Development of Rat Cerebellar Purkinje Cells: Electrophysiological Properties Following Acute Isolation and in Long-Term Culture.

The objectives of this study were twofold: (1) to characterize the electrical properties of Purkinje cells (PCs) acutely isolated from rat cerebella at different stages of development, and (2) to determine whether PCs grown in long-term culture develop electrophysiological properties similar to the isolated cells. PCs were identified with the aid of immunocytochemical staining techniques, and the electrical properties were analyzed using whole-cell patch recording. Acutely isolated cells displayed a progressive differentiation between late embryonic and early postnatal periods. We were able to identify four stages of differentiation. Stage 1 cells (embryonic days 20-22) were electrically inexcitable, did not respond to glutamate, and displayed only small outward currents using voltage clamp protocols. Stage 2 cells (postnatal days 1-4) generated non-overshooting action potentials with current stimulation, and displayed small inward and outward currents under voltage clamp. The membrane potential of these cells were depolarized by glutamate resulting in an increase in intracellular free calcium (measured with fura-2). Stage 3 and 4 cells spanned postnatal days 5-9 and 10-14, respectively, and the cells displayed progressively larger voltage-dependent conductances and greater sensitivity to glutamate. We found no evidence for spontaneous excitability in PCs isolated at any of these stages. Two conditions were found to be critical for getting dissociated PCs to survive and differentiate in long-term culture: the cells needed to be plated no later than P3, and the media required 25mM potassium chloride. Under these conditions we found that late embryonic PCs cultured for 3-4 weeks developed electrophysiological properties similar to the relatively mature PCs isolated from two week- old animals. Our results demonstrate that PCs can develop normal electrophysiological properties in the absence of the highly structured architecture of the intact cerebellum.