Imaging extracellular waves of glutamate during calcium signaling in cultured astrocytes

A growing body of evidence proposes that glial cells have the potential to play a role as modulators of neuronal activity and synaptic transmission by releasing the neurotransmitter glutamate (Araque et al., 1999). We explore the spatial nature of glutamate release from astrocytes with an enzyme-lin ked assay system and CCD imaging technology. In the presence of glutamate, L-glutamic dehydrogenase (GDH) reduces NAD(+) to NADH, a product that fluor esces when excited with UV light. Theoretically, provided that GDH and NAD( +) are present in the bathing saline, the release of glutamate from stimula ted astrocytes can be optically detected by monitoring the accumulation of NADH. Indeed, stimuli that induce a wave of elevated calcium among astrocyt es produced a corresponding spread of extracellular NADH fluorescence. Trea tment of cultures either with thapsigargin, to deplete internal calcium sto res, or with the membrane-permeant calcium chelator BAPTA AM significantly decreased the accumulation of NADH, demonstrating that this fluorometric as say effectively monitors calcium-dependent glutamate release. With a tempor al resolution of 500 msec and spatial resolution of similar to 20 mu m, dis crete regions of glutamate release were not reliably resolved. The wave of glutamate release that underlies the NADH fluorescence propagated at an ave rage speed of similar to 26 mu m/sec, correlating with the rate of calcium wave progression (10-30 mu m/sec), and caused a localized accumulation of g lutamate in the range of 1-100 mu M. Further analysis of the fluorescence a ccumulation clearly demonstrated that glutamate is released in a regenerati ve manner, with subsequent cells that are involved in the calcium wave rele asing additional glutamate.