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.