Effect of Zolpidem on Miniature IPSCs and Occupancy of Postsynaptic GABA_AReceptors in Central Synapses

Abstract

GABAA-mediated miniature IPSCs (mIPSCs) were recorded from layer V pyramidal neurons of the visual cortex using whole-cell patch-clamp recording in rat brain slices. At room temperature, the benzodiazepine site agonist zolpidem enhanced both the amplitude (to 138 +/- 26% of control value at 10 microM) and the duration (163 +/- 14%) of mIPSCs. The enhancement of mIPSC amplitude was not caused by an increase of the single-channel conductance of the postsynaptic receptors, as determined by peak-scaled non-stationary fluctuation analysis of mIPSCs. The effect of zolpidem on fast, synaptic-like (1 msec duration) applications of GABA to outside-out patches was also investigated. The EC50 for fast GABA applications was 310 microM. In patches, zolpidem enhanced the amplitude of currents elicited by subsaturating GABA applications (100-300 microM) but not by saturating applications (10 mM). The increase of mIPSC amplitude by zolpidem provides evidence that the GABAA receptors are not saturated during miniature synaptic transmission in the recorded cells. By comparing the facilitation induced by 1 microM zolpidem on outside-out patches and mIPSCs, we estimated the concentration of GABA seen by the postsynaptic GABAA receptors to be approximately 300 microM after single vesicle release. We have estimated a similar degree of receptor occupancy at room and physiological temperature. However, at 35 degreesC, zolpidem did not enhance the amplitude of mIPSCs or of subsaturating GABA applications on patches, implying that, in these neurons, zolpidem cannot be used to probe the degree of receptor occupancy at physiological temperature.

Alterations of GABAA-Receptor Function and Allosteric Modulation During Development of Status Epilepticus

“…It would be interesting to know whether the enhanced GABAergic function contributed to the periodic reduction of seizure severity. The weighted decay time constant of mIPSCs in the present study was larger than previously reported values at room temperature (McIntyre et al 2002;Perrais and Ropert 1999). However, our recordings were performed at a temperature range (19 -23°C) lower than that of the above-cited studies (22-26°C).…”

“…Diazepam modulation of mIPSC amplitude may be age dependent; potentiation of GABA-evoked currents (Zhang et al 2004) and mIPSC amplitudes (Cohen et al 2000;Defazio and Hablitz 1998;Hajos et al 2000;Nusser et al 1997;Perrais and Ropert 1999) has been observed in juvenile neurons including hippocampal dentate granule cells. In addition, the potentiating effect of diazepam may also be temperature dependent (Perrais and Ropert 1999).…”

Contributions of the GABAAReceptor α6 Subunit to Phasic and Tonic Inhibition Revealed by a Naturally Occurring Polymorphism in the α6 Gene

“…The GABA concentration dependence correlated with the relative affinities of ␥2 and ␦ subunit-containing GABARs for GABA: EC 50 values for ␣6/␦ subunit-containing GABARs are ϳ500 -700 nM, whereas for ␣6/␥2 subunit-containing GABARs, they are 2-20 M (Hanchar et al, 2005). This correlation between GABA affinity and benzodiazepine modulation is consistent with proposed mechanisms of benzodiazepine action in which the modulators increase the effective affinity of GABA (Perrais and Ropert, 1999). At higher steady-state GABA concentrations, benzodiazepines become less effective because the binding sites are fully occupied by GABA.…”

Presynaptically Silent GABA Synapses in Hippocampus

“…4C). However, the concentration of synaptically released GABA in the inhibitory cleft is thought to be only 300 -500 M (Jones and Westbrook, 1995;Perrais and Ropert, 1999), and the concentration that spills over to nearby glutamatergic synapses, and so is available for endocytosis, would be even lower (Rusakov et al, 1999).…”