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Trevor S. Lee, Deniz Dalkara, Leah Byrne, Stephan G. Jarjisian, Meike Visel, Lance J. Kriegsfeld, John G. Flannery; Circadian Rhythm in a Photo-Insensitive Mouse Model with Retinal Expression of Exogenous Light-Gated Ion Channels. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1898.
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Gnat1-/-, Cnga3-/-, Opn4-/- triple-knockout (TKO) mice exhibit disrupted signal transduction in their rod-, cone- and melanopsin-based photoreception pathways. The activity-rest patterns of these mice have been shown to be insensitive to environmental light-dark conditions and their circadian rhythm (CR) does not entrain even to intense illumination. In this study, we investigate the potential for therapeutic recovery of light-induced CR modulation via AAV-mediated expression of light-gated ion channels (LGICs) in retinal ganglion cells (RGCs) of TKO mice.
TKO mice were injected either (1) intravascularly as neonates, with an AAV9 vector or (2) intravitreally at P15, with an AAV2 vector. The vectors delivered cDNA encoding for GFP-conjugated I107V-mutated Chimera EF (ChIEF) or an engineered photoswitch-coupling iGluR6 channel (LiGluR), driven by hSyn promoter.Wheel-running actograms were recorded from singly-housed adult mice, under bright constant light and 12-hour biphasic light-dark conditions. In the final phase of behavior data collection, the mice were kept under constant dim red (non-entraining) light conditions before being light-pulsed at their circadian time 16 and immediately perfused. Immunohistochemistry was performed to map retinal expression of the exogenous LGICs and to measure neuronal activity in the suprachiasmatic nucleus (SCN) via c-fos labeling.
The treated mice predominantly failed to respond to the presented light conditions. Some treated mice showed actogram phase shifts, but not entrainment, coincident with the transition between light-cycle programs. Fluorescence labeling revealed extensive expression of the exogenous LGICs across RGCs. However, SCN c-fos levels were not elevated relative to uninjected TKO mice.
Broad expression of hSyn-driven LGICs across RGCs is insufficient to enable CR photoregulation in genetically light-insensitive mice under the illumination conditions tested. LiGluR may transiently influence CR regulation under dramatic changes in bright light conditions, but confirmation was confounded by photoswitch turnover and re-administration. The lack of SCN activation could also indicate promoter or vector tropism incompatibility in the intrinsically photosensitive RGCs or other cell types critical for CR photoregulation.
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