Regulation of Intracellular Calcium in Cerebellar Granule Neurons: Effects of Depolarization and of Glutamatergic and Cholinergic Stimulation

The regulation of the cytosolic free Ca 2+ concentration ([Ca 2+ ] i ]) was investigated by microfluorimetry in single cerebellar granule neurons exposed to various treatments (high K + , glutamate, or acetylcholine) and drugs. The responses to the treatments developed asynchronously during cell culture, with high K + and glutamate reaching their maxima at 6 and 7 days in vitro and acetylcholine at 9 days in vitro. The biphasic [Ca 2+ ] i ] transients induced by high K + (an initial peak, followed by a plateau 30–40% of the peak, both sustained by dihydropyridine‐sensitive voltage‐gated Ca 2+ channels) were dissipated by washing with fresh medium or, more rapidly, by addition of excess EGTA ( t 1/2 = 11 ± 2 and 3 ± 0.6 s, respectively). Compared to those induced by high K + , the [Ca 2+ ] i ] transients induced by glutamate administered in Mg 2+ ‐free medium were much more variable. An initial peak, sustained by voltage‐gated Ca 2+ channels, was visible in only ∼50% of the cells and disappeared when multiple glutamate pulses were administered. In the rest of the population, the transients were monophasic, with persistent plateaus sustained only in part (30–40%) by voltage‐gated Ca 2+ ] channels. These plateaus were either stable or (in 10% of the cells) slowly growing to high [Ca 2+ ] i ] values. Addition of excess EGTA caused (Ca 2+ ] i ] to recover almost as fast as in the cells depolarized with K + ( t 1/2 = 5 ± 1.8s), whereas after washing or N ‐methyl‐D‐aspartate (NMDA) receptor blockade the recovery occurred, but was slow. These results indicate that, in glutamate‐stimulated cells, Ca 2+ extrusion is affected only slightly, whereas Ca 2+ influx is stimulated persistently via multiple pathways [voltage‐gated channels, NMDA receptors, and possibly other(s) to be characterized]. A third type of stimulatory response, a rapid Ca 2+ ] i ] spike (resembling spontaneous fluctuations observed in a few cells only), was elicited by acetylcholine administered in Mg 2+ ‐free medium. Because this response was prevented by the blockade of not only muscarinic, but also NMDA receptors, as well as Na + and Ca 2+ channels, it appears to require the activation of action potentials and synaptic connections between the cells of the culture. An additional effect of acetylcholine was inhibitory and consisted in a depression of the glutamate‐ and K + ‐induced plateaus, possibly occurring via a negative modulation of voltage‐gated Ca 2+ channels.