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 rather limited. Here, we used two-photon laser scanning microscopy (TPLSM) to investigate: i) real-time light-triggered calcium responses in ON and OFF RGCs, as well as distinct RGC compartments (dendrites, soma, axons), and ii) alterations in light-evoked calcium transients during ocular hypertension damage.

Methods : Live calcium imaging in RGCs was performed by TPLSM after intraocular administration of an adeno-associated virus (AAV) carrying GCaMP6, a genetically-encoded calcium indicator. Ocular hypertension was induced by intracameral injection of magnetic microbeads. Light-evoked calcium responses were analyzed using a customized Python program, ImageJ and R code. A semi-automated algorithm was used to calculate baseline fluorescence (F₀), peak fluorescence (DF/F₀), rise time (Tr): time to reach half peak DF/F₀, decay time (Td): time to fall to half peak DF/F₀, and signal-to-noise ratio. Student’s t-test or ANOVA with Bonferroni corrections 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 (DF/F₀) fluorescence and faster (low Tr) responses than OFF cells (N=3 mice/group, n=4-9 cells/mice, Student’s t-test, 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 (DF/F₀) in axons relative to soma (N=5 mice/group, n=5-7 cells, ANOVA, p<0.001, p<0.05). Furthermore, we found that RGC calcium responses were altered soon after induction of ocular hypertension. For example, ON RGCs displayed a significant increase in Td values relative to controls (N=3 mice/group, n=8 cells/group, p<0.05) suggesting delayed calcium recovery in glaucoma.

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 and might be a sign of impaired calcium homeostasis in vulnerable RGCs.

This is a 2020 ARVO Annual Meeting abstract.