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Benzodiazepines (BDZs) are known to increase the amplitude and duration of IPSCs. Moreover, at low [GABA], BDZs strongly enhance GABAergic currents suggesting the up-regulation of agonist binding while their action on gating remains a matter of debate. In the present study we have examined the impact of flurazepam and zolpidem on mIPSCs by investigating their effects on GABA(A)R binding and gating and by considering dynamic conditions of synaptic receptor activation. Flurazepam and zolpidem enhanced the amplitude and prolonged decay of mIPSCs. Both compounds strongly enhanced responses to low [GABA] but, surprisingly, decreased the currents evoked by saturating or half-saturating [GABA]. Analysis of current responses to ultrafast GABA applications indicated that these compounds enhanced binding and desensitization of GABA(A) receptors. Flurazepam and zolpidem markedly prolonged deactivation of responses to low [GABA] but had almost no effect on deactivation at saturating or half-saturating [GABA]. Moreover, at low [GABA], flurazepam enhanced desensitization-deactivation coupling but zolpidem did not. Recordings of responses to half-saturating [GABA] applications revealed that appropriate timing of agonist exposure was sufficient to reproduce either a decrease or enhancement of currents by flurazepam or zolpidem. Recordings of currents mediated by recombinant ('synaptic') alpha1beta2gamma2 receptors reproduced all major findings observed for neuronal GABA(A)Rs. We conclude that an extremely brief agonist transient renders IPSCs particularly sensitive to the up-regulation of agonist binding by BDZs.

Estrogens exert a variety of modulatory effects on the structure and function of the nervous system. In particular, 17 beta-estradiol was found to affect GABAergic inhibition in adult animals but its action on GABAergic currents during development has not been elucidated. In the present study, we investigated the effect of 17 beta-estradiol on hippocampal neurons developing in vitro. In this model, mIPSC kinetics showed acceleration with age along with increased alpha1 subunit expression, similarly as in vivo. Long-term treatment with 17 beta-estradiol increased mIPSC amplitudes in neurons cultured for 6-8 and 9-11DIV and prolonged the mIPSC decaying phase only in the 9-11DIV group. The time needed for the onset of 17 beta-estradiol effect on mIPSC amplitude was approximately 48 h. In the period of 9-11DIV, treatment with 17 beta-estradiol strongly reduced the tonic conductance activated by low GABA concentrations. The effects of 17 beta-estradiol on mIPSCs and tonic conductance were not correlated with any change in expression of considered GABAAR subunits (alpha1-3, alpha5-6, gamma2) while alpha4 and delta subunits were at the detection limit. In conclusion, we provide evidence that 17 beta-estradiol differentially affects the phasic and tonic components of GABAergic currents in neurons developing in vitro.

Protons are the most ubiquitous and very potent modulators of the biological systems. Hydrogen ions are known to modulate GABA(A) receptors (GABA(A)Rs), but the mechanism whereby these ions affect IPSCs and the gating of GABA(A)Rs is not clear. In the present study we examined the effect of protons on miniature IPSCs (mIPSCs) and found that hydrogen ions strongly affected both their amplitude and time course. To explore the underlying mechanisms with resolution adequate to the time scale of synaptic transmission, we recorded current responses to ultrafast GABA applications at various pH. These experiments revealed that the major effect of protons on GABA(A)R gating is a strong enhancement of desensitization and binding rates at increasing pH. This analysis also indicated that desensitization rate is the fastest ligand-independent transition in the GABA(A)R gating scheme. Although proton effects on the time course of mIPSCs and current responses to saturating [GABA] were similar, the pH dependencies of amplitudes were almost opposite. Our quantitative analysis, based on model simulations, indicated that this difference resulted from a much shorter receptor exposure to agonist in the case of mIPSCs. Modeling of IPSCs as current responses to brief exponentially decaying GABA applications was sufficient to reproduce correctly the pH dependence of mIPSCs, and optimal fit was obtained for peak [GABA] of 1.5-3 mm and a clearance time constant of 0.075-0.125 msec. Our analysis indicates that, for these parameters of GABA transient, in control conditions (pH 7.2) mIPSCs are not saturated.

The time course of GABA-evoked currents is the main source of information on the GABA(A) receptor gating. Since the kinetics of these currents depends on the transitions between several receptor conformations, it is a major challenge to define the relations between current kinetics and the respective rate constants of the microscopic gating scheme. The aim of this study was to further explore the impact of different GABA(A) receptor conformations on the kinetics of currents elicited by ultra-fast GABA applications. We show that the rising phase and amplitude of GABA-evoked currents depend on desensitization and singly bound states. The occupancy of bound receptors depends not only on binding properties but also on opening/closing and desensitization. The impact of such functional coupling between channel states is critical in conditions of high non-equilibrium typical for synaptic transmission. The concentration dependence of the rising phase of the GABA-elicited current indicates positive cooperativity between agonist binding sites. We provide evidence that preequilibration at low GABA concentrations reduce GABA-evoked currents due to receptor trapping in a singly bound desensitized state.

Current responses to ultrafast gamma-aminobutyric acid (GABA) applications were recorded from excised patches in rat hippocampal neurons to study the gating properties of GABA(A) receptors at GABA concentrations close to saturating ones and higher. The amplitude of currents saturated at approximately 1 mm, while the onset rate of responses reached saturation at 4-6 mm GABA. At high GABA concentrations (> 10 mm), the amplitude of current responses was reduced in a dose-dependent manner with a half-blocking GABA concentration of approximately 50 mm. The peak reduction at high GABA doses was accompanied by a tendency to increase the steady-state to peak ratio. At concentrations higher than 30 mm, this effect took the form of a rebound current, i.e. during the prolonged GABA applications, the current firstly declined due to desensitization onset and then, instead of decreasing towards a steady-state value, clearly increased. Both the self-inhibition of GABA(A) receptors by high GABA doses and rebound were clearly voltage dependent, being larger at positive holding potentials. The fast desensitization component accelerated with depolarization at all saturating [GABA] tested. The rebound phenomenon indicates that the self-block of GABAA receptors is state dependent, and suggests that the sojourn in the desensitized conformation provides a 'rescue' from the block. We propose that high GABA concentrations inhibit the receptors by direct occlusion of the channel pore having no effect on the receptor gating.