Collybistin promotes submembrane clustering of gephyrin and is essential for the postsynaptic localization of gephyrin and ␥-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 nontransfected 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.A fundamental issue in the GABAergic synapse field is to understand the mechanisms that regulate the postsynaptic clustering of GABA A 3 receptors (GABA A Rs) and GABAergic synaptic strength during inhibitory synapse formation. Collybistin (CB) is a cytoplasmic protein that binds to gephyrin, helping the latter to cluster and translocate to the submembranous compartment (1-4). CB is a guanine nucleotide exchange factor (GEF) that catalyzes GDP-GTP exchange on the small GTPase Cdc42 of the Rho family (5). CB is essential for the initial synaptic localization and maintenance of gephyrin and GABA A Rs at GABAergic synapses in the hippocampus and amygdala (6, 7).In adult rat or mouse brain, two alternative spliced forms are expressed, CB2 and CB3, which are identical except for the C termini (3). There is also CB1 with a different C terminus, but this isoform is not expressed in neurons or in the adult brain. In humans, CB3 is called hPEM2. However, CB2 has not been detected in humans (3). There are also splice variants of CB2 and CB3 (or hPEM2) with or without an Src homology 3 (SH3) domain (1, 3). Alt...

We have found that the γ2 subunit of the GABAA receptor (γ2-GABAAR) specifically interacts with protocadherin γ-C5 (Pcdh-γC5) in the rat brain. The interaction occurs between the large intracellular loop of the γ2-GABAAR and the cytoplasmic domain of Pcdh-γC5. In brain extracts, Pcdh-γC5 co-immunoprecipitates with GABAARs. In co-transfected HEK293 cells, Pcdh-γC5 promotes the transfer of γ2-GABAAR to the cell surface. We have previously shown that in cultured hippocampal neurons, endogenous Pcdh-γC5 forms clusters, some of which associate with GABAergic synapses. Overexpression of Pcdh-γC5 in hippocampal neurons increases the density ofγ2-GABAAR clusters but has no significant effect on the number of GABAergic contacts that these neurons receive, indicating that Pcdh-γC5 is not synaptogenic. Deletion of the cytoplasmic domain of Pcdh-γC5 enhanced its surface expression but decreased the association with both γ2-GABAAR clusters and presynaptic GABAergic contacts. Cultured hippocampal neurons from the Pcdh-γ triple C-type isoform knockout (TCKO) mouse (Pcdhgtcko/tcko) showed plenty of GABAergic synaptic contacts, although their density was reduced compared with sister cultures from wild type and heterozygous mice. Knocking down Pcdh-γC5 expression with shRNA decreased γ2-GABAAR cluster density and GABAergic innervation. The results indicate that although Pcdh-γC5 is not essential for GABAergic synapse formation or GABAAR clustering, i) Pcdh-γC5 regulates the surface expression of GABAARs via cis-cytoplasmic interaction with γ2-GABAAR; and ii) Pcdh-γC5 plays a role in the stabilization and maintenance of some GABAergic synapses.

Background:The number of GABA type-A receptors (GABA A Rs) affects the strength of GABAergic synapses. Results: The E3 ubiquitin ligase RNF34 binds to and ubiquitinates the ␥2 GABA A R subunit, tagging GABA A Rs for degradation. Conclusion: RNF34 regulates the number of postsynaptic GABA A Rs. Significance: This is the first identification of an E3 ubiquitin ligase involved in GABA A R trafficking.

The potential role of taurine on epilepsy and acupuncture anticonvulsion was addressed in the present study. Epilepsy was induced by micro-injection of penicillin into hippocampus of Wistar rats. Taurine was applied by intraperitioneal (i.p.) injection. Electro-acupuncture (EA) was performed on acupoints of DU 20 "Bai Hui" and DU 16 "Feng Fu" along DU channel. Epileptic grades were evaluated by electro-encephalography (EEG) and behavior score. We featured the dose-response relationship between taurine-treated epilepsy and epilepsy-only subjects, detected the effect of exogenous taurine on epilepsy and acupuncture treatment, and investigated taurine transporter immuno-activity in hippocampus using immunohistochemistry. It was found that; I), taurine had a significant antiepileptic effect as applied at i.p. 20 mg/kg, 40mg/kg, 80mg/k.g, especially at 40mg/kg in the rat model of penicillin-induced seizure. Animals were improved by one to three Racine grades in behavior and in frequency and amplitude of EEG 2), Exogenous taurine enhanced the anti-convulsive effect of EA. Both behavior and EEG were improved in taurine-treated rats. EA inhibited 2 Ll, Q. GUO, J-C, JIN, H-B , CHENG, J-S.. YANG, R.epilepsy. Exogenous taurine impToved epilepsy in a synergistic manner to EA. 3), EA increased the concentration of laurine tTansporter in hippocampus by comparing EA-treated epilepsy with normal control and penicillin only, or EA-treated plus taurine-treated epilepsy with taurine-treated only epilepsy and penicillin only. The resulting data suggested that taurine may play an inhibitory role against epilepsy as an inhibitory amino acid in the central nervous system and EA may inhibit epilepsy via upregulating the concentration of taurine transporter to increase the release of taurine.

Eleetro-acupuncture (EA) partially inhibited epilepsy with great success. The biological basis underlying EA anti-convulsion remained uncertain, which resulted in limited application and slow improvement of acupuncture. Our previous study indicated that taurine may play an inhibitory role against epilepsy as an inhibitory aniino acid in the central nervous system and EA may inhibit epilepsy via up-regulating the expression of taurine transporter to increase the release of taurine. Involvement of taurine in kainic acid (KA)-induced epilepsy and anti-convulsion of EA was further addressed on taurine deficiency animal in the present work. We instituted endogenous taurine-deficiency model by supplementation of beta-alanine (3%) in drinking water for continuous 10 days initially, injected KA into lateral cerebral ventricle to induce epileptic seizure, and performed EA treatment on DU26 "RenZhong" and Kl "YongQuan" acupoints by an EA apparatus (Model G6805-2) using successive waves with the frequency 64Hz and Ihe current intensity 0.8-1.0mA for 30 minutes in Sprague-Dawley (SD) rats. 208Taurine levels markedly decreased in cortex, hippocampus, striatum and cerebellum of rats after beta-alanine administralion by fluore-HPLC measurement-EA alleviated epileptie activity in rals at 3.5h time point after KA injection, whereas beta-alanine-indueed laurine depletion rendered rats more susceptible to KA-induced epilepsy. TauHne transporter level increased after EA treatment. These results suggested thai taurine partieipated in epileptogenesis and EA may be related to taurine in controliing epileptic seizure.