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[GABA.sub.A] receptors ([GABA.sub.A]RS), the principal sites of synaptic inhibition in the brain, are dynamic entities on the neuronal cell surface, but the role their membrane trafficking plays in shaping neuronal activity remains obscure. Here, we examined this by using mutant receptor [beta]3 subunits ([beta]3S408/9A), which have reduced binding to the clathrin adaptor protein-2, a critical regulator of [GABA.sub.A]R endocytosis. Neurons expressing [beta]3S408/9A subunits exhibited increases in the number and size of inhibitory synapses, together with enhanced inhibitory synaptic transmission due to reduced [GABA.sub.A]R endocytosis. Furthermore, neurons expressing [beta]3S408/9A subunits had deficits in the number of mature spines and reduced accumulation of postsynaptic density protein-95 at excitatory synapses. This deficit in spine maturity was reversed by pharmacological blockade of [GABA.sub.A]Rs. Therefore, regulating the efficacy of synaptic inhibition by modulating [GABA.sub.A]R membrane trafficking may play a critical role in regulating spine maturity with significant implications for synaptic plasticity together with behavior. endocytosis | inhibition | phosphorylation more...

Benzodiazepines potentiate γ-aminobutyric acid type A receptor (GABAAR) activity and are widely prescribed to treat anxiety, insomnia, and seizure disorders. Unfortunately, clinical use of benzodiazepines (BZs) is severely limited by tolerance. The mechanisms leading to BZ tolerance are unknown. BZs bind at the interface between an a and γ subunit of GABAARs, preferentially enhancing synaptic receptors largely composed of α(1-3, 5), β3, and γ2 subunits. Using confocal imaging and patch-clamp approaches, we show that treatment with the BZ flurazepam decreases GABAAR surface levels and the efficacy of neuronal inhibition in hippocampal neurons. A dramatic decrease in surface and total levels of α2 subunit-containing GABAARs occurred within 24 h of flurazepam treatment, whereas GABAARs incorporating α1 subunits showed little alteration. The GABAAR surface depletion could be reversed by treatment with the BZ antagonist Ro 15-1788. Coincident with decreased GABAAR surface levels, flurazepam treatment reduced miniature inhibitory postsynaptic current amplitude, which returned to control levels with acute Ro 15-1788 treatment. GABAAR endocytosis and insertion rates were unchanged by flurazepam treatment. Treatment with leupeptin restored flurazepam lowered receptor surface levels, strongly suggesting that flurazepam increases lysosomal degradation of GABAARs. Together, these data suggest that flurazepam exposure enhances degradation of α2 subunit-containing GABAARs after their removal from the plasma membrane, leading to a reduction in inhibitory synapse size and number along with a decrease in the efficacy of synaptic inhibition. These reported subtype-specific changes in GABAAR trafficking provide significant mechanistic insight into the initial neuroadaptive responses occurring with BZ treatment. [ABSTRACT FROM AUTHOR] more...

To reach the open state, the GABAA receptor (GABAAR) is assumed to bind two agonist molecules. Although it is currently believed that GABAAsubscript>R could also operate in the monoliganded state, the gating properties of singly bound GABAAR are poorly understood and their physiological role is still obscure. In the present study, we characterize for the first time the gating properties of singly bound GABAARs by using a mutagenesis approach and we propose that monoliganded GABAAR contribute in shaping synaptic responses. At saturating GABA concentrations, currents mediated by recombinant GABAARs with a single functional binding site display slow onset, fast deactivation kinetics, and slow rate of desensitization-resensitization. GABAARs with two binding sites activated by brief pulses of subsaturating GABA concentrations (in the range of the GABA concentration profile in the synaptic cleft) could also mediate fast deactivating currents, displaying deactivation kinetics similar to those mediated by GABAARs with a single functional binding site. Model simulations of receptors activated by realistic synaptic GABA waves revealed that a considerable proportion of GABAA receptors open in the monoliganded state during synaptic transmission, therefore contributing in shaping IPSCs. [ABSTRACT FROM AUTHOR] more...

Gliotransmission, the release of molecules from astrocytes, regulates neuronal excitability and synaptic transmission in situ. Whether this process affects neuronal network activity in vivo is not known. Using a combination of astrocyte-specific molecular genetics, with in vivo electrophysiology and pharmacology, we determined that gliotransmission modulates cortical slow oscillations, a rhythm characterizing nonrapid eye movement sleep. Inhibition of gliotransmission by the expression of a dominant negative SNARE domain in astrocytes affected cortical slow oscillations, reducing the duration of neuronal depolarizations and causing prolonged hyperpolarizations. These network effects result from the astrocytic modulation of intracortical synaptic transmission at two sites: a hypofunction of postsynaptic NMDA receptors, and by reducing extracellular adenosine, a loss of tonic Al receptor-mediated inhibition. These results demonstrate that rhythmic brain activity is generated by the coordinated action of the neuronal and glial networks. [ABSTRACT FROM AUTHOR] more...