Functional Properties of Ryanodine Receptors in Hippocampal Neurons Change During Early Differentiation in Culture

Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892 Sukhareva, Manana, Susan V. Smith, Dragan Maric, and Jeffery L. Barker. Functional Properties of Ryanodine Receptors in Hippocampal Neurons Change During Early Differentiation in Culture. J. Neurophysiol. 88: 1077-1087, 2002. 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza- s -indacene-3-propionyl)amino)hexanoic acid ryanodine (BODIPY-ryanodine) binding and Ca 2+ imaging were used to study the properties of ryanodine receptors (RyRs) and cytoplasmic Ca 2+ (Ca ) changes in neurons cultured from the embryonic rat hippocampus during the earliest stages of differentiation. Baseline Ca levels declined from 164 ± 5 (SD) nM at early stages to 70 ± 4 nM in differentiated neurons. Fluorescent BODIPY-ryanodine binding signals identified activated RyRs in somata, which were eliminated by removal of external Ca 2+ or by blockage of Ca 2+ entry through L-type but not N-type Ca 2+ channels. The GABA synthesis inhibitor 3-mercaptopropionic acid completely abolished ryanodine binding. Caffeine or K + -depolarization inhibited the activity of RyRs at very early stages of differentiation but had stimulatory effects at later stages after a network of processes had formed. BayK-8644 stimulated RyRs throughout all regions of all differentiating cells. The results suggest that in differentiating embryonic hippocampal neurons the activity of RyRs is maintained via Ca 2+ entering through L-type Ca 2+ channels. The mode of activation of L-type voltage-gated Ca 2+ channels with either membrane depolarization or specific pharmacological agents affects the coupled activity of RyRs differently as neurons differentiate processes and networks.