 4 reduction and the impairment of GABAergic system. Furthermore, overexpressing neuronal PAS domain protein 4 could also restore the damage of GABAergic system caused by single prolonged stress while interfering with neuronal PAS domain protein 4 caused an opposite effect. Finally, after stimulation of rat primary spinal cord neurons with exogenous corticosterone in vitro, neuronal PAS domain protein 4 and GABAergic markers were also downregulated, and RU486 reversed that. Together, our results demonstrated that preoperative anxiety led to GABAergic system impairment in spinal cord and thus caused hyperalgesia due to glucocorticoid-induced downregulation of neuronal PAS domain protein 4.

Adeno-associated viral overexpression of neuroligin 2 in the mouse hippocampus enhances GABAergic synapses and impairs hippocampal-dependent behaviors

The cell adhesion molecule neuroligin2 (NLGN2) regulates GABAergic synapse development, but its role in neural circuit function in the adult hippocampus is unclear. We investigated GABAergic synapses and hippocampus-dependent behaviors following viral-vector-mediated overexpression of NLGN2. Transducing hippocampal neurons with AAV-NLGN2 increased neuronal expression of NLGN2 and membrane localization of GABAergic postsynaptic proteins gephyrin and GABAARγ2, and presynaptic vesicular GABA transporter protein (VGAT) suggesting trans-synaptic enhancement of GABAergic synapses. In contrast, glutamatergic postsynaptic density protein-95 (PSD-95) and presynaptic vesicular glutamate transporter (VGLUT) protein were unaltered. Moreover, AAV-NLGN2 significantly increased parvalbumin immunoreactive (PV+) synaptic boutons co-localized with postsynaptic gephyrin+ puncta. Furthermore, these changes were demonstrated to lead to cognitive impairments as shown in a battery of hippocampal-dependent mnemonic tasks and social behaviors.

Class 4 Semaphorins and Plexin-B receptors regulate GABAergic and glutamatergic synapse development in the mammalian hippocampus

To understand how proper circuit formation and function is established in the mammalian brain, it is necessary to define the genes and signaling pathways that instruct excitatory and inhibitory synapse development. We previously demonstrated that the ligand-receptor pair, Sema4D and Plexin-B1, regulates inhibitory synapse development on an unprecedentedly fast time-scale while having no effect on excitatory synapse development. Here, we report previously undescribed synaptogenic roles for Sema4A and Plexin-B2 and provide new insight into Sema4D and Plexin-B1 regulation of synapse development in rodent hippocampus. First, we show that Sema4a, Sema4d, Plxnb1, and Plxnb2 have distinct and overlapping expression patterns in neurons and glia in the developing hippocampus. Second, we describe a requirement for Plexin-B1 in both the presynaptic axon of inhibitory interneurons as well as the postsynaptic dendrites of excitatory neurons for Sema4D-dependent inhibitory synapse development. Third, we define a new synaptogenic activity for Sema4A in mediating inhibitory and excitatory synapse development. Specifically, we demonstrate that Sema4A signals through the same pathway as Sema4D, via the postsynaptic Plexin-B1 receptor, to promote inhibitory synapse development. However, Sema4A also signals through the Plexin-B2 receptor to promote excitatory synapse development. Our results shed new light on the molecular cues that promote the development of either inhibitory or excitatory synapses in the mammalian hippocampus.

Genetic deletion of NMDA receptors suppresses GABAergic synaptic transmission in two distinct types of central neurons

NMDA-type ionotropic glutamate receptors (NMDARs) play an important role in the regulation of synapse development and function in the brain. Recently we have shown that NMDARs are critical for GABAergic synapse development in developing hippocampal neurons. However, it remains unclear whether NMDARs are important for establishment of GABAergic synaptic transmission in other types of neurons in the brain. Here we report that in both cortical pyramidal neurons and midbrain dopamine neurons in ventral tegmental area (VTA), genetic deletion of the GluN1 subunit, which is required for assembly of functional NMDARs, leads to a strong reduction of GABAergic synaptic transmission. These data demonstrate that NMDARs play an important role in the development of GABAergic synaptic transmission in two types of neurons with distinct developmental origins, and suggest that NMDARs are commonly involved in development of GABAergic synaptic transmission in different types of neurons in the brain.

Molecular Dissection of Neuroligin 2 and Slitrk3 Reveals an Essential Framework for GABAergic Synapse Development

In the brain, many types of interneurons make functionally diverse inhibitory synapses onto principal neurons. Although numerous molecules have been identified to function in inhibitory synapse development, it remains unknown whether there is a unifying mechanism for development of diverse inhibitory synapses. Here we report a general molecular mechanism underlying hippocampal inhibitory synapse development. In developing neurons, the establishment of GABAergic transmission depends on Neuroligin 2 (NL2), a synaptic cell adhesion molecule (CAM). During maturation, inhibitory synapse development requires both NL2 and Slitrk3 (ST3), another CAM. Importantly, NL2 and ST3 interact with nanomolar affinity through their extracellular domains tosynergistically promote synapse development. Selective perturbation of the NL2-ST3 interactionimpairs inhibitory synapse development with consequent disruptions in hippocampal network activity and increased seizure susceptibility. Our findings reveal how unique postsynaptic CAMs work inconcert to control synaptogenesis and establish a general framework for GABAergic synapse development.

