Fig. 4
Cytosolic calcium homeostasis is dysregulated in response to the excitatory neurotransmitter glutamate in OPA1 R445H iPSC-RGCs. (a) Representative live cell confocal images of cytosolic calcium levels in isogenic control and R445H neurons stained with Fluo4 at baseline, and after stimulation with 5 µM glutamate and 10 µM ionomycin. Scale bar, 20 μm. (b & c) Quantification of fold-change in Fluo4 fluorescence intensity over time normalised to baseline values, denoted F0. 5 µM glutamate and 10 µM ionomycin were added at the indicated time points. Neurons were maintained in magnesium-free recording buffer throughout the experiment. Grey lines show Fluo4 fluorescence intensity in individual cells from a representative experiment, blue/red lines the mean average normalised Fluo4 fluorescence intensity. (d-f) Quantification of initial slope, amplitude and decay slope of Fluo4 fluorescence intensity in response to 5 µM glutamate in isogenic control R445H and iPSC-RGCs. Isogenic control, n = 167; R445H, n = 210 cells sampled from three independent differentiations. **** = p < 0.0001, Mann-Whitney tests. Box plots show median (middle line), 25th-75th percentile (box) and min/max values (whiskers)