Abstracts

The tonic GABA current is a GABA receptor-mediated current that is persistently active. This is thought to be distinct from the [ldquo]phasic[rdquo] synaptic GABA current and mediated by extrasynaptic receptors. The tonic GABA current is regarded as an important contributor to the baseline level of neuronal excitability and a potential target for pharmacological therapy. The purpose of this study was to explore tonic GABA current in a simplified experimental model to better understand the contribution of the tonic current to normal neuronal excitability and the role of the tonic GABA current in epilepsy. Primary hippocampal cell microcultures (100-200 [mu]M diameter) were prepared from 1-to-3-day postnatal Sprague-Dawley rats as previously described (Mennerick [italic]et al. [/italic]1995). Cells were plated onto 35 mm plastic culture dishes pre-coated with collagen microdroplets sprayed onto a layer of agarose. Whole-cell patch-clamp recordings were performed on solitary neurons with only autaptic (recurrent) synaptic innervation 10-22 days after plating. Exogenous drugs were applied with a multi-barrel pipette coupled to miniature solenoid valves for rapid switching between solutions. Solitary excitatory neurons were identified by autaptic response polarity, kinetics and pharmacology. The tonic current was measured as the change in the holding current in the presence of 10 [mu]M bicuculline. In an initial characterization of the tonic current, we found no relationship between time in culture and tonic current density. We then explored the source of the tonic current in this system where receptor activation by GABA from synaptic spillover is not possible as no inhibitory synapses are nearby. We tested the hypothesis that the current is mediated by non-synaptic GABA or another agonist released by cells on the microculture. First we used stop flow and rapid flow protocols to manipulate accumulation of the putative agonist and were unable to change the amplitude of the current. We then hypothesized that unliganded spontaneous openings of GABA receptor channels may underlie the tonic current. To test this idea, we used gabazine which is an ineffective antagonist of unliganded, spontaneous openings but an effective blocker of GABA-gated responses. We found that saturating gabazine concentrations had no effect on the tonic current in excitatory neurons. In contrast, in solitary inhibitory (GABAergic) neuron microcultures where synaptic overspill could possibly contribute to the tonic current, gabazine partially blocked the tonic current and completely blocked inhibitory postsynaptic currents. These results suggest that the tonic GABA current in excitatory single neuron hippocampal microisland cultures is mediated by spontaneous, unliganded activation of GABA channels. This implies that spontaneous activity may contribute to the tonic GABA current [italic]in vivo[/italic], particularly in regions with low ambient GABA concentrations. (Supported by NIH)