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Jared D Chrispell, Shoji Osawa, Ellen R Weiss; Role of cAMP in the Recovery of the Cone Photoresponse in Zebrafish Larvae. Invest. Ophthalmol. Vis. Sci. 2014;55(13):424.
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In the vertebrate retina, cAMP is known to play a role in circadian rhythm and dysregulation of cAMP has been associated with retinal degeneration. Light-dependent changes in cAMP levels have been observed in rod inner segments in mice and cone inner segments in goldfish, where they can influence the phosphorylation state of proteins involved in phototransduction. The retina-specific G protein-coupled receptor kinases Grk1 and Grk7 are substrates of Protein kinase A (PKA), the major downstream effector of cAMP. Elevated cAMP levels leads to increased phosphorylation of Grk1 in mice and Grk 7 in frogs - an indication of a link between cAMP and proteins involved in phototransduction in cones. cAMP has been studied most extensively in rods, where levels are found to be high in the dark and low in the light. In zebrafish, cones mature prior to rods and electroretinogram (ERG) analyses detect cone photoresponses as early as 4 days post fertilization (dpf). In contrast, ERG responses in rods appear between 15 and 21 dpf. Therefore, the zebrafish retina at 4-7 dpf serves functionally as an all-cone model. In order to better understand how changes in cAMP influence the cone photoresponse in vertebrates, we employed the use of drugs that increase intracellular cAMP levels and measured their effects on cone recovery by ERG analysis of zebrafish larvae.
Zebrafish larvae (5 dpf) were incubated for 30 min in forskolin (50 μM) or IBMX (1 mM). This was followed by co-incubation for 5 min with L-AP4 (500 μM) to block inner retina signaling and allow recording of the cone mass receptor potential. Recovery was measured using a dual flash paradigm with increasing interstimulus intervals.
Zebrafish larvae exposed to forskolin, an activator of adenylyl cyclase, display a decreased rate of recovery when compared to untreated larvae. A similar result is obtained when larvae are exposed to IBMX, a phosphodiesterase inhibitor.
Increased levels of intracellular cAMP in the zebrafish all-cone retina result in decreased visual function, in the form a lower recovery rate in response to bright flashes of light. This observation is in agreement with our hypothesis that intracellular levels of cAMP affect proteins critical for efficient recovery of the photoresponse in cones. Further studies will be carried out to identify the specific proteins involved and the mechanism by which they are affected.
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