Distribution and Activation of Intracellular Ca2+Stores in Cultured Olfactory Bulb Neurons

Carlson, Greg C., Melissa L. Slawecki, Eric Lancaster, and Asaf Keller. Distribution and activation of intracellular Ca2+stores in cultured olfactory bulb neurons. J. Neurophysiol. 78: 2176–2185, 1997. 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 β-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.