Clock gene expression in the retina of melatonin‐proficient (C3H) and melatonin‐deficient (C57BL) mice

Abstract

In several mammalian species, the retina contains an autonomous circadian clock and is capable of synthesizing melatonin. The function of circadian clocks depends on interlocking transcriptional/translational feedback loops involving several clock genes. Here we investigated the expression of two clock genes (Per1, Cry2) and the level of phosphorylated (p) cyclic AMP response element binding protein (CREB) in retinae of melatonin-deficient (C57BL) with an intact retina and melatonin-proficient (C3H) mice with degenerated outer nuclear layer. RNase protection assay and in situ hybridization revealed in both strains a rhythm in transcript levels for Per1 with a peak at zeitgeber time (ZT) 08, but not for Cry2. Immunoreactions for PER1, CRY2 and pCREB were localized to the nuclei of cells in the inner nuclear layer (INL) and ganglion cell layer (GC) of both strains and to the outer nuclear layer of C57BL. In C3H, protein levels of PER1 and CRY2 followed a clear day/night rhythm in the INL and the GC with a peak at the end of the day (ZT14). pCREB levels peaked at the beginning of the day. Noteably, in melatonin-deficient C57BL mice, protein levels of PER1, CRY2 and pCREB did not show significant changes over a 16L/8D cycle. These data suggest that melatonin influences PER1 and CRY2 protein levels via post-transcriptional mechanisms and also plays a role in rhythmic regulation of pCREB levels in the mammalian retina.

Impact of melatonin receptors on pCREB and clock-gene protein levels in the murine retina

“…These are three period genes (Per), two clock/cycle-related genes (Clock and Bmal1), two cryptochrome genes (Cry) and casein kinase 1ε (Okamura et al 2002;Allada et al 2001;Reppert and Weaver 2001;Young and Kay 2001). Several investigations have shown circadian expression of these clock genes also in the retina of mice (Sun et al 1997;Shearman et al 1997;Miyamoto and Sancar 1998;Dinet et al 2007).…”

Expression and light sensitivity of clock genes Per1 and Per2 and immediate‐early gene c‐fos within the retina of early postnatal Wistar rats

“…At P1, P3, and P5, Per1 was expressed throughout the retina with no specific layer localization. At P10, the expression increased in the outer part of the retina, the site of developing photoreceptors, although, in the adult rat retina, spontaneous Per1 and Per2 mRNA expression and PER1 protein were found mainly in the inner nuclear layer (INL) and in a small group of ganglion cells (Namihira et al, 2001;Witkovsky et al, 2003;García-Fernández et al, 2007;Dinet et al, 2007;Ruan et al, 2008). Our preliminary data suggest that Per1 gene expression may predominate in the INL at P15, i.e., immediately after eye opening (unpublished results).…”

The retinal clock in mammals: role in health and disease

“…In early research, retinal degenerative animal models such as the rd mouse and the Royal College of Surgeons rat, both characterized by progressive degeneration of photoreceptors, showed arrhythmic melatonin release, whereas the daily rhythms of DA were retained, 5,59 suggesting that photoreceptors are necessary to maintain rhythmic melatonin production, but their integrity was not required for DA signaling. With the discovery of clock genes, photoreceptors have been the site of the most intensive controversy because initial reports claimed that expression of Clock, Bmal1, Per1-2, or Cry2 takes place in the outer nuclear layer (ONL) of rodents, [60][61][62] whereas others failed to detect them. 17,52,63 More recently, by using ONL isolated by laser microdissection, vibratome sectioning, or following kainic acid treatment, several studies demonstrated clock property in rat photoreceptors under an LD cycle, but these rhythms were severely damped in constant darkness, indicating that the photoreceptor clock is not robust, [64][65][66] although data obtained in the mouse support the opposite conclusion.…”

“…10 Responsible cell types have not been identified yet, although DAergic amacrine cells, which have been shown to express the whole set of clock genes, 68 are a good candidate. Although their contribution to clock gene oscillations in the retina has been poorly evaluated, ganglion cells have also been reported to harbor clock gene transcripts or products, 52,[61][62][63]69 sometimes with sparse distribution. The most extensive ex vivo analysis of the distribution of clock gene expression in the retina was conducted by immunohistochemistry in the mouse.…”