Regulation of GABAergic synapse development by postsynaptic membrane proteins.

In the adult mammalian brain, GABAergic neurotransmission provides the majority of synaptic inhibition that balances glutamatergic excitatory drive and thereby controls neuronal output. It is generally accepted that synaptogenesis is initiated through highly specific protein-protein interactions mediated by membrane proteins expressed in developing presynaptic terminals and postsynaptic membranes. Accumulating studies have uncovered a number of membrane proteins that regulate different aspects of GABAergic synapse development. In this review, we summarize recent advances in understanding of GABAergic synapse development with a focus on postsynaptic membrane molecules, including receptors, synaptogenic cell adhesion molecules and immunoglobulin superfamily proteins.

An NMDA Receptor-Dependent Mechanism Underlies Inhibitory Synapse Development.

In the mammalian brain, GABAergic synaptic transmission provides inhibitory balance to glutamatergicexcitatory drive and controls neuronal output. The molecular mechanisms underlying the development of GABAergic synapses remain largely unclear. Here, we report that NMDA-type ionotropic glutamate receptors (NMDARs) in individual immature neurons are the upstream signaling moleculesessential for GABAergic synapse development, which requires signaling via Calmodulin binding motif in the C0 domain of the NMDAR GluN1 subunit. Interestingly, in neurons lacking NMDARs, whereas GABAergic synaptic transmission is strongly reduced, the tonic inhibition mediated by extrasynaptic GABAA receptors is increased, suggesting a compensatory mechanism for the lack of synaptic inhibition. These results demonstrate a crucial role for NMDARs in specifying the development of inhibitory synapses, and suggest an important mechanism for controlling the establishment of the balance between synaptic excitation and inhibition in the developing brain.

The Specific α-Neurexin Interactor Calsyntenin-3 Promotes Excitatory and Inhibitory Synapse Development

Perturbations of cell surface synapse-organizing proteins, particularly α-neurexins, contribute to neurodevelopmental and psychiatric disorders. From an unbiased screen, we identify calsyntenin-3 (alcadein-β) as a synapse-organizing protein unique in binding and recruiting α-neurexins, but not β-neurexins. Calsyntenin-3 is present in many pyramidal neurons throughout cortex and hippocampus but is most highly expressed in interneurons. The transmembrane form of calsyntenin-3 can trigger excitatory and inhibitory presynapse differentiation in contacting axons. However, calsyntenin-3-shed ectodomain, which represents about half the calsyntenin-3 pool in brain, suppresses the ability of multiple α-neurexin partners including neuroligin 2 and LRRTM2 to induce presynapse differentiation. Clstn3⁻/⁻ mice show reductions in excitatory and inhibitory synapse density by confocal and electron microscopy and corresponding deficits in synaptic transmission. These results identify calsyntenin-3 as an α-neurexin-specific binding partner required for normal functional GABAergic and glutamatergic synapse development.

Sema4D localizes to synapses and regulates GABAergic synapse development as a membrane-bound molecule in the mammalian hippocampus

While numerous recent advances have contributed to our understanding of excitatory synapse formation, the processes that mediate inhibitory synapse formation remain poorly defined. Previously, we discovered that RNAi-mediated knockdown of a Class 4 Semaphorin, Sema4D, led to a decrease in the density of inhibitory synapses without an apparent effect on excitatory synapse formation. Our current work has led us to new insights about the molecular mechanisms by which Sema4D regulates GABAergic synapse development. Specifically, we report that the extracellular domain of Sema4D is proteolytically cleaved from the surface of neurons. However, despite this cleavage event, Sema4D signals through its extracellular domain as a membrane-bound, synaptically localized protein required in the postsynaptic membrane for proper GABAergic synapse formation. Thus, as Sema4D is one of only a few molecules identified thus far that preferentially regulates GABAergic synapse formation, these findings have important implications for our mechanistic understanding of this process.

The Class 4 Semaphorin Sema4D Promotes the Rapid Assembly of GABAergic Synapses in Rodent Hippocampus

Proper circuit function in the mammalian nervous system depends on the precise assembly and development of excitatory and inhibitory synaptic connections between neurons. Through a loss-of-function genetic screen in cultured hippocampal neurons, we previously identified the class 4 Semaphorin Sema4D as being required for proper GABAergic synapse development. Here we demonstrate that Sema4D is sufficient to promote GABAergic synapse formation in rodent hippocampus and investigate the kinetics of this activity. We find that Sema4D treatment of rat hippocampal neurons increases the density of GABAergic synapses as detected by immunocytochemistry within 30 min, much more rapidly than has been previously described for a prosynaptogenic molecule, and show that this effect is dependent on the Sema4D receptor PlexinB1 using PlxnB1(-/-) mice. Live imaging studies reveal that Sema4D elicits a rapid enhancement (within 10 min) in the rate of addition of synaptic proteins. Therefore, we demonstrate that Sema4D, via PlexinB1, acts to initiate synapse formation by recruiting molecules to both the presynaptic and the postsynaptic terminals; these nascent synapses subsequently become fully functional by 2 h after Sema4D treatment. In addition, acute treatment of an organotypic hippocampal slice epilepsy model with Sema4D reveals that Sema4D rapidly and dramatically alters epileptiform activity, which is consistent with a Sema4D-mediated shift in the balance of excitation and inhibition within the circuit. These data demonstrate an ability to quickly assemble GABAergic synapses in response to an appropriate signal and suggest a potential area of exploration for the development of novel antiepileptic drugs.

