Propagation of intercellular calcium waves in retinal astrocytes and müler 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 Müller cells. Octanol (0.5 mM), which blocks coupling between astrocytes and Müller cells, did not reduce propagation into Müller cells. Purinergic receptor antagonists suramin (100 μM), PPADS (20-50 μM), and apyrase (80 U/ml), in contrast, substantially reduced wave propagation into Müller cells (wave radii reduced to 16-61% of control). Suramin also reduced wave propagation from Müller cell to Müller 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 μM at the stimulation site and 6.8 μM at a distance of 100μm. ATP release propagated outward from the stimulation site with a velocity of 41 μm/sec, somewhat faster than the 28 μm/sec velocity of Ca²⁺ waves. Ejection of 3 μM 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 Müller cells and from Müller cells to other Müller cells primarily by the release of ATP.