Optogenetic Investigation of Ca2+ Handling In Early Diabetic Retinopathy

Abstract

Background: Previous work showed that electrically-evoked inhibition to Rod Bipolar Cells (RBC) is reduced in a mouse model of early diabetes. It is hypothesized that this is due to impaired Ca2+ handling in the presynaptic amacrine cell, either through increased Ca2+ buffering or decreased influx. To test this hypothesis and develop a mechanism for this effect, a model where direct optogenetic activation of inhibitory amacrine cells that expressed the light-activated channel ChR2 was used to isolate amacrine cell inputs to RBCs. Application of selective Ca2+ channel blockers could then assess potential locations of amacrine Ca2+ disruption. Using whole cell patch clamp electrophysiology, recordings were made from a 6 week diabetic population and vehicle injected control animals. Results: Robust inhibitory currents were recorded from RBCs after ChR2 stimulus that were significantly diminished by the application of nifedipine to block L-type Ca2+ channels in both diabetic and control conditions. There were no significant differences in the magnitude of these responses between conditional groups. However, in the control group the decay tau of the response to the 50ms stimulus was significantly diminished by nifedipine (? p =0.0498, n = 5), but this was not seen in the diabetic group (? p = 0.9498, n=7). A 1s nifedipine-reduced response saw its decay tau increase in the diabetic group but not the control. Ca2+ - induced Ca2+ release (CICR) blockade with ryanodine decreased responsivity equally between groups in the 1s stimulus, but showed no significant kinetic changes. CICR blockade for a 50ms stimulus response showed significant kinetic changes in diabetes, but otherwise reduced the response equally between diabetic and control. Blockade of the mitochondrial Ca2+ uniporter (MCU) had little effect on the optogenetic response. Conclusion: This study presents evidence that diabetes alters amacrine cell output to the rod bipolar cell unmasked through blockade of the L-type calcium channel, and the Endoplasmic Reticulum (ER). An apparent explanation for our results is that diabetic calcium buffering is dysregulated, leading to prolonged responses. The underlying mechanism for this alteration is complex and not yet clearly elucidated. Significance: Using optogenetic isolation of amacrine to RBC inputs, we show nifedipine unmasks slower decay kinetics (1s), and delayed onset in diabetes at 50ms, that ryanodine blockade modulates kinetics in control, but not diabetic (50ms), while MCU blockade has minimal effect.