GABAergic terminals are required for postsynaptic clustering of dystrophin but not of GABA(A) receptors and gephyrin.

In rat hippocampal cultures, we show by multilabeling immunocytochemistry that pyramidal cells, which receive little or no GABAergic input, mistarget alpha2-GABA(A) receptors and gephyrin to glutamatergic terminals. This mismatch does not occur in neurons innervated by numerous GABAergic terminals. A similar phenomenon has been reported for isolated autaptic hippocampal neurons (Rao et al., 2000). GABAergic synapses typically form multiple release sites apposed to GABA(A) receptor and gephyrin clusters. Remarkably, dystrophin, a protein highly abundant in skeletal muscle membranes, is extensively colocalized with alpha2-GABA(A) receptors exclusively opposite GABAergic terminals. In addition, selective apposition of syntrophin and beta-dystroglycan to GABAergic presynaptic terminals suggests that the entire dystrophin-associated protein complex (DPC) clusters at GABAergic synapses. In contrast to gephyrin and GABA(A) receptors, DPC proteins are not mistargeted to glutamatergic synapses, indicating independent clustering mechanisms. This was confirmed in hippocampal neurons cultured from GABA(A) receptor gamma2 subunit-deficient mice. Clustering of GABA(A) receptor and gephyrin in these neurons was strongly impaired, whereas clustering of dystrophin and associated proteins was unaffected by the absence of the gamma2 subunit. Our results indicate that accumulation of dystrophin and DPC proteins at GABAergic synapses occurs independently of postsynaptic GABA(A) receptors and gephyrin. We suggest that selective signaling from GABAergic terminals contributes to postsynaptic clustering of dystrophin.

Formation and plasticity of GABAergic synapses: physiological mechanisms and pathophysiological implications.

gamma-Aminobutyric acid(A) (GABA(A)) receptors mediate most of the fast inhibitory neurotransmission in the CNS. They represent a major site of action for clinically relevant drugs, such as benzodiazepines and ethanol, and endogenous modulators, including neuroactive steroids. Alterations in GABA(A) receptor expression and function are thought to contribute to prevalent neurological and psychiatric diseases. Molecular cloning and immunochemical characterization of GABA(A) receptor subunits revealed a multiplicity of receptor subtypes with specific functional and pharmacological properties. A major tenet of these studies is that GABA(A) receptor heterogeneity represents a key factor for fine-tuning of inhibitory transmission under physiological and pathophysiological conditions. The aim of this review is to highlight recent findings on the regulation of GABA(A) receptor expression and function, focusing on the mechanisms of sorting, targeting, and synaptic clustering of GABA(A) receptor subtypes and their associated proteins, on trafficking of cell-surface receptors as a means of regulating synaptic (and extrasynaptic) transmission on a short-time basis, on the role of endogenous neurosteroids for GABA(A) receptor plasticity, and on alterations of GABA(A) receptor expression and localization in major neurological disorders. Altogether, the findings presented in this review underscore the necessity of considering GABA(A) receptor-mediated neurotransmission as a dynamic and highly flexible process controlled by multiple mechanisms operating at the molecular, cellular, and systemic level. Furthermore, the selected topics highlight the relevance of concepts derived from experimental studies for understanding GABA(A) receptor alterations in disease states and for designing improved therapeutic strategies based on subtype-selective drugs.

Intact sorting, targeting, and clustering of gamma-aminobutyric acid A receptor subtypes in hippocampal neurons in vitro.

The cellular and subcellular distribution of four GABA(A) receptor subtypes, identified by the presence of the alpha1, alpha2, alpha3, or alpha5 subunit, was investigated immunocytochemically in dissociated cultures of hippocampal neurons. We addressed the questions whether (1) cell-type specific expression, (2) axonal/somatodendritic targeting, and (3) synaptic/extrasynaptic clustering of GABA(A) receptor subtypes was retained in vitro. For comparison, the in vivo distribution pattern was assessed in sections from adult rat brain. The differential expression of GABA(A) receptor subunits allowed to identify five morphologically distinct cell types in culture: the alpha1 subunit was observed in glutamic acid decarboxylase-positive interneurons, the alpha2 and alpha5 subunits marked pyramidal-like cells, and the alpha3 subunit labeled three additional cell types, including presumptive hilar cells. All subunits were found in the somatodendritic compartment. In addition, appropriate axonal targeting was evidenced by the intense alpha2, and sometimes alpha3 subunit labeling of axon-initial segments (AIS) of pyramidal cells and hilar cells, respectively. Accordingly, both receptor subtypes were targeted to AIS in vivo, as well. Synaptic receptors were identified by colocalization with gephyrin, a postsynaptic clustering protein, and apposition to presynaptic terminals labeled with synapsin I. In vitro and in vivo, alpha1- and alpha2-receptor subtypes formed numerous synaptic clusters, alpha3-GABA(A) receptors were located either synaptically or extrasynaptically depending on the cell type, whereas alpha5-GABA(A) receptors were extrasynaptic. We conclude that receptor targeting to broad subcellular locations does not require specific GABAergic innervation patterns, which are disturbed in vitro, but depends on protein-protein interactions in the postsynaptic cell that are both subunit- and neuron-specific.

Influx of extracellular calcium regulates actin-dependent morphological plasticity in dendritic spines.

Dendritic spines contain a specialized cytoskeleton composed of dynamic actin filaments capable of producing rapid changes in their motility and morphology. Transient changes in Ca2+ levels in the spine cytoplasm have been associated with the modulation of these effects in a variety of ways. To characterize the contribution of Ca2+ fluxes originating through different pathways to these phenomena, we used time-lapse imaging of cultured hippocampal neurons expressing GFP-actin to follow the influence of postsynaptic neurotransmitter receptors, voltage-activated Ca2+ channels and release from internal Ca2+ stores on spine actin dynamics. Stimulation of AMPA receptors produced a rapid blockade of actin-dependent spine motility that was immediately reversible when AMPA was removed. Stimulation of NMDA receptors also blocked spine motility but in this case suppression of actin dynamics was delayed by up to 30 min depending on NMDA concentration and motility was never seen to recover when NMDA was removed. These effects could be mimicked by depolarizing neurons under appropriate circumstances demonstrating the involvement of voltage-activated Ca2+ channels in AMPA receptor-mediated effects and the receptor associated Ca2+ channel in the effects of NMDA. Caffeine, an agent that releases Ca2+ from internal stores, had no immediate effect on spine actin, a result compatible with the lack of caffeine-releasable Ca2+ in cultured hippocampal neurons under resting conditions. Blocking internal store function by thapsigargin led to a delayed suppression of spine actin dynamics that was dependent on extracellular Ca2+. Together these results indicate the common involvement of changes in Ca2+ levels in modulating actin-dependent effects on dendritic spine motility and morphology through several modes of electrophysiological activation.