Voltage Clamp and Fura-2 Measurements of Cultured Rat Purkinje Neurons Show Dendritic Localization of Ca sup (2+) Influx.

We have used whole-cell patch recording and fura-2 imaging techniques to analyze calcium fluxes in cultured cerebellar Purkinje cells (PCs). The specific objectives of this study were the following: (1) to characterize the types of voltage- dependent and glutamate-activated conductances present in cultured PCs; (2) to monitor intracellular Ca sup (2+) levels in these cells during application of high potassium, glutamate, or glutamate analogs; and (3) to evaluate the types of calcium channels contributing to the calcium responses using pharmacological blocking agents. Voltage clamp experiments were performed on cell bodies and demonstrated that cultured PCs had several types of voltage-dependent and glutamate-activated conductances. The cells displayed TTX-sensitive inward and La- /Ba-sensitive outward currents, but we found no evidence for either high- or low-threshold calcium currents under conditions that should have maximized the signal, i.e., in TTX-TEA-Ba saline. Glutamate and its analogs, kainate, quisqualate, and N- methyl-D-aspartate (NMDA), each depolarized the membrane potential of PCs when applied to either the cell bodies or dendrites. The time course and voltage dependence of the underlying currents were unaffected by extracellular Mg sup (2+) Fura-2 imaging experiments demonstrated that free Ca sup (2+) levels in cell bodies and dendrites were elevated following local application of either high potassium saline or glutamate. When individuals cells were injected with fura-2 and analyzed in TTX-containing saline, the Ca sup (2+) elevation was primarily in the dendrites. The Ca response to high potassium was reduced in a dose-dependent manner by nifedipine (ED sub 50 = 5 X 10 sup -7 M ) . The Ca response to glutamate (or NMDA) was reduced by 2-amino-5- phosphonovaleric acid (APV). The response to glutamate was also reduced by nifedipine or LaCl sub 3 indicating that both voltage-dependent and glutamate-coupled Ca channels were activated by glutamate application. The voltage clamp and imaging results suggest that the Ca channels were located primarily in the dendrites, results which support and extend what has been demonstrated for PCs in cerebellar slices.