Characterization of electrically evoked [3H]-D-aspartate release from hippocampal slices.
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abstract
Electrical stimulation has certain advantages over chemical stimulation methods for the study of neurotransmitter release in brain slices. However, measuring detectable quantities of electrically evoked release of endogenous or radiolabeled markers of excitatory amino acid neurotransmitters has required current intensities or frequencies much higher than those usually required to study other transmitter systems. We demonstrate here that [3H]-D-aspartate (D-ASP) release can be detected from hippocampal slices at lower stimulation intensities in the presence of a glutamate reuptake inhibitor. Subsequently, we optimized the electrical stimulus parameters for characterizing electrically evoked D-ASP release. Under the experimental conditions described, greater than 90% of electrically evoked D-ASP release is calcium-dependent. Evoked D-ASP release is markedly reduced by pre-treating slices with the synaptic vesicle toxin bafilomycin A1 (BAF A1) or in the presence of 10-mM magnesium. Evoked D-ASP release is also reduced to variable degrees by N- and P/Q type voltage-sensitive calcium channel antagonists. Neither spontaneous efflux nor evoked D-ASP release were affected by NMDA, AMPA or group I metabotropic glutamate receptor (mGluR) antagonists. Evoked D-ASP release was reduced in the presence of an adenosine A1 receptor agonist and potentiated by treatment with a group I mGluR5 agonist. Evoked [3H]-D-ASP release was similar in magnitude to evoked [3H]-L-glutamate (L-GLU) release. Finally, in separate experiments using the same electrical stimulus parameters, more than 90% of electrically evoked endogenous L-GLU release was calcium dependent, a pattern similar to that observed for evoked [3H]-D-ASP release. Taken together, these results indicate that electrically evoked [3H]-D-ASP release mimics evoked glutamate release in brain slices under the experimental conditions employed in these studies.
