Development of rat cerebellar Purkinje cells: electrophysiological properties following acute isolation and in long-term culture.

The objectives of this study were 2-fold: (1) to characterize the electrical properties of Purkinje cells (PCs) acutely isolated from rat cerebella at different stages of development, and (2) to compare these properties with those recorded from PCs grown in long-term culture. PCs under both conditions were identified with the aid of cell-specific immunocytochemical staining, and the electrical properties were analyzed using whole-cell-recording techniques. PCs acutely isolated during late embryonic and early postnatal periods displayed a progressive change in electrical properties. Between embryonic days 20 and 22 (stage 1), PCs were inexcitable, did not respond to glutamate, and displayed only small outward currents under voltage clamp. During postnatal days 1-4 (stage 2), current stimulation elicited nonovershooting action potentials, and small inward and outward currents were evoked under voltage clamp. Glutamate application depolarized the cells 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 showed progressively larger voltage-dependent conductances and greater sensitivity to glutamate. We found no evidence for either spontaneous or complex spikes in PCs isolated at any of these stages. In agreement with previous studies, we found that PCs dissociated from postnatal rats did not survive well in culture. On the other hand, PCs from embryonic rats cultured for 2-3 weeks in high-potassium, serum-supplemented medium developed extensive dendritic processes and excitability. Current stimulation or glutamate application elicited depolarizing waveforms reminiscent of climbing fiber-evoked responses in vivo. The results suggest that dendritic processes are important in the generation of complex spikes and that PC excitability can develop in the absence of the highly structured architecture of the intact cerebellum.