Intracellular calcium stores in chick cerebellum Purkinje neurons: ontogenetic and functional studies

The molecular composition of intracellular Ca 2+ stores in developing chicken cerebellum Purkinje neurons from embryonic day 11 (El1 ) to posthatching day 2 (p2) was studied by immunocytochemistry using specific antibodies for three molecular constituents, the receptor (R) and/or channel sensitive to inositol 1,4,5- trisphosphate (IP,), Ca 2+-adenosinetriphosphatase (ATPase), and calsequestrin (CS). CS, IP3R, and Ca2+-ATPase were first detected by light-microscopic immunofluorescence in migrating Purkinje cells at El l-El2 and throughout late phases of embryonic development. Ontogenesis of CS, IPSR, and Ca2+- ATPase accompanied well-defined stages of cerebellum histogenesis and cytogenesis and was accomplished before hatching. High-resolution immunogold electronmicroscopy revealed that, at ElB-Pl, CS was still largely distributed to the endoplasmic reticulum (ER) lumen and began to be segregated to ER subcompartments (calciosomes) only by p2, whereas the IP3R was concentrated into ER cisternal stacks as early as El8. Both ionotropic and metabotropic plasma membrane receptors were present in dissociated single chicken Purkinje cells from El6 onward, as indicated by measurements of membrane currents (whole cell recording mode) and of cytoplasmic Ca2+ transients monitored with the cell-trappable fluorescent indicator fura 2-acetoxymethyl ester, respectively. Cytoplasmic Ca 2+ transients were detected after either activation of glutamate metabotropic receptors, i.e., evidence of IP3-sensitive Ca2+ channels, or application of caffeine, i.e., evidence of ryanodine-sensitive Ca2+ channels. Intracellular Ca2+ stores appear to be functional during embryonic development.