Release of endogenous glutamate and aspartate from the frog spinal cord in vitro

Abstract

The release of the endogenous excitatory amino acids aspartate and glutamate from an in vitro slice preparation of the frog spinal cord was investigated by using high performance liquid chromatography (HPLC). Spinal slices were incubated at 4°C to minimize amino acid uptake; samples of the bathing solution were collected at 10 min intervals for amino acid assay. In each experiment electrophysiological responses were recorded continuously from ventral roots after stimulation of an adjacent dorsal root, so that changes in the profile of amino acid release could be correlated with electrophysiological responses. At rest the release of glutamate and aspartate was 52.9 ± 5.8 and 66.9 ± 5.6 pmol/10 min, respectively, and was unaffected by low Ca 2+ media or tetrodotoxin. After trains of high frequency stimulation applied to a dorsal root the release of glutamate and aspartate was significantly and reproducibly enhanced by 33 ± 13 and 49 ± 18%, respectively. The stimulus-dependent release was blocked by low Ca 2+ media, tetrodotoxin or topical application of the neuropeptide thyrotropin releasing hormone. The present study provides direct evidence to support the role of glutamate and aspartate as neurotransmitters in the spinal cord.