Treatment of enriched Müller glia cultures with 50 mM KCl resulted in reduced intracellular levels of quinacrine staining after a 10 min period of stimulation (Fig. 3). No difference in quinacrine staining could Temozolomide supplier be detected in control cultures after the same period of incubation, discarding the possibility that the decrease in quinacrine staining observed in KCl-treated cultures was due to fluorescence fading. The effect of 50 mM KCl on the accumulation of extracellular ATP was also investigated in these cultures (Fig. 3E). The amount of ATP in the bathing solution of the cultures was measured after an incubation of 5 min in Hank’s

balanced salt solution and after an additional incubation of 5 min in Hank’s solution containing 50 mM KCl. An increase of ∼77% over the basal levels of ATP was observed when cultures were incubated with 50 mM KCl. While non-stimulated levels of ATP were 1.68 ± 0.3 pmol/culture, levels of ATP observed in KCl-treated cultures were 2.97 ± 0.45 pmol/culture. Both NMDA and AMPA/KA ionotropic glutamate receptors were shown to be expressed in chick Müller glial cells

(Lamas et al., 2005, López et al., 1994 and López et al., 1997). Ruxolitinib Activation of these sites was shown to elicit the release of ATP from astrocytes, although activation of NMDA receptors elicits ATP release to a lesser extent (Pangršič et al., 2007, Queiroz et al., 1997 and Queiroz et al., 1999). To investigate if glutamate could also induce the release of ATP from retinal Müller glia cells in culture, the effect of 1 mM glutamate on quinacrine staining of glial cultures was evaluated (Fig. 4). Treatment of the cultures with glutamate for 10 min induced a decrease in both the intensity of

quinacrine fluorescence as in the number of quinacrine stained granules (Fig. 4D). No reduction in quinacrine staining was observed in control, non-treated cultures (Fig. 4B). Also, for no decrease in quinacrine staining was observed when glutamate-treated cultures were co-incubated with 50 μM of the antagonists DNQX (Fig. 4F) or MK-801 (Fig. 4H). The amount of extracellular ATP in glutamate-stimulated glial cultures was also evaluated using the luciferin–luciferase assay (Fig. 5). Consistent with the reduction in quinacrine staining, incubation with 1 mM glutamate, for 5 min, at 37 °C, induced a significant increase in the extracellular levels of ATP in the cultures. While the medium of control cultures showed ATP levels of 1.81 ± 0.15 pmol/culture, glutamate-treated cultures showed ATP levels of 3.43 ± 0.33 pmol/culture. The effect of glutamate selective agonists for NMDA and non-NMDA receptors on quinacrine staining of retina glial cells or extracellular levels of ATP is shown in Fig. 6.