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Christopher Kessler, Edward Pugh, Marie Burns; Electrical measurements of pigment bleaching in vivo. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6125.
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The dark noise consequent to bleaching and the slow regeneration of rhodopsin limits the signaling of rods under mesopic conditions. The reduced bleaching noise and faster regeneration rates of cone opsins contributes to the cone's ability to signal at high light intensities and to avoid electrical signal saturation. Understanding the processes that rate-limit pigment regeneration in rods and cones requires developing methods to measure functional pigment levels in the intact eye. Our purpose was to develop an electrophysiological assay for pigment bleaching and regeneration in living mice.
Mice (c57Bl/6 or Gnat1-/-) were dark-adapted overnight and anesthetized with ketamine/xylazine in darkness using infrared night vision goggles. ERGs were recorded using a platinum loop corneal electrode referenced to a bite bar, and elicited using a Xenon flash lamp that delivered brief (~2 µs) flashes at preset intervals.
A single intense flash elicited a bi-phasic potential that reached a corneal-negative peak amplitude of 120-150 µV within 0.55 ms of the flash, and preceded the classic a-wave and b-wave components of the ERG. Repeated flashes caused a progressive amplitude decline of ~15%/flash, consistent with this signal being the early receptor potential (ERP) arising from isomerization of the opsins located in patent discs of photoreceptors. The amplitude and time course of the decline in ERP amplitude were indistinguishable in wild-type mice and in Gnat1-/- mice, which lack the rod a-wave. Both wild-type and Gnat1-/- ERP signals recovered initially with a rate of about 2% of the pigment per minute, which is comparable to the rate of rhodopsin regeneration measured previously in unanesthetized mice (Lyubarsky et al., 2005, Biochem 44:9880).
The early receptor potential can be reliably measured in mouse corneal ERGs, if the optical configuration and electrical recordings are optimized. Our findings also suggest that the rate of rhodopsin regeneration in vivo is relatively unaffected by ketamine/xylazine anesthesia.
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