The proposed research will use quantitative electron microscope (EM) autoradiography in conjunction with electrophysiological and biochemical procedures to study acetylcholine receptors (AChR) and esterases (AChE) at the vertebrate neuromuscular junction (nmj). The control of turnover rates and steady state site densities of these molecules (especially of AChR) will be investigated in normal, denervated and reinnervated junctions, and in junctions manipulated experimentally. Experimental manipulations will include the use of metabolic inhibitors and specific antibodies. Attempts will be made to improve the sensitivity of (DNA/RNA) in situ hybridization procedures, in order to study the control of AChR synthesis. Physiological studies will utilize voltage clamp techniques to correlate miniature endplate currents (MEPCs) with AChR and AChE site densities. Specific inhibitors will be used to change the AChR and AChE site densities. Bath-applied ACh, at different concentrations, will be used to partially ligate the AChR-channel complex (causing some desensitization). The resultant changes in the shape and amplitude of the MEPCs will allow us to derive various kinetic parameters for the interaction between ACh and receptor in the intact nmj.
