The long term goals of this proposed research project are to understand the mechanisms of action on ventral horn spinal cord neurons of candidate excitatory amino acid neurotransmitters. The putative neurotransmitters will include the glutamate analogs quisqualic acid, kainic acid and n- methyl-d-aspartate. These studies encompass both an electrophysiological analysis of cell membrane currents recorded under voltage clamp conditions in the presence of specific agonists and antagonists as well as the sue of fluorescent probes to monitor receptor/channel distribution and changes in intracellular ion concentration. Agonist and antagonist application will employ a rapid superfusion technique in which the cell is repositioned following electrode insertion into a small chamber of approximate volume 100 mul. Solution changes occur within 20-30 msec thus achieving an effective concentration clamp of the cell environment. These studies will be done on cells acutely isolated from the ventral horns of 7 day old chick embryos using enzymatic dissociation methods. The cells will not be maintained in a tissue culture system to avoid the possibility that receptor alterations may occur with cell growth. Apart from identifying the complement of amino acid receptors on isolated neurons, and to positively identify the number of receptor types associated with different ion channels, we plan to examine the types of functional groups on the receptor/channel protein by using pH titration and protein modifying reagents. In addition, the possibility than any of the agonists can alter intracellular calcium either by calcium permeation through an agonist gated channel or by an agonist-receptor interaction that triggers a second messenger system will be investigated using fluorescent probes and immunohistochemistry. The focus will be one the inositol phospholipid and cyclic nucleotide pathways. Fluorescent probes will also be used to determine the distribution of receptor/channel proteins on the cell surface in particular the n-methyl-d-aspartate receptor. It is expected that this research will supply basic knowledge about the physiological importance and role that excitatory amino acid receptors play in the intact nervous system.
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