Developmental maturation of synaptic and extrasynaptic GABAA receptors in mouse thalamic ventrobasal neurones.

Thalamic ventrobasal (VB) relay neurones express multiple GABA(A) receptor subtypes mediating phasic and tonic inhibition. During postnatal development, marked changes in subunit expression occur, presumably reflecting changes in functional properties of neuronal networks. The aims of this study were to characterize the properties of synaptic and extrasynaptic GABA(A) receptors of developing VB neurones and investigate the role of the alpha(1) subunit during maturation of GABA-ergic transmission, using electrophysiology and immunohistochemistry in wild type (WT) and alpha(1)(0/0) mice and mice engineered to express diazepam-insensitive receptors (alpha(1H101R), alpha(2H101R)). In immature brain, rapid (P8/9-P10/11) developmental change to mIPSC kinetics and increased expression of extrasynaptic receptors (P8-27) formed by the alpha(4) and delta subunit occurred independently of the alpha(1) subunit. Subsequently (> or = P15), synaptic alpha(2) subunit/gephyrin clusters of WT VB neurones were replaced by those containing the alpha(1) subunit. Surprisingly, in alpha(1)(0/0) VB neurones the frequency of mIPSCs decreased between P12 and P27, because the alpha(2) subunit also disappeared from these cells. The loss of synaptic GABA(A) receptors led to a delayed disruption of gephyrin clusters. Despite these alterations, GABA-ergic terminals were preserved, perhaps maintaining tonic inhibition. These results demonstrate that maturation of synaptic and extrasynaptic GABA(A) receptors in VB follows a developmental programme independent of the alpha(1) subunit. Changes to synaptic GABA(A) receptor function and the increased expression of extrasynaptic GABA(A) receptors represent two distinct mechanisms for fine-tuning GABA-ergic control of thalamic relay neurone activity during development.

Vertebrate-specific sequences in the gephyrin E-domain regulate cytosolic aggregation and postsynaptic clustering.

Gephyrin is a multifunctional protein contributing to molybdenum cofactor (Moco) synthesis and postsynaptic clustering of glycine and GABA(A) receptors. It contains three major functional domains (G-C-E) and forms cytosolic aggregates and postsynaptic clusters by unknown mechanisms. Here, structural determinants of gephyrin aggregation and clustering were investigated by neuronal transfection of EGFP-tagged deletion and mutant gephyrin constructs. EGFP-gephyrin formed postsynaptic clusters containing endogenous gephyrin and GABA(A)-receptors. Isolated GC- or E-domains failed to aggregate and exerted dominant-negative effects on endogenous gephyrin clustering. A construct interfering with intermolecular E-domain dimerization readily auto-aggregated but showed impaired postsynaptic clustering. Finally, two mutant constructs with substitution of vertebrate-specific E-domain sequences with homologue bacterial MoeA sequences uncovered a region crucial for gephyrin clustering. One construct failed to aggregate, but retained Moco biosynthesis capacity, demonstrating the independence of gephyrin enzymatic activity and aggregation. Reinserting two vertebrate-specific residues restored gephyrin aggregation and increased formation of postsynaptic clusters containing GABA(A) receptors at the expense of PSD-95 clusters - a marker of glutamatergic synapses. These results underscore the key role of specific E-domain regions distinct from the known dimerization interface for controlling gephyrin aggregation and postsynaptic clustering and suggest that formation of gephyrin clusters influences the homeostatic balance between inhibitory and excitatory synapses.