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R.D. Ralston, J. Bilotta, H.M. Petry; Circadian Cycles of Caspase–3 Activation in Zebrafish Cone Photoreceptors . Invest. Ophthalmol. Vis. Sci. 2004;45(13):5287.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Previously we reported that activated caspase–3, a critical apoptotic protease, is present in cone photoreceptors in the dark–adapted zebrafish retina (Shepherd et al. 2002). The apparently non–apoptotic role of this protease in photoreceptor function is unknown. To determine whether its activation is governed by circadian cycles or related to retinomotor movements, we examined its distribution at various times in the daily lighting cycle. Methods: Adult zebrafish were sacrificed at six time points in a 14:10 L:D cycle: 1–hr prior to light–on, 1–hr post light–on, mid–light, 1–hr prior to light–off, 1–hr post light offset and mid–dark. Fish were fixed in Bouin's fixative, embedded, and sectioned coronally at 5um. Sections were processed for activated caspase–3 (Cell Signaling Technology) using standard post–embed immunocytochemical methods and were compared to adjacent nissl–stained sections for morphological identification of photoreceptor compartments. Results: Pale caspase–3 immunoreactivity (IR+) was routinely observed in cone ellipsoids, but the location of dark IR+ that was seen in cone myoids and nuclei varied with time of day and with cone type. At all time points dark IR+ was located at the distal nucleus/myoid junction of the Short Single (SS) cones. In fish sacrificed 1–hr prior to light offset, 1–hr post light offset, and at mid–dark cycle, dark IR+ was observed in the proximal portion of the Long Single (LS) and Double Cone (DC) nuclei. However at 1 hr prior to light onset, dark IR+ was seen in the distal portion of these cones. At 1 hr post light onset, dark IR+ was observed in LS myoids and in the distal portion of the DC myoids. A similar pattern was seen at mid–light except that label was present throughout the DC myoids. IR+ was not observed in rods at any time point Conclusions: The location of activated caspase–3 immunoreactivity in the inner segments and nuclei of all zebrafish cones suggests a non–apoptotic mechanism. The fact that the movement of caspase–3 IR+ within and between cone cell compartments varied with time–of–day, and not with the presence or absence of light, suggests that changes in caspase–3 activation are associated with circadian cycles and not in response to light. Possible functions of the protease may be related to daily photoreceptor renewal or to the control of retinomotor movements. This last possibility is strengthened by findings that nitric oxide is involved in retinomotor movements in lower vertebrates (Angotzi et al, 2002), and our previous report of co–localization (in dark–adapted zebrafish) of caspase–3 and nitric oxide synthase in cone nuclei.
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