Abstracts

It has been proposed that the development of benzodiazepine (BDZ)-pharmacoresistance during status epilepticus (SE) is the result of a rapid alteration of the functional properties of postsynaptic GABA[sub]A[/sub] receptors (GABARs). However the acute effects of SE on the properties of synaptic GABARs have not been investigated in detail. In this study, the effects of SE on GABAergic synaptic transmission were studied by characterizing the effect of the BDZ diazepam (DZP) on miniature inhibitory postsynaptic currents (mIPSCs) recorded from control neurons and SE-treated neurons. Whole-cell patch clamp recordings of GABAR currents from cultured hippocampal neurons, 14-18 days in culture, and from dentate granule cells (DGCs) in hippocampal slices were performed at room temperature. The hippocampal neurons were cultured per the methods of Goslin and Banker. The removal of MgCl[sub]2 [/sub]from the extracellular media of these cultures results in sustained epileptiform bursting ([italic]in vitro[/italic] SE). Hippocampal slices were obtained from 5 naive rats and from 4 rats in which SE was induced by continuous hippocampal stimulation ([italic]in vivo[/italic] SE). [italic][underline]In vitro[/underline][/italic][underline] SE[/underline]: mIPSCs were recorded from 20 control cultured neurons and 6 cultured neurons exposed to a magnesium-free extracellular environment for 3 hours (SE-treated neurons). For control neurons, bath application of DZP 100 nM resulted in a prolongation of mIPSC decay (59.1 [plusmn] 3.0 vs. 73.2 [plusmn] 4.0 msec, p[lt]0.001) and a small increase in mIPSC amplitude (57.6 [plusmn] 2.7 vs. 62.1 [plusmn] 3.7 pA, p[lt]0.05). In contrast, the bath application of DZP had no effect on the decay (80.0 [plusmn] 12.5 vs. 68.17 [plusmn] 7.6 msec, p[gt]0.05) or amplitude (35.5 [plusmn] 4.0 vs. 37.8 [plusmn] 1.4 pA, p[gt]0.05) of mIPSCs recorded from SE-treated neurons. [underline][italic]In vivo[/italic] SE:[/underline] mIPSCs were recorded from 13 control DGCs in hippocampal slices obtained from naive rats and from 11 DGCs in hippocampal slices acutely obtained from rats in self-sustaining SE of 60 minutes in duration (SE-treated neurons). For naive DGCs, bath application of DZP 30 nM resulted in a prolongation of mIPSC decay (fast time constant: 8.1 [plusmn] 0.2 vs. 9.8 [plusmn] 0.2 msec, p[lt]0.01; slow time constant: 90.4 [plusmn] 8.2 vs. 133.5 [plusmn] 12.0 msec, p[lt]0.01) but no change in mIPSC amplitude (43.1 [plusmn] 4.6 vs. 46.2 [plusmn] 3.5 pA, p[gt]0.05). For SE-treated DGCs, following bath application of DZP 30 nM, there was a slight decrease in the fast time constant (14.6 [plusmn] 1.2 vs. 11.6 [plusmn] 0.7 msec, p[lt]0.05), no change in the slow time constant (108.3 [plusmn] 18.9 vs. 87.7 [plusmn] 12.4 msec, p[gt]0.05), and no change in mIPSC amplitude (30.7 [plusmn] 2.1 vs. 29.5 [plusmn] 2.0 pA, p[gt]0.05). These studies demonstrate the loss of benzodiazepine sensitivity of synaptic GABARs during SE. The development of pharmacoresistance was observed in both an [italic]in vitro[/italic] and an [italic]in vivo[/italic] model of SE. Additional studies will be required to determine the cellular mechanisms underlying this change in GABAergic synaptic transmission. (Supported by NS-040337, NS-044370, NS-048413, HD-001421.)