Fura-2 measurements of cultured rat Purkinje neurons show dendritic localization of Ca2+ influx.
The specific objectives of this study were the following: (1) to characterize the types of calcium currents in cultured PCs using whole-cell voltage-clamp techniques; (2) using fura-2 imaging techniques, to monitor intracellular Ca2+ levels during application of high potassium, glutamate, or glutamate analogs; and (3) to evaluate the types of calcium channels contributing to the calcium fluxes using pharmacological blocking agents. Voltage-clamp analysis of calcium currents proved to be difficult due to space-clamping problems. The latter was presumably due to the unfavorable geometry of cultured PCs. Nevertheless, we found no evidence for inward currents in cells bathed in TTX-TEA-BaCl2 saline. On the other hand, fura-2 measurements demonstrated that free Ca2+ levels were elevated in PCs following local application of either high-potassium saline or glutamate. When individual cells were injected with fura-2 and analyzed in TTX-containing saline, the Ca2+ elevation was usually greater in the dendrites. Since Ca2+ levels were not elevated in all dendrites of the same cell, the smaller responses in the soma wre not simply due to volumetric differences. Together with the voltage-clamp results, the fura-2 data indicate that calcium channels were localized to certain dendrites. Using selective calcium channel blockers, we found evidence for 2 types of calcium conductances in the dendrites of cultured PCs. The Ca conductance induced by high potassium was reduced in a dose-dependent manner by nifedipine (ED50 = 5 X 10(-7) M), indicating that a high-threshold voltage-dependent calcium channel was present. The Ca response to glutamate (or NMDA) was reduced by 2-amino-5-phosphonovaleric acid (ED50 = 10(-4) M), as well as by nifedipine or 10(-4) M LaCl3, indicating that both voltage-dependent and glutamate-coupled channels were opened by glutamate application.
Hockberger
P E
PE
Department of Molecular Biophysics, AT&T Bell Laboratories, Murray Hill, New Jersey 07974.
Tseng
H Y
HY
Connor
J A
JA
D000818:Animals; D001572:Benzofurans; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology; D002478:Cells, Cultured; D003712:Dendrites / Q000378:metabolism* / Q000648:ultrastructure; D004594:Electrophysiology; D005456:Fluorescent Dyes; D016257:Fura-2; D005971:Glutamates / Q000494:pharmacology; D011689:Purkinje Cells / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D051381:Rats
D001572:Benzofurans; D015220:Calcium Channels; D005456:Fluorescent Dyes; D005971:Glutamates; D002118:Calcium; D016257:Fura-2