Exposure of rod photoreceptors to bright (rhodopsin bleaching) light triggers operation of the retinoid visual cycle and markedly desensitizes the rod flash response. Subsequent dark adaptation of the rods requires, in addition to excitation decay (shut-off of activated transduction intermediates and cGMP replenishment), recovery from """"""""silent"""""""" desensitization processes that depress transduction signaling even after the near-complete recovery of circulating current. In vitro studies show that the magnitude of silent desensitization far exceeds that attributable to bleach-induced reduction in quantum catch, and have identified numerous likely contributing reactions. However, the lack of information on the operation of silent desensitization in the living eye is a major current obstacle to ultimately understanding in vivo mechanisms and relative roles of the contributing reactions. A main focus of the project is to determine the in vivo bleaching-dependence of the silent desensitizations's magnitude and timing in mouse and in human rods using paired-flash electroretinographic (ERG) recording, a recently developed technique that permits noninvasive determination of the full time course of the rod weak-flash response. In normally functioning rods of mice and of human subjects, and in abcr-/- mice that exhibit sluggish excitation decay due to lack of the ABCA4 transporter of all-trans retinal bleaching product, we will test the specific hypothesis that excitation decay rate-limits the duration of silent desensitization. The accuracy of the paired-flash method in quantitatively determining the rod flash response -- specifically, the possibility that ERG b-wave intrusion skews derivation of the rod response amplitude -- will be tested in experiments on the nob mouse, a b-wave deficient model. A further focus of the project is to test a recently raised hypothesis concerning retinoid metabolism in the retinal pigment epithelium (RPE), a process directly linked with the RPE's role in supporting rhodopsin regeneration in dark adapting rods. The hypothesis -- that 11-cis retinoid in the RPE regulates the efflux of all-trans retinol at the RPE basolateral membrane -- will be tested in radiolabeling experiments on mice that exhibit normal or impaired visual cycle operation. Results of the project will advance fundamental knowledge of rod dark adaptation and provide foundation for further studies of photoreceptor disease in human subjects and experimental animals.
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