Substance P, an undecapeptide, has been identified by combined immunohistochemical and radioimmunological techniques to be present in nerve fibers and terminals in amphibian autonomic ganglia. The peptide has been shown to depolarize some sympathetic neurons of frog and guinea pig and to modulate the activation of the acetylcholine receptor (AChR) in chromaffin cells and in embryonic chick sympathetic neurons. The physiological role of the peptide in ganglionic transmission remains uncertain, however. The long-range objective of this work is to define the precise physiologic effects of substance P and other peptides in the modulation of cholinergic transmission in developing chick autonomic ganglia. The specific aims of this proposal focus on first, a determination of the effect of substance P on the activation of the cholinergic receptor. This includes a detailed electrophysiological characterization of substance P's inhibition of ACh-induced inward currents in embryonic sympathetic neurons in vitro. Second, the pharmacology of the effect will be studied to examine the action of substance P on the dose dependence of ACh's interaction with the AChR as well as to characterize the substance P receptor in detail. Third, studies will be initiated to examine the mechanism of substance P action at the cholinergic receptor using voltage clamp recording for fluctuation analysis of receptor kinetics as well as single channel (patch clamp) recording. A fourth aim of this work is to extend the observation of substance P effects on applied ACh to studies of the peptide at cholinergic synapses on the neurons. Preliminary studies indicate that sympathetic neurons can be innervated in vitro by dorsal spinal cord explants containing the preganglionic neurons. The studies that comprise this proposal constitute the first aspect of defining the role of substance P in the modulation of cholinergic synapses in autonomic ganglia in vivo. Since the peptide is distributed throughout the nervous system and yet its functional role is largely undefined, these studies will yield fundamental and important information about the physiological role and mechanism of action of substance P.