Digital imaging of the Ca indicator fura-2 has been used to study the responses of developing granule cells in culture to depolarization and transmitter action. Unstimulated cells bathed in Krebs saline exhibited cytoplasmic Ca ion concentrations, [Ca2+], that were generally in the 30-60 nM range. Exposure of cells to high-potassium (25 mM) saline depolarized the membrane potential and produced an immediate rise in [Ca2+] that recovered within 2-3 min in normal saline. The response grew progressively larger over the first 20 d in culture. Transient increases in [Ca2+] to levels greater than 1 microM were observed after 12-14 d in vitro, at which time the cells displayed intense electrical activity when exposed to high K. At this stage, the increases were attenuated by blocking action potential activity with TTX. In TTX-treated or immature cells, in which the transient phase of the Ca change was relatively small, a second exposure to high K typically produced a much larger Ca response that the initial exposure. The duration of this facilitation of the response persisted for periods longer than 5 min. Application of the neurotransmitter GABA induced a transient increase in membrane conductance, with a reversal potential near resting potential (approx. -60 mV), and caused an intracellular Ca2+ increase that outlasted the exposure to GABA by several minutes. Glutamate, or kainate, induced an increase in membrane conductance but with a reversal potential more positive than spike threshold. These agents also elevated intracellular Ca2+, but unlike the case with GABA, this Ca response reversed rapidly upon removal of the transmitter. The facilitatory effect of repeated exposures to high-K saline, as well as the persistent Ca elevation following a brief GABA application, suggests that granule cells possess the capability of displaying activity-dependent changes in Ca levels in culture.

We have used a combination of immunocytochemical and electrophysiological measurements to monitor the differentiation of cerebellar granule cells in vitro. We present immunocytochemical evidence showing that several characteristic features of developing rat cerebellar tissue were retained in postnatal explant cultures. Most notably the cultures expressed radiating GFAP-positive (Bergmann) glia processes, proliferating NSE-negative neuroblasts, and migrating NSE-positive granule cells. The latter were subdivided into 3 developmental stages--i.e., immature, intermediate, and mature granule cells, based upon cell differences in location from the explant, intensity of NSE staining, excitability, and the amplitude of voltage-dependent conductances. Immature cells were identifiable during the first week in culture and were located up to 140 micron from the explant. These cells stained lightly for NSE and displayed conductances of insufficient magnitude to generate action potentials. Intermediate cells were present after 1-2 weeks in culture and were located up to 500 micron from the explant. These cells were also NSE positive and were characterized by the presence of soma action potentials. Intermediate cells displayed 3 large voltage-dependent conductances: a transient, TTX-sensitive inward current; a delayed, TEA-sensitive outward current; and a transient, 4AP-sensitive outward current. Mature cells were present after 1 month in culture and, like intermediate cells, were no more than 500 micron from the explant. However, mature cells stained more intensely for NSE, and the somata of these cells were devoid of voltage-dependent conductances (although axonal currents were usually present). These results indicate that granule cells undergo a stereotypic sequence of differentiation in postnatal explant cultures. These stages may correspond to developmental changes in granule cells during migration into the (internal) granular cell layer in vivo.

No abstract