Clock gene expression in the rat retina: effects of lighting conditions and photoreceptor degeneration.

Circadian Rhythms and Sleep Disorders Program, Neuroscience Institute, Morehouse School of Medicine, 720 Westview Dr, Atlanta, GA 30310-1495, USA.
Brain Research (Impact Factor: 2.84). 08/2007; 1159(1):134-40. DOI: 10.1016/j.brainres.2007.05.023
Source: PubMed


Previous studies have shown that, in the Royal College of Surgeon rat, circadian rhythms in the retinal dopaminergic and melatonergic systems are still present after the photoreceptors have degenerated, thus demonstrating that circadian rhythmicity in the mammalian retina can be generated independently from the photoreceptors. The aim of the present study was to investigate the pattern of expression of the clock genes in the retina of the Royal College of Surgeons rat under different lighting conditions. Expression of clock genes was investigated in the retina of normal and dystrophic Royal College of Surgeons rats under 12 h of light/12 h of dark (LD), constant darkness (DD) and constant light (LL) using Real Time Quantitative RT-PCR. Our data indicate that, in control animals, Period1, Period2, Cryptochrome1, Cryptochrome2, Clock, Rora, Rev-Erb alpha and Npas2 mRNA levels showed a significant variation over the sampling period in LD cycles and in DD, whereas Bmal1 mRNA did not show any significant variation. In LL, the transcripts for Per1, Per2, Clock and Rev-Erb alpha showed significant temporal variations. In the dystrophic retina, only Per1 and Per2 mRNA levels showed a temporal variation over the 20-h period. Our work indicates that degeneration of the photoreceptor cells dramatically affected the expression levels and patterns of many clock genes. Finally, the present study suggests that investigating the expression pattern of clock genes using the whole retina or animals with photoreceptor degeneration may not provide any definitive answers about the working of the retinal circadian clock system.

