Disruption of postsynaptic GABA_A receptor clusters leads to decreased GABAergic innervation of pyramidal neurons

Abstract

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...

Molecular and synaptic organization of GABAA receptors in the cerebellum: Effects of targeted subunit gene deletions

“…In these mice, there is an almost complete loss of a1 subunit and gephyrin clusters in the cerebellum, in line with the requirement of the c2 subunit for postsynaptic aggregation of GABA A receptors and gephyrin (71,72). No specific compensation with another c2 subunit variant was observed, indicating indirectly that the c1 and c3 subunits do not contribute significantly to neuronal GABA A receptors in the mouse cerebellum.…”

Calcium-modulating cyclophilin ligand regulates membrane trafficking of postsynaptic GABAA receptors

“…Such presynaptic deficits have recently been demonstrated upon shRNA-mediated knockdown of the γ2 subunit (Li et al, 2005), or postsynaptic depletion of the γ2 subunit by knock down of GODZ . Interestingly, significant deficits in GABAergic innervation are also seen in γ2 +/-knockout neurons, provided they are cocultured with an excess of wildtype neurons (R. Lal and B.L., unpublished results).…”

“…4D). Reduced GABAergic innervation is also seen upon postsynaptic knockdown of the γ2 subunit (Li et al, 2005) or GODZ , a palmitoyltransferase required for normal accumulation of GABA A Rs at synapses. Collectively, the data indicate that CAML is required for normal exocytosis and/or membrane stability of γ2 subunit containing GABA A Rs.…”

“…Of particular interest are postsynaptic receptor subtypes, which contain the γ2 subunit in diverse combinations with α1-3 and β1-3 subunits and carry GABAergic fast synaptic currents (Fritschy and Brunig, 2003;Luscher and Keller, 2004;Moss and Smart, 2001). The γ2 subunit is essential for trafficking and accumulation of GABA A Rs in the postsynaptic membrane and for GABAergic synaptic transmission (Essrich et al, 1998;Li et al, 2005;Schweizer et al, 2003). Moreover, modest developmental deficits in γ2 subunit-containing GABA A Rs are implicated in the etiology of anxiety and depressive disorders (Crestani et al, 1999;Earnheart et al, 2007).…”

GABA and neuroligin signaling: linking synaptic activity and adhesion in inhibitory synapse development

“…The γ2 subunit is essential for accumulation of cell surface GABA A Rs at postsynaptic sites [41,42]. Interestingly, acute suppression of γ2 expression in cultured hippocampal neurons not only disrupts GABA A R clustering but also results in a profound reduction of GABAergic innervation of γ2 deficient neurons [43,44]. Moreover, when palmitoylation of the γ2 subunit was suppressed by knockdown of the DHHC-family palmitoyltransferase GODZ, trafficking of GABA A Rs to postsynaptic sites was perturbed and GABAergic innervation was reduced [43].…”