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Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABAARs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABAARs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region (Gabra2-1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents. Gabra2–1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. Surviving Gabra2-1 mice show anxiety and elevations in electroencephalogram δ power, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development.

Knowledge of the functional organization of the GABAergic system, the main inhibitory neurotransmitter system, in the CNS has increased remarkably in recent years. In particular, substantial progress has been made in elucidating the molecular mechanisms underlying the formation and plasticity of GABAergic synapses. Evidence available ascribes a key role to the cytoplasmic protein gephyrin to form a postsynaptic scaffold anchoring GABA(A) receptors along with other transmembrane proteins and signaling molecules in the postsynaptic density. However, the mechanisms of gephyrin scaffolding remain elusive, notably because gephyrin can auto-aggregate spontaneously and lacks PDZ protein interaction domains found in a majority of scaffolding proteins. In addition, the structural diversity of GABA(A) receptors, which are pentameric channels encoded by a large family of subunits, has been largely overlooked in these studies. Finally, the role of the dystrophin-glycoprotein complex, present in a subset of GABAergic synapses in cortical structures, remains ill-defined. In this review, we discuss recent results derived mainly from the analysis of mutant mice lacking a specific GABA(A) receptor subtype or a core protein of the GABAergic postsynaptic density (neuroligin-2, collybistin), highlighting the molecular diversity of GABAergic synapses and its relevance for brain plasticity and function. In addition, we discuss the contribution of the dystrophin-glycoprotein complex to the molecular and functional heterogeneity of GABAergic synapses.

Elucidating the molecular organization of synapses is essential for understanding brain function and plasticity. Immunofluorescence, combined with various fluorescent probes, is a sensitive and versatile method for morphological studies. However, analysis of synaptic proteins in situ is limited by epitope-masking after tissue fixation. Furthermore, postsynaptic proteins (such as ionotropic receptors and scaffolding proteins) often require weaker fixation for optimal detection than most intracellular markers, thereby hindering simultaneous visualization of these molecules. We present three protocols, which are alternatives to perfusion fixation, to overcome these restrictions. Brief tissue fixation shortly after interruption of vital functions preserves morphology and antigenicity. Combined with specific neuronal markers, selective detection of gamma-aminobutyric acid A (GABA(A)) receptors and the scaffolding protein gephyrin in relation to identified inhibitory presynaptic terminals in the rodent brain is feasible by confocal laser scanning microscopy. The most sophisticated of these protocols can be associated with electrophysiology for correlative studies of synapse structure and function. These protocols require 2-3 consecutive days for completion.

In this study, we investigated the distribution and developmental expression of the GABA(B) receptor subunits, GABA(B1) and GABA(B2), in the main and accessory olfactory bulbs of the rat. Antibodies raised against these subunits strongly labelled the glomerular layer, suggesting that olfactory and vomeronasal nerve fibers express functional GABA(B) receptors. Using postembedding immunogold cytochemistry, we found that GABA(B) receptors can be present at both extrasynaptic and presynaptic sites of olfactory nerve terminals, and in the latter case they are preferentially associated with the peripheral part of the synaptic specialization. Olfactory nerve fibers expressed GABA(B1) and GABA(B2) at early developmental stages, suggesting that GABA(B) receptors may play a role in olfactory development. Output and local neurons of the main and accessory olfactory bulbs were also labelled for GABA(B1) and GABA(B2), although the subcellular distribution patterns of the two subunits were not completely overlapping. These results indicate that presynaptically located GABA(B) receptors modulate neurotransmitter release from olfactory and vomeronasal nerve fibers and that, in addition to this presynaptic role, GABA(B) receptors may regulate neuronal excitability in infraglomerular circuits.