Work over the last twenty years has identified a remarkable number of proteins that form ion channels in the mammalian brain. In many cases, we have detailed information about the molecular characteristics of the channels and on how they can by modulated by G proteins and second messengers. However, we understand much less about how the many voltage-dependent channels expressed in a single central neuron work together to produce the firing patterns characteristic of that particular neuron. The goal of the proposed research is to understand how the firing properties of cerebellar Purkinje neurons are produced by particular combinations of ion channels. The work will combine current clamp recordings of action potential firing with a voltage-clamp analysis of the voltage-dependent sodium, potassium, and calcium channels underlying the action potentials. Studies on Purkinje neurons in cerebellar slices will examine ion channels and intrinsic membrane properties of dendrites and soma and will investigate how these interact under physiological conditions. The electrophysiological characterization will be complemented by immunocytochemical localization of particular sodium, calcium, and potassium channels. A preparation of dissociated cell bodies will allow a high-quality voltage-clamp of voltage-activated currents. Ionic substitution and specific channel blockers, especially peptide toxins, will be used to distinguish the contributions of particular channels. Action potential waveforms will be used as command voltages to determine the contribution of particular ion channels to particular firing patterns, with a special focus on understanding pacemaking activity and complex spikes. Understanding the mechanisms involved in regulating the electrical excitability of central neurons will help in understanding the normal function of the nervous sytem as well as pathophysiological states resulting from stroke, intoxication, and epilepsy.
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