Live two-photon calcium imaging in retinal ganglion cells: characterization of early changes in a mouse glaucoma model.

Abstract

Purpose : Calcium plays a critical role in the regulation of neuronal activity. Our current understanding of calcium dynamics in living retinal ganglion cells (RGCs) and how they are altered in glaucoma is limited. Here, we used two-photon laser scanning microscopy (TPLSM) to investigate i) real-time light-triggered calcium responses in ON and OFF RGCs and their compartments (dendrites, soma, axons), and ii) alterations in light-evoked calcium responses during ocular hypertension damage.

Methods : Live calcium imaging in RGCs was performed by TPLSM in transgenic mice carrying the calcium indicator CGaMP6 (Thy1.GCaMP6) or after intraocular administration of an adeno-associated virus (AAV) encoding GCaMP6. Ocular hypertension was induced by intracameral injection of magnetic microbeads. The following light-evoked calcium responses were measured: i) baseline fluorescence (F₀), ii) peak fluorescence (ΔF/F₀), ii) rise time (Tr: time to reach 1/3 peak ΔF/F₀), and iv) decay time (Td: time to fall to 1/3 peak ΔF/F₀). Student’s t-test or ANOVA were applied (significance = p < 0.05).

Results : TPLSM imaging demonstrated distinct light-evoked calcium dynamics among RGC subtypes, with ON cells characterized by higher peak (ΔF/F₀) fluorescence and faster (low Tr) responses than OFF cells (N=8 mice/group, n=~70 cells/group, p<0.001, p<0.01). TPLSM also revealed distinct compartment-dependent calcium responses including lower baseline (F₀) in axons and dendrites relative to soma, and higher peak fluorescence (ΔF/F0) in axons relative to soma (N=5 mice/group, n=5-7 cells, ANOVA, p<0.001, p<0.05). RGC calcium responses were altered soon after induction of ocular hypertension. For example, ON RGCs transduced with AAV.GCaMP6 displayed a significant increase in Td values relative to controls (N=4-8 mice/group, n=48-61 cells/group, p<0.05) suggesting delayed calcium signal decay in glaucoma. These results were consistent with those observed in Thy1.GCaMP6 mice (N=7-8 mice/group, n=76-90 cells/group, p<0.05).

Conclusions : Our data support that: i) TPLSM is a powerful tool to assess calcium dynamics in living RGCs with unprecedented spatiotemporal resolution, ii) calcium responses differ among RGC subtypes and subcellular compartments, and iii) calcium dynamics are altered in glaucoma indicating impaired calcium homeostasis in vulnerable RGCs.

This is a 2021 ARVO Annual Meeting abstract.