Neurons in the mammalian brain possess a rich variety of voltage-dependent ion channels, but there has been little detailed analysis of how particular ion channels work together to regulate the firing patterns of mammalian central neurons. In part, this has been due to limitations in voltage-clamping central neurons, especially for studying the large voltage-activated currents that flow during the action potential. The goal of the proposed research is to understand how the distinctive firing properties of cerebellar Purkinge neurons are produced by particular ion channels. The work is based on using a preparation of dissociated Purkinje neurons that allows a high-quality voltage-clamp of voltage- activated currents. Preliminary data show that the dissociated cells retain two of the distinctive firing properties of Purkinje cells in vivo, spontaneous firing and formation of complex action potentials. The experimental design will combine current clamp recordings of action potential firing with a voltage-clamp analysis of the voltage-dependent sodium, potassium, and calcium channels that underlie the action potentials. Voltage clamp experiments will use ionic substitution and specific channel blockers, especially peptide toxins, to distinguish the contributions of particular channel types to the overall sodium, calcium, and potassium currents. Action potential waveforms will be used as command voltages to determine the contribution of particular ion channels to firing patterns. A particular focus will be to characterize a novel repolarization-gated sodium current using single channel and whole-cell recordings, and to understand the role of the current in spontaneous firing and in the formation of multi-spike action potentials. Understanding the mechanisms involved in regulating the electrical excitability of central neurons will help in understanding the normal function of the nervous system as well as pathophysiological states resulting form stroke, intoxication, and epilepsy.
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