Abstract
Abstract: :
Purpose: Our studies showed that isorhodopsin mediates rod function in Rpe65–/– mice (Fan et al., 2003), which explains the minimal rod electroretinograms (ERG) response in Rpe65–/– mice, leading us to the conclusion that endogenous isorhodopsin is generated through an RPE65–independent pathway. Isorhodopsin was found to accumulate linearly with prolonged dark–rearing, eliminating the light–driven RGR–pathway as a source for 9–cis retinal. Interestingly, a subset of Rpe65–/– mice having a tan coat color exhibit enhanced ERG responses. We hypothesized that this due to the higher levels of isorhodopsin accumulation than in agouti Rpe65–/– mice rather than the more efficient light absorption in a less pigmented background. Methods: Rpe65–/– mice (tan and agouti) were maintained in darkness from 1 day to 18 weeks. Rod function was analyzed in vivo using scotopic single–flash ERGs. Pigment levels were determined by extraction of retinal homogenates in 1% dodecylmaltoside and measurement of the absorption spectrum. Retinoids were extracted in methanol and analyzed by high performance liquid chromatography. Results: Isorhodopsin in agouti Rpe65–/– mice accumulated linearly during the 18 weeks of dark–rearing. After overnight dark adaptation, the tan color animals had similar ERG responses as the agouti animal obtained after 4 weeks of dark–rearing. The quantity of isorhodopsin and 9–cis retinal extracted from the retinas of 2–week dark–reared tan colored mice was comparable to the 4–week dark–reared agouti Rpe65–/– mice. The pigment measurements and retinoid extraction experiments confirmed that the 9–cis retinal accumulated 2–fold faster in tan than in agouti Rpe65–/– animals. Conclusions: In addition to its important precursor role in cell differentiation and development, 9–cis retinal also acts as the chromophore of the functional pigment isorhodopsin in Rpe65–/– mice. The difference in kinetics of 9–cis retinal accumulation in Rpe65–/– mice with varied pigmentation is expected to be a useful tool in the elucidation of this retinoid metabolic pathway.
Keywords: regeneration • opsins • electroretinography: non–clinical