The proposed research has two main objectives. The first is to attempt to establish the identity of amino acid neurotransmitters in the rat spinal cord. The second is to study the mechanisms involved in the release of amino acids from the spinal cord in vivo. Previous studies from this laboratory have demonstrated the presence of group-specific transport systems which mediate the efflux of amino acids from pre-loaded brain tissue slices. Electrical stimulation of slices evoked release of amino acids which have known excitatory and inhibitory properties, but failed to release other amino acids. Amino acid efflux could be accelerated by exchange diffusion, or by exposure to medium containing ouabain or elevated potassium. When spontaneous efflux was maximally accelerated, then electrical stimulation evoked no further release, suggesting that membrane transport might be rate limiting in stimulus-coupled release. A method for superfusion of the rat spinal subarachnoid space in vivo has been developed. The volume characteristics of the spinal compartments have been studied, and amino acid transport out of the spinal fluid has been quantitated. In addition, application of a radioactive dansylation method for amino acid analysis has been successful for the quantitation of amino acids in 0.1 ul of spinal fluid. Through the use of the spinal perfusion model and the sensitive assay procedure, a systematic study of amino acid release into spinal fluid will be carried out. The specificity of amino acid release under various conditions (e.g. gycine release with activation of inhibitory cells) will be studied. The influence of electrical stimulation of dorsal and ventral roots and of various agents added to the spinal fluid will be investigated. Such agents will include strychnine, bicuculline, pentobarbital, acetylcholine, as well as ionic modifications. The release of endogenous, exogenous, and newly-synthesized amino acids will be studied. Attempts will be made to define the morphological boundaries of the transmitter pool by subcellular fractionation of spinal cord.