The overall objective of this study is to increase understanding of synaptic transmission in the peripheral nervous system by studying the ability of several neuropeptides to modulate cholinergic transmission between nerve cells and by determining the mechanisms by which cholinergic neurotransmission can be modulated.
The specific aims for the coming year are: 1) to test the hypothesis that Substance P, enkephalin and VIP modulate cholinergic transmission by altering calcium currents, either directly or indirectly, in presynaptic neurons; 2) to test the hypothesis that subsets of cholinergic enteric neurons contain additional neuroactive molecules; and 3) to develop procedures that permit active terminals of living neurons to be visualized without being damaged. The experiments designed to achieve these aims will be conducted on neurons that are grown in cultures after having been dissociated from the myenteric plexus of the small intestines of newborn rats.
Aim 1 will involve using the whole cell patch clamp method to measure neuronal currents before, during and after exposure to the test agents.
Aim 2 will involve: a) immunohistochemical examination of neurons that have been demonstrated to be cholinergic by their ability to cause cholinergic synaptic potentials in other neurons and b) double immunohistochemical staining for choline acetyltransferase and several neuropeptides that have been reported to be in enteric neurons.
Aim 3 will involve using a SIT camera to view, with low light levels, terminals of neurons that: 1) have been stimulated in the presence of small fluorescent molecules or 2) have been injected with fluorescent dyes.
