Intracellular blockade of GABA<inf>A</inf> receptors in the rat hippocampal neurons

Abstract

The intracellular blockade of GABA<inf>A</inf>-receptor-mediated currents is a useful approach to suppress the GABAergic conductance in a single cell and to isolate the glutamatergic component of network-driven activities. Previously an approach has been described allowing intracellular blockade of GABA<inf>A</inf> receptors by means of intracellular dialysis of a neuron with the pipette-filling solution, in which fluoride ions that hardly pass through the GABA<inf>A</inf> receptor channels substitute for Cl^- and in which Mg²⁺ and ATP are omitted to induce rundown of the GABA<inf>A</inf> receptors during whole-cell patch-clamp recordings. However, the kinetics of suppression of GABAergic conductance and the effect on the currents mediated by glutamate receptors remain unknown. Here, using whole-cell recordings with fluoride-based, Mg²⁺- and ATP-free solution on CA3 hippocampal neurons of neonatal rats, we show that after 1 h of such dialysis, both spontaneous and evoked GABA<inf>A</inf>-receptor-mediated synaptic currents and responses induced by the GABA<inf>A</inf> receptor agonist isoguvacine were completely suppressed. Inward GABAergic postsynaptic currents were suppressed prior to outward currents. Synaptic responses mediated by AM PA receptors were not affected by the dialysis, whereas the NM DA-receptor-mediated postsynaptic currents were reduced by approximately 20%. Dialysis with fluoride-based Mg²⁺, ATP-free solution either fully blocked giant depolarizing potentials (G DPs) in CA3 pyramidal cells (n = 2) or reduced the charge crossing the membrane during G DPs and shifted the G DP reversal potential to more positive values (n = 5). The dialysis-resistant component of G DPs was mediated by glutamate receptors, since: (i) it reversed around 0 mV; (ii) it demonstrated a negative slope conductance at negative membrane voltages, which is characteristic of NM DA receptor-mediated responses; (iii) kinetics of the individual events composing the dialysis-resistant component of G DPs at negative voltages were very similar to those of AM PA receptor-mediated synaptic currents. Thus, this procedure can be useful to isolate the glutamate receptor-mediated component of neuronal network-driven activities.