The Journal of Neurophysiology Vol. 78 No. 4 October 1997, pp. 2176-2185
Copyright ©1997 The American Physiological Society

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

Greg C. Carlson, Melissa L. Slawecki, Eric Lancaster, and Asaf Keller

Department of Anatomy and Neurobiology and The Neuroscience Program, University of Maryland School of Medicine, Baltimore, Maryland 21201

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 -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.

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