Abstracts

Rationale: Zinc is abundant within the glutamatergic mossy fibers that project from the dentate gyrus to the CA3 region of the hippocampus. Zinc is a negative allosteric modulator of GABAA receptors. Extrasynaptic d-containing GABAA receptors are more sensitive to Zn2+ inhibitory blockade than g-containing receptors. Release of Zn2+ into the extrasynaptic space presents interesting considerations for the GABAergic channel tone of d-containing receptors, which are responsible for modulating hippocampal excitability. Neurosteroids are endogenous regulators of neuronal excitability and seizure susceptibility. Allopregnanolone (AP) exhibits powerful anticonvulsant activity in a variety of seizure types. Neurosteroids are allosteric modulators of both synaptic (aβg2-containing) and extrasynaptic (aβd-containing) GABAA receptors, but they display greater sensitivity towards d-subunit receptors in dentate gyrus granule cells (DGGCs). Here we report a novel role of zinc in selective inhibition of neurosteroid responses at extrasynaptic d-containing GABAA receptors in the dentate gyrus.Methods: Adult, male wildtype and d-subunit knockout mice were used in the study to investigate the mechanisms of tonic current and drug sensitivity in brain slice recording and in vivo seizure susceptibility. Tonic and phasic currents were recorded from brain slices using whole-cell patch clamp electrophysiology. Zn2+-induced, negative modulation of AP-sensitive, GABAergic currents were analyzed. Antiseizure effect of AP and its modulation by zinc was evaluated in fully-kindled mice expressing stage 5 seizures. Zinc was infused by intrahippocampal route 30-min prior to AP treatment. AP was administered 15-min prior to kindling stimulation.Results: Patch-clamp studies confirmed enhanced sensitivity to AP potentiation of GABA-gated currents in DGGCs. Zn2+ perfusion displayed a concentration-dependent block of tonic current activity due to d-containing, extrasynaptic GABAA receptors, and at 100 μM it completely blocked tonic currents, similar to the competitive antagonist gabazine. Phasic currents remained intact in Zn2+ concentrations below saturating conditions (10-50 μM). Moreover, Zn2+ sensitivity on tonic current was not evident in d-subunit knockout mice. In seizure studies, AP (5 mg/kg, sc) completely protected mice against kindling seizures. Pretreatment with Zn2+, however, significantly blocked the anticonvulsant activity of AP in fully-kindled mice. This response was reversible as animals showed stage 5 seizures 24 h post-zinc perfusion.Conclusions: These results indicate that zinc inhibits neurosteroid allosteric effects at extrasynaptic d-containing GABAA receptors in the DGGCs. This is also evident in vivo in blocking antiseizure effect of AP. Zinc inhibition of anticonvulsant activity of neurosteroids has clinical implications because of likely interactions between zinc and endogenous neurosteroids in the brain. ** Supported by NIH grant NS051398 **