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R. H. Kramer, K. Borges, K. P. Greenberg, M. Banghart, D. Trauner, J. G. Flannery; A Photochemical Switch for Conferring Light Sensitivity Onto Endogenous Ion Channels and Retinal Neuron Firing. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1935.
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Our goal is to develop a photochemical method for bestowing light-sensitivity on native retinal ion channels, thereby allowing action potential firing to be controlled by light. This could allow optical information input into the visual system of animals that have lost their photoreceptors from retinal degeneration. In contrast to other light-activated channels that depend on exogenous gene expression (e.g. channelrhodopsin-2), our method targets endogenous channels.
We chemically synthesized and injected into the vitreous of young rat eyes a photoisomerizable compound that covalently attaches to native K+ channels and acts as a light-regulated open channel blocker. This "Photoswitchable Affinity Label" (PAL) blocks K+ current flow in darkness or visible light, but allows current flow when exposed to 380-400 nm light. Light-adapted retinas were removed from eyes 30-60 min after PAL injection and extracellular recordings were obtained from retinal ganglion cells (RGCs) in wholemount preparations to assess the affect of light on spontaneous firing.
Intravitreal injection of PAL caused >50% of RGCs to dramatically change firing frequency, usually within 200 msec of a light flash. Exposure to 390 nm light was expected to open K+ channels of RGC’s and inhibit their firing, but in most cases it had the opposite effect, suggesting a dominant action on inhibitory amacrine cells. Consistent with this, the GABA receptor antagonist bicuculline eliminated enhanced firing to 390 nm light, revealing a smaller, and perhaps direct, suppression of RGC firing. In vivo PAL injection had no deleterious effects on retinal histology or on ERG responses recorded up to 1 week later.
PAL injection into the intact eye imparts light responses onto retinal neurons with no apparent toxic effects. PAL confers light-sensitivity onto multiple types of retinal neurons. RGC firing is rapidly switched on and off with different wavelengths of light, allowing precise bidirectional control. Ongoing behavioral experiments are testing whether PAL-injected rat models of retinal degeneration can perceive visual stimuli.
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