Neural Cell Adhesion Molecule-Mediated Fyn Activation Promotes GABAergic Synapse Maturation in Postnatal Mouse Cortex

GABAergic basket interneurons form perisomatic synapses, which are essential for regulating neural networks, and their alterations are linked to various cognitive dysfunction. Maturation of basket synapses in postnatal cortex is activity dependent. In particular, activity-dependent downregulation of polysialiac acid carried by the neural cell adhesion molecule (NCAM) regulates the timing of their maturation. Whether and how NCAM per se affects GABAergic synapse development is unknown. Using single-cell genetics to knock out NCAM in individual basket interneurons in mouse cortical slice cultures, at specific developmental time periods, we found that NCAM loss during perisomatic synapse formation impairs the process of basket cell axonal branching and bouton formation. However, loss of NCAM once the synapses are already formed did not show any effect. We further show that NCAM120 and NCAM140, but not the NCAM180 isoform, rescue the phenotype. Finally, we demonstrate that a dominant-negative form of Fyn kinase mimics, whereas a constitutively active form of Fyn kinase rescues, the effects of NCAM knockdown. Altogether, our data suggest that NCAM120/NCAM140-mediated Fyn activation promotes GABAergic synapse maturation in postnatal cortex.

Selective control of inhibitory synapse development by Slitrk3-PTPδ trans-synaptic interaction

Balanced development of excitatory and inhibitory synapses is required for normal brain function, and their imbalance may underlie pathogenesis of neuropsychiatric disorders. Compared with many identified trans-synaptic adhesion complexes that organize excitatory synapses, little is known about organizers specific for inhibitory synapses. Here we report Slit and NTRK-like family member 3 (Slitrk3) as a postsynaptic adhesion molecule that selectively regulates inhibitory synapse development via trans-interaction with axonal tyrosine phosphatase receptor PTPδ. Slitrk3 expressed in fibroblasts triggers only inhibitory presynaptic differentiation in contacting axons of cocultured rat hippocampal neurons. Recombinant Slitrk3 preferentially localizes to inhibitory postsynaptic sites. Slitrk3-deficient mice exhibit decreases in inhibitory but not excitatory synapse number and function in hippocampal CA1 neurons and exhibit increased seizure susceptibility and spontaneous epileptiform activity. Slitrk3 requires trans-interaction with axonal PTPδ to induce inhibitory presynaptic differentiation. These results identify Slitrk3-PTPδ as an inhibitory-specific trans-synaptic organizing complex required for normal functional GABAergic synapse development.

Differential Regulation of the Postsynaptic Clustering of γ-Aminobutyric Acid Type A (GABAA) Receptors by Collybistin Isoforms

Collybistin promotes submembrane clustering of gephyrin and is essential for the postsynaptic localization of gephyrin and γ-aminobutyric acid type A (GABA(A)) receptors at GABAergic synapses in hippocampus and amygdala. Four collybistin isoforms are expressed in brain neurons; CB2 and CB3 differ in the C terminus and occur with and without the Src homology 3 (SH3) domain. We have found that in transfected hippocampal neurons, all collybistin isoforms (CB2(SH3+), CB2(SH3-), CB3(SH3+), and CB3(SH3-)) target to and concentrate at GABAergic postsynapses. Moreover, in non-transfected neurons, collybistin concentrates at GABAergic synapses. Hippocampal neurons co-transfected with CB2(SH3-) and gephyrin developed very large postsynaptic gephyrin and GABA(A) receptor clusters (superclusters). This effect was accompanied by a significant increase in the amplitude of miniature inhibitory postsynaptic currents. Co-transfection with CB2(SH3+) and gephyrin induced the formation of many (supernumerary) non-synaptic clusters. Transfection with gephyrin alone did not affect cluster number or size, but gephyrin potentiated the clustering effect of CB2(SH3-) or CB2(SH3+). Co-transfection with CB2(SH3-) or CB2(SH3+) and gephyrin did not affect the density of presynaptic GABAergic terminals contacting the transfected cells, indicating that collybistin is not synaptogenic. Nevertheless, the synaptic superclusters induced by CB2(SH3-) and gephyrin were accompanied by enlarged presynaptic GABAergic terminals. The enhanced clustering of gephyrin and GABA(A) receptors induced by collybistin isoforms was not accompanied by enhanced clustering of neuroligin 2. Moreover, during the development of GABAergic synapses, the clustering of gephyrin and GABA(A) receptors preceded the clustering of neuroligin 2. We propose a model in which the SH3- isoforms play a major role in the postsynaptic accumulation of GABA(A) receptors and in GABAergic synaptic strength.