The overall goal of the proposed experiments is to learn more about the dynamic contribution of the internode to the electrical behavior of myelinated axons, and about mechanisms modulating transmitter release from motor nerve terminals. Electrophysiological and imaging techniques will be applied to lizard and rat neuromuscular preparations. Changes in [Ca2+] and [Na+] within axons and terminals will be measured by injecting into the axon the Ca-sensitive dye fura- 2 or the Na-sensitive dye SBFI, and then ratio-imaging fluorescence emissions using an ultra-sensitive charge-coupled device (CCD) camera. One series of experiments will study activation of delayed (K) and inward rectifier (Na+K) ion channels in the internodal (submyelin) axolemma of intact motor axons. We have demonstrated that internodal delayed rectifier channels are activated during axonal activity, and will use electrophysiological and imaging techniques to determine whether the resulting accumulation of K outside the internodal axolemma increases the likelihood of ectopic discharge, a mechanism proposed for post-ischemic discharge of human axons in vivo. Current through inward rectifier channels and electrogenic pump activity will be detected and localized by measuring post-stimulation changes in intra-axonal [Na+]. A second series of experiments will study movement of fluorescent dyes injected into regions of compact myelin. We will use ion- sensitive dyes to study the cation composition of the dye-filled compartments, both at rest and following axonal stimulation. We will determine how dye distribution varies with the charge and size of the injected dye, and in response to applied currents. These experiments will give new information concerning myelin sheath permeability. A final series of experiments will measure stimulation-induced changes in [Ca2+] and [Na+] within motor nerve terminals, along with the correlated end-plate potentials to measure quantal transmitter release. These experiments, which include intra-axonal injection of GDP and GTP analogs, will elucidate mechanisms of hormonal modulation of transmitter release, and test hypotheses concerning relationships between intracellular cations and transmitter release. Information gained from these studies may contribute to understanding the etiology of diseases characterized by dysfunction of myelin (multiple sclerosis, Guillian-Barre syndrome) or motor nerve terminals.