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Jessica Carr, William H Baldridge; Calcium dynamics of ganglion cell layer neurons in rd1 mice. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4847.
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© ARVO (1962-2015); The Authors (2016-present)
Prolonged activation of P2X7 purinoreceptors during retinal degeneration has been suggested to lead to the formation of pores permeable to large molecules in retinal ganglion cells (RGCs). P2X7 pores could increase intracellular calcium concentration ([Ca2+]i) and possibly cell death, but RGC calcium dynamics in animal models of retinal degeneration have not been studied. The presence of P2X7 pores may also permit loading with calcium-indicator dye. Using an experimental model of retinal degeneration (rd1 mouse), we assessed calcium dye permeability and calcium dynamics of ganglion cell layer (GCL) neurons in comparison to wildtype control mice.
Freshly enucleated eyes of rd1 mice (C3H/HeJ strain, Charles River Laboratories; 70-XX days old) and C57BL/6 (control) mice were intravitreally injected with 750 nl of fura-2 pentapotassium salt (20 mM), a membrane impermeable calcium indicator. In some cases, eyes were electroporated. Retinas were isolated, flat-mounted, superfused with oxygenated Hank’s solution, and fura-2 ratiometric calcium imaging of the GCL performed. Kainic acid (KA; 50 µM) superfusion was used to evoke transient increases of fura-2 ratio (an indicator of [Ca2+]i) in GCL neurons. A P2X7 receptor antagonist, A740003 (10 µM), was used to assess the potential influence of P2X7 receptors on KA-mediated increases in [Ca2+]i.
The number of GCL neurons loaded by fura-2 salt was significantly greater in rd1 retinas (85 ± 14, n=6) compared to control retinas (20 ± 13, n=5; p<0.0001). KA-evoked peak fura-2 ratio was significantly greater in rd1 cells (0.54 ± 0.12, n=110 from 5 retinas) compared to electroporated control cells (0.41 ± 0.07, n=101, 8 retinas, p<0.0001). A740003 significantly (p<0.001) reduced the KA-evoked peak fura-2 ratio in both rd1 and control retinas; but A740003 had a greater effect in rd1 retinas (22% reduction) compared to controls (7% reduction).
Membrane impermeable fura-2 salt loaded numerous neurons in the GCL of rd1 mice retinas but produced less labelling of GCL neurons in control retinas. This result is consistent with the presence of P2X7 receptor-mediated pores in RGCs of animal models of retinal degeneration. Increases of [Ca2+]i produced by KA and the effect of A740003 were both greater in rd1 mice compared to control mice. This suggests that the calcium dynamics of GCL neurons in rd1 retina are altered compared to controls.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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