Time-lapse confocal calcium imaging in an intact unrolled hippocampus preparation

Abstract

The excitatory neurotransmitter glutamate (100 μM) induces intracellular calcium transients in cultured hippocampal astrocytes that can be imaged using the calcium indicator (Fluo3AM) and time-lapse microscopy. In response to glutamate (Fig. 1A), cultured astrocytes exhibit distinct patterns of intracellular Ca2+ oscillations and long-distance intercellular waves. Two distinct types of intercellular Ca2+ waves are attributed to excitation by different agonists of the glutamate receptor subtypes. A long-distance regenerative intercellular wave is induced by the ionotropic glutamate receptor, kainate (Fig. 1B). This is a true wave lasting for 50-125 sec with a constant velocity of 10-20 μm/sec. This wave requires extracellular Ca2+ and Na+ and is driven by the Na+/Ca2+ exchanger. A fast Ca2+ wave which travels at speeds from 10 to 200 μm/sec is dependent upon the metabotropic glutamate receptor, is inducible by t-ACPD and is dependent upon cytoplasmic release of Ca2+ regulated by IP3 (Fig. 1C). This wave is not dependent upon extracellular Ca2+ and is stopped by MCPG, a specific inhibitor of IP3-mediated intracellular Ca2+ release.