Figure 3.
NPY attenuates glutamate-induced [Ca2+]i increase in purified RGCs. (a) [Ca2+]i trace from a cultured RGC illustrating the response of RGCs to increasing concentrations of glutamate (10–1000 µM). (b) Example of cultured RGCs. Bright field image (ba) and pseudocolor representation of Fura-2 ratio on basal condition (bb) and after stimulation with 30 µM glutamate (bc) are shown. (c) [Ca2+]i traces showing RGC responses to two consecutive glutamate stimuli (30 µM) for 30 s each stimulus. After the first glutamate stimulus, 1 µM NPY or a drug-free solution (control) was applied to RGCs during 10 min followed by a second glutamate stimulus. For illustration, two experiments (control and NPY) are shown. The increase above basal Fura-2 ratio (340 nm/380 nm) was quantified for each stimulus as a Delta value. (d) The Delta 2 (second stimulus)/Delta 1 (first stimulus) ratios are presented for different drug applications for 10 min: 1 µM NPY, Figure 3. Continued. 1 µM (Leu31, Pro34) − NPY, 300 nM NPY13–36, 1 µM (Gly1, . . . Aib32)-PP, or a drug-free solution (control). A small decrease in Delta 2/Delta 1 ratios may be apparent in RGCs exposed to NPY and (Leu31, Pro34) − NPY. (e) Scatter plots for two populations of RGCs from the same cell culture showing the dispersion of Delta 2/Delta 1 ratio values among cells. Each point represents an individual RGC. The effect of NPY is evident in some RGCs. Cells were treated with a drug-free solution (control) or NPY. Note that in NPY-treated population, a small downward shift may be observed. Dashed line indicates 0.9 ratio value. (f) Percentage of cells presenting Delta 2/Delta 1 ratio below 0.9. The application of NPY or (Leu31, Pro34) − NPY for 10 min increased the percentage of cells with Delta 2/Delta 1 ratio below 0.9. Data are presented as mean ± SEM of n = 5 to 10 independent experiments. *p < .05, compared with control. Kruskal-Wallis followed by Dunn’s test.