The alpha5 subunit of the GABA(A) receptors (GABA(A)Rs) has a restricted expression in the brain. Maximum expression of this subunit occurs in the hippocampus, cerebral cortex, and olfactory bulb. Hippocampal pyramidal cells show high expression of alpha5 subunit-containing GABA(A)Rs (alpha5-GABA(A)Rs) both in culture and in the intact brain. A large pool of alpha5-GABA(A)Rs is extrasynaptic and it has been proposed to be involved in the tonic GABAergic inhibition of the hippocampus. Nevertheless, there are no studies on the localization of the alpha5-GABA(A)Rs at the electron microscope (EM) level. By using both immunofluorescence of cultured hippocampal pyramidal cells and EM postembedding immunogold of the intact hippocampus we show that, in addition to the extrasynaptic pool, there is a pool of alpha5-GABA(A)Rs that concentrates at the GABAergic synapses in dendrites of hippocampal pyramidal cells. The results suggest that the synaptic alpha5-GABA(A)Rs might play a role in the phasic GABAergic inhibition of pyramidal neurons in hippocampus and cerebral cortex.

We have studied the effects of GABAergic innervation on the clustering of GABA A receptors (GABA A Rs) in cultured hippocampal neurons. In the absence of GABAergic innervation, pyramidal cells form small (0.36 Ϯ 0.01 m diameter) GABA A R clusters at their surface in the dendrites and soma. When receiving GABAergic innervation from glutamic acid decarboxylase-containing interneurons, pyramidal cells form large (1.62 Ϯ 0.08 m breadth) GABA A R clusters at GABAergic synapses. This is accompanied by a disappearance of the small GABA A R clusters in the local area surrounding each GABAergic synapse. Although the large synaptic GABA A R clusters of any neuron contained all GABA A R subunits and isoforms expressed by that neuron, the small clusters not localized at GABAergic synapses showed significant heterogeneity in subunit and isoform composition. Another difference between large GABAergic and small non-GABAergic GABA A R clusters was that a significant proportion of the latter was juxtaposed to postsynaptic markers of glutamatergic synapses such as PSD-95 and AMPA receptor GluR1 subunit. The densities of both the glutamate receptor-associated and non-associated small GABA A R clusters were decreased in areas surrounding GABAergic synapses. However, no effect on the density or distribution of glutamate receptor clusters was observed. The results suggest that there are local signals generated at GABAergic synapses that induce both assembly of large synaptic GABA A R clusters at the synapse and disappearance of the small GABA A R clusters in the surrounding area. In the absence of GABAergic innervation, weaker GABA A R-clustering signals, generated at glutamatergic synapses, induce the formation of small postsynaptic GABA A R clusters that remain juxtaposed to glutamate receptors at glutamatergic 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 transynaptic 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.

We have used RNA interference (RNAi) to knock down the expression of the c2 subunit of the GABA A receptors (GABA A Rs) in pyramidal neurons in culture and in the intact brain. Two hairpin small interference RNAs (shRNAs) for the c2 subunit, one targeting the coding region and the other one the 3¢-untranslated region (UTR) of the c2 mRNA, when introduced into cultured rat hippocampal pyramidal neurons, efficiently inhibited the synthesis of the GABA A receptor c2 subunit and the clustering of other GABA A R subunits and gephyrin in these cells. More significantly, this effect was accompanied by a reduction of the GABAergic innervation that these neurons received. In contrast, the c2 shRNAs had no effect on the clustering of postsynaptic a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, postsynaptic density protein 95 (PSD-95) or presynaptic glutamatergic innervation. A c2-enhanced green fluorescent protein (EGFP) subunit construct, whose mRNA did not contain the 3¢-UTR targeted by c2 RNAi, rescued both the postsynaptic clustering of GABA A Rs and the GABAergic innervation. Decreased GABA A R clustering and GABAergic innervation of pyramidal neurons in the post-natal rat cerebral cortex was also observed after in utero transfection of these neurons with the c2 shRNAs. The results indicate that the postsynaptic clustering of GABA A Rs in pyramidal neurons is involved in the stabilization of the presynaptic GABAergic contacts. Studies of c2 subunit-deficient mouse mutants have shown that the c2 subunit of the GABA A receptor (GABA A R) is necessary for the postsynaptic clustering of the GABA A Rs and for the maintenance of GABA A R clusters at GABAergic synapses (Essrich et al. 1998;Schweizer et al. 2003). The c2-/-mouse mutant shows a severe deficit in GABAergic synaptic transmission and dies soon after birth (Günther et al. 1995). GABA A Rs play a morphogenic role during embryonic development (Rudolph and Mohler 2004;Vicini and Ortinski 2004). Thus, some of the observed phenotypes in these and other mutant mice might result from developmental alterations, while the absence of phenotype might be because of compensatory mechanisms. RNA interference (RNAi, Dykxhoorn et al. 2003;Huppi et al. 2005) is a simpler alternative to the gene knockout technology that can also overcome some of the limitations inherent to the use of mouse mutants. In this study, we have used c2 RNAi to study GABA A R clustering in loss-of-function experiments, both in neuronal cultures and in the intact brain after in utero electroporation. Our results support the notion that the c2 subunit is necessary for the postsynaptic clustering and maintenance of GABA A Rs and gephyrin (a postsynaptic scaffolding protein that is present at inhibitory GABAergic and glycinergic synapses). More interesting, because we revealed it with RNAi technology but it had not been previously observed with the c2 mouse mutants, is the observation that the disruption of the postsynaptic clustering Abbreviations used: AIS, axon initial segmen...

We have isolated, from a rat brain cDNA library, a clone corresponding to a 2779-bp cDNA encoding a novel splice form of the glutamate receptor interacting protein-1 (GRIP1). We call this 696-amino acid splice form GRIP1c 4-7 to differentiate it from longer splice forms of GRIP1a/b containing seven PDZ domains. The four PDZ domains of GRIP1c 4-7 are identical to PDZ domains 4 -7 of GRIP1a/b. GRIP1c 4-7 also contains 35 amino acids at the N terminus and 12 amino acids at the C terminus that are different from GRIP1a/b. In transfected HEK293 cells, a majority of GRIP1c 4-7 was associated with the plasma membrane. GRIP1c 4-7 interacted with GluR2/3 subunits of the ␣-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor. In low density hippocampal cultures, GRIP1c 4-7 clusters colocalized with GABAergic (where GABA is ␥-aminobutyric acid) and glutamatergic synapses, although a higher percentage of GRIP1c 4-7 clusters colocalized with ␥-aminobutyric acid, type A, receptor (GABA A R) clusters than with ␣-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor clusters. Transfection of hippocampal neurons with hemagglutinin-tagged GRIP1c 4-7 showed that it could target to the postsynaptic complex of GABAergic synapses colocalizing with GABA A R clusters. GRIP1c 4-7-specific antibodies, which did not recognize previously described splice forms of GRIP1, recognized a 75-kDa protein that is enriched in a postsynaptic density fraction isolated from rat brain. EM immunocytochemistry experiments showed that in intact brain GRIP1c 4-7 concentrates at postsynaptic complexes of both type I glutamatergic and type II GABAergic synapses although it is also presynaptically localized. These results indicate that GRIP1c 4-7 plays a role not only in glutamatergic synapses but also in GABAergic synapses.