Synergic effect of diazepam and muscimol via presynaptic GABA(A) receptors on glutamatergic evoked EPSCs

Abstract

We investigated the functional roles of diazepam (DZP) at presynaptic GABA(A) receptors on glutamatergic nerve terminals in contributing to glutamatergic transmission evoked by single and/or paired-pulse focal electrical stimulation. In mechanically dissociated rat hippocampal CA3 neurons with adherent glutamatergic nerve terminals (boutons), namely 'synaptic bouton' preparation, action potential-evoked excitatory postsynaptic currents (eEPSCs) were recorded using conventional whole-cell patch configuration under voltage-clamp condition. Selective activation of presynaptic GABA(A) receptors by muscimol (3-30μM) induced presynaptic inhibition: i.e. the decrease of amplitude and increase of failure rate (Rf) and paired-pulse ratio (PPR) of eEPSCs which are sensitive to bicuculline. DZP (10-100μM) also induced such presynaptic inhibition, but the bicuculline-insensitive effects were caused by inhibition of both voltage-dependent Na(+) and Ca(2+) channels. Muscimol (0.01-0.3μM) or DZP (0.1-3μM) itself did not induce any currents at the low concentration used. However, simultaneous application of muscimol and DZP at low concentrations induced a significant bicuculline-sensitive presynaptic inhibition. Marked desensitization of presynaptic inhibition was also caused by muscimol at higher concentrations than 10μM. The results suggest that in vivo conditions, activation of presynaptic GABA(A) receptors could be readily available with a tiny amount of DZP.