Figure 2.

Endogenous NOS activity and NO production regulate the survival of cultured retinal neurons. Endogenous NO production in cultured retinal neurons was regulated using L-arginine (L-Arg) and L-NAME and monitored by DAF-FM-DA labeling (a, E6 and b, E8). Calibration bar in a and b=10 μm, E6 (c and d) or E8 (e and f) cells were treated with L-arginine or L-NAME in C0, cultured up to C4, fixed and counted. Neuronal survival in (c–f) was evaluated by counting viable neurons in purified cultures. Total cell numbers in (a): CT=612 cells, n=6; in (b): CT=780 cells, n=6; in (c): CT=486 cells, n=6; in (d): CT=540 cells, n=6. Data represent the mean±S.E.M. (error bars). **P<0.01, ***P<0.001 in relation to control, one-way ANOVA. In g, E6 or E8 purified cultures were pulsed in C1 with radiolabeled calcium for 2 min. Cells were lysed and the homogenates were processed for liquid scintillation. In h, E6 or E8 retinas were incubated with DAF-FM-DA (5 μM) in HBSS for 30 min. Retinas were washed, flat-mounted and visualized in a confocal microscope. Calibration bars=50 μm. Histograms display the pixel count versus pixel intensity in E6 or E8 retinas labeled with DAF-FM-DA. (i) Dose-response curve of SNAP in E6 and E8 mixed cultures. Cell death was evaluated by EthD1 labeling. EC₅₀ for SNAP in E6=68.4 μM. EC₅₀ for SNAP in E8=148.2 μM. Total EthD1-positive cells in (i): CT E6=1456 cells, n=3. In j and k, DETA NONOate also regulates neuronal survival differentially. E6 (j) or E8 (k) mixed culture was treated with DETA NONOate (500 nM). EthD1 measured cell death. EthD1-positive cells: CT E6=237, n=3; CT E8=156, n=3. Data represent the mean±S.E.M. (error bars). *P<0.05 relative to CT in j and k, t-test