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dc.contributor.authorCarr, Jessica
dc.date.accessioned2020-07-21T16:53:03Z
dc.date.available2020-07-21T16:53:03Z
dc.identifier.urihttp://hdl.handle.net/10222/79540
dc.description.abstractIn the rd1 mouse model of inherited retinal degeneration, activation of P2X7 purinoreceptors (P2X7r) has been suggested to lead to the formation of pores permeable to large molecules in retinal ganglion cells (RGCs). This suggests that retinal neurons downstream from degenerating photoreceptors are also affected during retinal degeneration, a phenomenon that could limit the effectiveness of treatment modalities for vision restoration that replace or restore photoreceptor function alone. P2X7r pores could increase intracellular calcium concentration ([Ca2+]i) and possibly cause cell death, but RGC calcium dynamics in the rd1 mouse have not been studied. The objective of this thesis was to examine Ca2+ indicator dye loading and the calcium dynamics (resting [Ca2+]i and kainic acid (KA)-induced Ca2+ response) of ganglion cell layer neurons (GCLn) in rd1 (C3H/HeJ) mice in comparison with wt (C57BL/6) mice. Freshly enucleated eyes were used and fura-2 loaded into GCLn either by intravitreal injection or applied to isolated retinas. In some cases, eyes were electroporated or P2Xr antagonists applied (A740003, P2X7r antagonist, 10 µM; PPADS, P2r antagonist, 1 mM; TNP-ATP, P2Xr antagonist, 500 µM). The fura-2-loading was compared with loading of the known P2X7r-permeable dye YO-PRO-1 (100 nM) in rd1 GCLn. Fura-2-loaded retinas were superfused with oxygenated Hank’s solution and fura-2 ratiometric calcium imaging of GCLn performed. To assess GCLn calcium dynamics, the resting [Ca2+]i was recorded and KA (AMPAr agonist, 50 µM) was superfused to evoke transient increases of fura-2 ratio (indicating an increase of [Ca2+]i). Purinergic drugs (A740003, P2X7r antagonist, 10 µM; apyrase, ATP-degrading enzyme, 5 units/ml; BzATP, P2X7r agonist, 250 µM; ATPγS, P2 agonist, 185 µM) were applied to assess the influence of the purinergic signalling pathway on resting [Ca2+]i and on KA-induced Ca2+ responses. In rd1 retinas, fura-2 loaded GCLn without the need for electroporation; however, loading was not reduced with P2Xr antagonists and fura-2 did not load the same population of GCLn as YO-PRO-1. Baseline [Ca2+]i was similar in GCLn in rd1 and wt retinas and purinergic drugs application had no effect. The KA-induced Ca2+ response was significantly greater in rd1 GCLn than wt GCLn. The enhanced response was blocked by P2X7r antagonist (A740003) but recovered by subsequent treatment with ATPγS. There was no effect of purinergic agonists on KA-induced Ca2+ responses when applied alone in either rd1 or wt GCLn. These results suggest that the enhanced KA-induced Ca2+ response in rd1 GCLn depends on P2X7r but does not involve ATP acting on P2X7r. A possible model is that P2X7r on other cell types in the retina release ATP through P2X7r pores and this ATP then affects the fura-2-loaded GCLn either directly, or indirectly, by acting on other P2r. Broadly, these data suggest that there is altered purinergic signalling in the rd1 retina, adding to the growing evidence that retinal neuron types other than photoreceptors are affected during retinal degeneration.en_US
dc.language.isoenen_US
dc.subjectP2X7en_US
dc.subjectRetinaen_US
dc.subjectGanglion Cell Layer Neuronen_US
dc.subjectInherited retinal degenerationen_US
dc.subjectCalciumen_US
dc.titleCalcium Dynamics of Ganglion Cell Layer Neurons in rd1 Miceen_US
dc.date.defence2020-06-11
dc.contributor.departmentDepartment of Clinical Vision Scienceen_US
dc.contributor.degreeMaster of Scienceen_US
dc.contributor.external-examinerDr. Spring Farrellen_US
dc.contributor.graduate-coordinatorDr. Darren Oystrecken_US
dc.contributor.thesis-readerStephanie Smithen_US
dc.contributor.thesis-readerDr. Francois Tremblayen_US
dc.contributor.thesis-supervisorDr. William Baldridgeen_US
dc.contributor.ethics-approvalNot Applicableen_US
dc.contributor.manuscriptsNot Applicableen_US
dc.contributor.copyright-releaseNot Applicableen_US
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