Propagation of intercellular calcium waves in retinal astrocytes and Muller cells

Intercellular Ca2+ waves are believed to propagate through networks of glial cells in culture in one of two ways: by diffusion of IP3 between cells through gap junctions or by release of ATP, which functions as an extracellular messenger. Experiments were conducted to determine the mechanism of Ca2+ wave propagation between glial cells in an intact CNS tissue. Calcium waves were imaged in the acutely isolated rat retina with the Ca2+ indicator dye fluo-4. Mechanical stimulation of astrocyte somata evoked Ca2+ waves that propagated through both astrocytes and Muller cells. Octanol (0.5 mM), which blocks coupling between astrocytes and Muller cells, did not reduce propagation into Muller cells. Purinergic receptor antagonists suramin (100 muM), PPADS (20-50 muM). and apyrase (80 U/ml), in contrast, substantially reduced wave propagation into Muller cells (wave radii reduced to 16-61% of control). Suramin also reduced wave propagation from Muller cell to Muller cell (51% of control). Purinergic antagonists reduced wave propagation through astrocytes to a lesser extent (64-81% of control). Mechanical stimulation evoked the release of ATP, imaged with the luciferin-luciferase bioluminescence assay. Peak ATP concentration at the surface of the retina averaged 78 muM at the stimulation site and 6.8 muM at a distance of 100 mum. ATP release propagated outward from the stimulation site with a velocity of 41 mum/sec, somewhat faster than the 28 mum/sec velocity of Ca2+ waves. Ejection of 3 muM ATP onto the retinal surface evoked propagated glial Ca2+ waves. Together, these results indicate that Ca2+ waves are propagated through retinal glial cells by two mechanisms. Waves are propagated through astrocytes principally by diffusion of an internal messenger, whereas waves are propagated from astrocytes to Muller cells and from Muller cells to other Muller cells primarily by the release of ATP.