Figure 3.

Figure 3.

Chemogenetic activation of HCs alters vesicular release of FM1-43 from cone terminals. A, Schematic representation of the mechanisms of negative (–) and positive (+) feedback of HCs onto cone photoreceptors (PR). VGCCs are voltage-gated Ca²⁺ channels, green circles are FM1-43-filled synaptic vesicles, orange dots are glutamate molecules in the synaptic cleft, AMPARs are AMPA receptors. Negative feedback is mediated by changes in the membrane potential of HCs, whereas positive feedback is mediated by increased intracellular Ca²⁺ in HCs, owing to influx of Ca²⁺ through Ca²⁺-permeant AMPA receptors. ΔCa_i is the change in intracellular Ca²⁺; ΔV_M is the change in HC membrane potential. B, Two-photon scanned images of the photoreceptor terminal layer in FaNaC zebrafish retinas. Retinas were pretreated with FM1-43 to load recycling synaptic vesicles and then treated for 20 min with the indicated receptor agonist. AMPA (20 μm) increased the rate of dye loss (destaining) and FMRFamide (10 μm) decreased destaining, but the AMPA-elicited increase was dominant when the two agonists were applied together. C, D, Time course of FM1-43 destaining in FaNaC fish. Without added agonists, cone terminals released FM1-43 at 0.87 ± 0.08% per minute (n = 10 retinas). Adding AMPA accelerated destaining rate (1.89 ± 0.13%, n = 9 retinas, p < 1 × 10−5). Adding FMRFamide (10 μm) decelerated destaining (0.54 ± 0.07% n = 9 retinas, p = 0.001). Adding AMPA and FMRFamide together resulted in a significant accelerated destaining rate from dark (p = 0.002), which was not significantly different from that with AMPA alone (1.96 ± 0.25%, n = 4, p = 0.88). Statistical differences were determined with one-way ANOVA (F(3,23) = 27.4, p = 8.9 × 10−8). E, F, Time course of FM1-43 destaining in PSAM fish. Adding PSEM^89S (30 μm) accelerated destaining (0.83 ± 0.19%, n = 9 retinas, p = 0.044). Adding AMPA accelerated destaining (2.19 ± 0.19%, n = 5 retinas, p = 0.0002). Adding PSEM^89S and AMPA together resulted in a destaining rate not significantly different from that with AMPA alone (2.05 ± 0.35%, n = 5, p = 0.82). Analysis was performed with one-way ANOVA (F(3,24) = 14.69, p = 1.2 × 10−5). For D and F open circles represent individual data points. Open boxes represent the interquartile range with whiskers. The mean is marked by x and the median by a line.