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    • "The retina was the first tissue outside the SCN to be shown to harbor a circadian clock, based on the ability of hamster retina cultures to display an autonomous and light-entrained rhythm of melatonin synthesis [7]. Accordingly, several clock genes analyzed at the level of the whole retina in vivo display circadian rhythmic patterns [16-21]. In addition, the Bmal1 gene was shown to be indispensable in the eye for optimal gene expression rhythms [14]. "
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    ABSTRACT: Purpose Circadian rhythms are central to vision and retinal physiology. A circadian clock located within the retina controls various rhythmic processes including melatonin synthesis in photoreceptors. In the present study, we evaluated the rhythmic expression of clock genes and clock output genes in retinal explants maintained for several days in darkness. Methods Retinas were dissected from Wistar rats, either wild-type or from the Per1-luciferase transgenic line housed under a daily 12 h:12 h light-dark cycle (LD12/12), and put in culture at zeitgeber time (ZT) 12 on semipermeable membranes. Explants from wild-type rats were collected every 4 h over 3 days, and total RNA was extracted, quantified, and reverse transcribed. Gene expression was assessed with quantitative PCR, and the periodicity of the relative mRNA amounts was assessed with nonlinear least squares fitting to sine wave functions. Bioluminescence in explants from Per1-luciferase rats was monitored for several days under three different culture protocols. Results Rhythmic expression was found for all studied clock genes and for clock downstream targets such as c-fos and arylalkylamine N-acetyltransferase (Aanat) genes. Clock and output genes cycled with relatively similar periods and acrophases (peaks of expression during subjective night, except c-fos, which peaked around the end of the subjective day). Data for Per1 were confirmed with bioluminescence monitoring, which also permitted culture conditions to be optimized to study the retina clock. Conclusions Our work shows the free-running expression profile of multiple clock genes and potential clock targets in mammalian retinal explants. This research further strengthens the notion that the retina contains a self-sustained oscillator that can be functionally characterized in organotypic culture.
    Molecular vision 06/2014; 20:742-52. · 1.99 Impact Factor
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    • "The ocular-mediated rhythm in SCN core NMDA and PAC1 receptor activation implies the existence of a retinal circadian oscillator that regulates the output of ipRGCs that project to the caudal SCN core. Indeed, it is well-established that a self-sustaining circadian oscillator is located in the eye [51], [52], [53], [54], [55], and circadian clock gene expression has been observed in most retinal cell types, including ganglion cells [56], [57], [58]. However, it remains unclear if ipRGCs are intrinsically rhythmic or if rhythms in these cells are induced by oscillators located elsewhere in the retina. "
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    ABSTRACT: The "core" region of the suprachiasmatic nucleus (SCN), a central clock responsible for coordinating circadian rhythms, shows a daily rhythm in phosphorylation of extracellular regulated kinase (pERK). This cellular rhythm persists under constant darkness and, despite the absence of light, is dependent upon inputs from the eye. The neural signals driving this rhythmicity remain unknown and here the roles of glutamate and PACAP are examined. First, rhythmic phosphorylation of the NR1 NMDA receptor subunit (pNR1, a marker for receptor activation) was shown to coincide with SCN core pERK, with a peak at circadian time (CT) 16. Enucleation and intraocular TTX administration attenuated the peak in the pERK and pNR1 rhythms, demonstrating that activation of the NMDA receptor and ERK in the SCN core at CT16 are dependent on retinal inputs. In contrast, ERK and NR1 phosphorylation in the SCN shell region were unaffected by these treatments. Intraventricular administration of the NMDA receptor antagonist MK-801 also attenuated the peak in SCN core pERK, indicating that ERK phosphorylation in this region requires NMDA receptor activation. As PACAP is implicated in photic entrainment and is known to modulate glutamate signaling, the effects of a PAC1 receptor antagonist (PACAP 6-38) on SCN core pERK and pNR1 also were examined. PACAP 6-38 administration attenuated SCN core pERK and pNR1, suggesting that PACAP induces pERK directly, and indirectly via a modulation of NMDA receptor signaling. Together, these data indicate that, in the absence of light, retinal-mediated NMDA and PAC1 receptor activation interact to induce cellular rhythms in the SCN core. These results highlight a novel function for glutamate and PACAP release in the hamster SCN apart from their well-known roles in the induction of photic circadian clock resetting.
    PLoS ONE 10/2013; 8(10):e76365. DOI:10.1371/journal.pone.0076365 · 3.23 Impact Factor
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    • "Within the mammalian retina, quantitative analyses of retinal clock gene expression as a function of daily hour have been performed for Mus musculus and Rattus norvegicus. There is considerable variation among published reports with respect to the rhythmicity of retinal clock gene expression, with some studies indicating cyclic expression of Bmal1 [30,44,45], Per1 [44,46-49], Cry1 [31,44,48,50], and Cry2 [30,44,48,50], and others reporting no rhythmic expression of the same transcription factors (Bmal1 [31,47,51]; Per1 [30,31,52]; Cry1 [30,53]; Cry2 [31,53]). Per2 was persistently seen as rhythmic in all studies, although some authors [54] were unable to demonstrate significant variations for any clock gene within the retina, once corrected for expression levels (which were high in the retina compared to the heart or liver). "
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    ABSTRACT: Prolonged periods of constant lighting are known to perturb circadian clock function at the molecular, physiological, and behavioral levels. However, the effects of ambient lighting regimes on clock gene expression and clock outputs in retinal photoreceptors-rods, cones and intrinsically photosensitive retinal ganglion cells-are only poorly understood. Cone-rich diurnal rodents (Muridae: Arvicanthis ansorgei) were maintained under and entrained to a 12 h:12 h light-dark cycle (LD; light: ~300 lux). Three groups were then examined: control (continued maintenance on LD); animals exposed to a 36 h dark period before sampling over an additional 24 h period of darkness (DD); and animals exposed to a 36 h light period before sampling over an additional 24 h period of light (~300 lux, LL). Animals were killed every 3 or 4 h over 24 h, their retinas dissected, and RNA extracted. Oligonucleotide primers were designed for the Arvicanthis clock genes Per1, Per2, Cry1, Cry2, and Bmal1, and for transcripts specific for rods (rhodopsin), cones (short- and mid-wavelength sensitive cone opsin, cone arrestin, arylalkylamine N-acetyltransferase) and intrinsically photosensitive retinal ganglion cells (melanopsin). Gene expression was analyzed by real-time PCR. In LD, expression of all genes except cone arrestin was rhythmic and coordinated, with acrophases of most genes at or shortly following the time of lights on (defined as zeitgeber time 0). Arylalkylamine N-acetyltransferase showed maximal expression at zeitgeber time 20. In DD conditions the respective profiles showed similar phase profiles, but were mostly attenuated in amplitude, or in the case of melanopsin, did not retain rhythmic expression. In LL, however, the expression profiles of all clock genes and most putative output genes were greatly altered, with either abolition of daily variation (mid-wavelength cone opsin) or peak expression shifted by 4-10 h. These data are the first to provide detailed measures of retinal clock gene and putative clock output gene expression in a diurnal mammal, and show the highly disruptive effects of inappropriate (nocturnal) lighting on circadian and photoreceptor gene regulation.
    Molecular vision 05/2013; 19:1060-73. · 1.99 Impact Factor
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