Does circadian efferent priming of transient rhabdom shedding in the lateral eye of Limulus involve changes in photoreceptor cell gene expression?

Abstract

Abstract: : Purpose: Transient rhabdom shedding (TRS), the invertebrate analog of rod outer segment disc shedding, is primed by a minimum of 3–5 h of overnight circadian efferent outflow and triggered by dawn in the horseshoe crab Limulus (J. Neurosci. 4:2792). Efferent input to the lateral eye is octopaminergic and activates adenylate cyclase through a G–protein coupled receptor leading to a rise in [cAMP]_i and activation of cAMP–dependent protein kinase (PKA). The long–term activation of PKA required to prime the retina for TRS suggests that efferent input may regulate photoreceptor gene expression. We investigated: 1) whether protein synthesis is required for octopaminergic priming of the lateral eye for TRS, and 2) expression and activation of the transcription factor CREB in the retina during priming. Methods/Results: Microinjection (1.4 µl/min) of 40 µM octopamine (OA) into lateral eyes (n=4) for 6 h in darkness during subjective day (no efferent input) primed 47.5% of 40 photoreceptors for TRS. TRS was triggered by a 20–min pulse of broad–spectrum artificial lighting (2400 cd/m²). Co–injection of 1 mM cycloheximide (CHX; protein synthesis inhibitor) and 40 µM OA primed 75% of 40 photoreceptors (n = 4 eyes) for TRS and increased the amount of rhabdom shed by 63% relative to OA alone. 10 mM CHX and 40 µM OA primed 95% of 40 photoreceptors (n = 4 eyes) for TRS and increased the amount of rhabdom shed by 253% relative to OA alone. Western blots of nuclear extracts from whole retina homogenates contained distinct 48 kDa bands of CREB– and phosphoCREB– (pCREB) like immunoreactivity. pCREB–like immunoreactivity is quenched by preabsorption of the antibody with the phosphopeptide antigen. Conclusions: CHX causes a dose–dependent increase in the probability that a photoreceptor will shed its rhabdom in response to a light trigger, suggesting that OA primes the retina for TRS by downregulating the expression of one or more photoreceptor proteins. We are investigating whether CREB plays a role in regulating gene expression during circadian efferent priming of the lateral eye for TRS.

Keywords: photoreceptors • circadian rhythms • gene/expression