Imaging of caffeine-inducible release of intracellular calcium in cultured embryonic mouse telencephalic neurons.
To gain a better understanding of Ca(2+)-induced Ca2+ release in central neurons, we have studied the increase in intracellular Ca2+ concentration ([Ca2+]i) induced by application of caffeine to cells cultured from embryonic mouse telencephalon (hippocampus or cortex). The magnitudes and distributions of changes in [Ca2+]i in neuron somata were measured by quantitative video microscopy. We observed that application of caffeine to pyramidally shaped neurons typically initiated an increase in [Ca2+]i in the cytoplasmic region between the nucleus and the base of a major dendrite. [Ca2+] in this region increased over a period of 3 to 6 s and was followed by, with a slight delay, a surge of Ca2+ that moved across the soma and into or over the nucleus. Similar Ca2+ responses to caffeine were observed in Ca(2+)-containing and nominally Ca(2+)-free external solutions, suggesting that caffeine was inducing Ca2+ release from intracellular stores. Ca2+ responses to caffeine were potentiated by inducing a tonic Ca2+ influx through N-methyl-D-aspartate (NMDA)-type glutamate receptors activated by 0.3 microM glutamate and multiple responses to caffeine could be elicited by using this Ca2+ influx to refill the intracellular stores. Ryanodine inhibition of caffeine-induced Ca2+ release was use- and concentration-dependent; the median effective concentration EC50 for ryanodine declined from 22 microM for the first application of caffeine to 20 nM for the fourth. We conclude, based on these responses to caffeine, that ryanodine-sensitive mechanisms of intracellular Ca2+ release are active in hippocampal and cortical neurons and may be involved in generation of directed Ca2+ waves that engulf the nucleus.
Tsai
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Division of Neurosciences, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Barish
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D000818:Animals; D002110:Caffeine / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D016257:Fura-2; D018698:Glutamic Acid / Q000494:pharmacology; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D007091:Image Processing, Computer-Assisted; D051379:Mice; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D009474:Neurons / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D012433:Ryanodine / Q000494:pharmacology; D013687:Telencephalon / Q000187:drug effects / Q000196:embryology / Q000378:metabolism* / Q000648:ultrastructure
D012433:Ryanodine; D002110:Caffeine; D018698:Glutamic Acid; D002118:Calcium; D016257:Fura-2