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

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Abstract
The regulation of the cytosolic free Ca2+ concentration ([Ca2+]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 [Ca2+]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 Ca2+ channels) were dissipated by washing with fresh medium or, more rapidly, by addition of excess EGTA (t1/2= 11 ± 2 and 3 ± 0.6 s, respectively). Compared to those induced by high K+, the [Ca2+]i] transients induced by glutamate administered in Mg2+-free medium were much more variable. An initial peak, sustained by voltage-gated Ca2+ 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 Ca2+] channels. These plateaus were either stable or (in 10% of the cells) slowly growing to high [Ca2+]i] values. Addition of excess EGTA caused (Ca2+]i] to recover almost as fast as in the cells depolarized with K+ (t1/2 = 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, Ca2+ extrusion is affected only slightly, whereas Ca2+ 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 Ca2+]i] spike (resembling spontaneous fluctuations observed in a few cells only), was elicited by acetylcholine administered in Mg2+-free medium. Because this response was prevented by the blockade of not only muscarinic, but also NMDA receptors, as well as Na+ and Ca2+ 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 Ca2+ channels.