Distribution and activation of intracellular Ca2+ stores in cultured olfactory bulb neurons

The presence and distribution of intracellular Ca2+ release pathways in olfactory bulb neurons were studied in dissociated cell cultures. Histochemical techniques and imaging of Ca2+ fluxes were used to identify two major intracellular Ca2+ release mechanisms: inositol 1, 4,5-triphosphate receptor (IP3R)-mediated release, and ryanodine receptor-mediated release. Cultured neurons were identified by immunocytochemistry for the neuron-specificmarker beta-tubulin III. Morphometric analyses and immunocytochemistry for glutamic acid-decarboxylase revealed a heterogeneous population of cultured neurons with phenotypes corresponding to both projection (mitral/tufted) and intrinsic (periglomerular/granule) neurons of the in vivo olfactory bulb. Immunocytochemistry for the IP3R, and labeling with fluorescent-tagged ryanodine, revealed that, irrespective of cell type, almost all cultured neurons express IP3R and ryanodine binding sites in both somata and dendrites. Functional imaging revealed that intracellular Ca2+ fluxes can be generated in the absence of external Ca2+, using agonists specific to each of the intracellular release pathways. Local pressure application of glutamate or quisqualate evoked Ca2+ fluxes in both somata and dendrites in nominally Ca2+ free extracellular solutions, suggesting the presence of IP3-dependent Ca2+ release. These fluxes were blocked by preincubation with thapsigargin and persisted in the presence of the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. Local application of caffeine, a ryanodine receptor agonist, also evoked intracellular Ca2+ fluxes in the absence of extracellular Ca2+. These Ca2+ fluxes were suppressed by preincubation with ryanodine. In all neurons, both IP3- and ryanodine-dependent release pathways coexisted, suggesting that they interact to modulate intracellular Ca2+ concentrations.