Circadian rhythms in the retina of rats with photoreceptor degeneration

Previous studies have demonstrated that the mammalian retina contains a circadian clock system that controls several retinal functions. In mammals the location of the retinal circadian clock is unknown whereas, in non-mammalian vertebrates, earlier work has demonstrated that photoreceptor cells contain the circadian clock. New experimental evidence has suggested that in mammals the retinal circadian clock may be located outside the photoreceptor cells. In this study we report that circadian rhythms in Aa-nat mRNA (in vivo) and melatonin synthesis (in vitro) are still present in the retina of rats lacking photoreceptors. The circadian pacemaker(s) controlling such rhythms is probably located in kainic acid sensitive neurons in the inner retina since kainic acid injections abolished the rhythmicity. These data are the first direct demonstration that circadian rhythmicity in the mammalian retina can be generated independently from the photoreceptors and the suprachiasmatic nuclei of the hypothalamus.     

Heterogeneous Expression of the Core Circadian Clock Proteins among Neuronal Cell Types in Mouse Retina

Circadian rhythms in metabolism, physiology, and behavior originate from cell-autonomous circadian clocks located in many organs and structures throughout the body and that share a common molecular mechanism based on the clock genes and their protein products. In the mammalian neural retina, despite evidence supporting the presence of several circadian clocks regulating many facets of retinal physiology and function, the exact cellular location and genetic signature of the retinal clock cells remain largely unknown. Here we examined the expression of the core circadian clock proteins CLOCK, BMAL1, NPAS2, PERIOD 1(PER1), PERIOD 2 (PER2), and CRYPTOCHROME2 (CRY2) in identified neurons of the mouse retina during daily and circadian cycles. We found concurrent clock protein expression in most retinal neurons, including cone photoreceptors, dopaminergic amacrine cells, and melanopsin-expressing intrinsically photosensitive ganglion cells. Remarkably, diurnal and circadian rhythms of expression of all clock proteins were observed in the cones whereas only CRY2 expression was found to be rhythmic in the dopaminergic amacrine cells. Only a low level of expression of the clock proteins was detected in the rods at any time of the daily or circadian cycle. Our observations provide evidence that cones and not rods are cell-autonomous circadian clocks and reveal an important disparity in the expression of the core clock components among neuronal cell types. We propose that the overall temporal architecture of the mammalian retina does not result from the synchronous activity of pervasive identical clocks but rather reflects the cellular and regional heterogeneity in clock function within retinal tissue.     

The mammalian retina as a clock

Many physiological, cellular, and biochemical parameters in the retina of vertebrates show daily rhythms that, in many cases, also persist under constant conditions. This demonstrates that they are driven by a circadian pacemaker. The presence of an autonomous circadian clock in the retina of vertebrates was first demonstrated in Xenopus laevis and then, several years later, in mammals. In X. laevis and in chicken, the retinal circadian pacemaker has been localized in the photoreceptor layer, whereas in mammals, such information is not yet available. Recent advances in molecular techniques have led to the identification of a group of genes that are believed to constitute the molecular core of the circadian clock. These genes are expressed in the retina, although with a slightly different 24-h profile from that observed in the central circadian pacemaker. This result suggests that some difference (at the molecular level) may exist between the retinal clock and the clock located in the suprachiasmatic nuclei of hypothalamus. The present review will focus on the current knowledge of the retinal rhythmicity and the mechanisms responsible for its control.     

Circadian clocks regulate adenylyl cyclase activity rhythms in human RPE cells

Genes and components of the circadian clock may represent relevant drug targets for diseases involving circadian dysfunctions. By exploiting an established cell line derived from human retinal pigment epithelium (HRPE), the cell constituting the blood-retinal barrier that is essential to maintain the visual functions of the sensorineural retina, we showed serum-shock induction of rhythmic changes in forskolin-evoked adenylyl cyclase (AC) activity. In the presence of Ca2+ and protein kinase A, the forskolin-induced AC activity is significantly, but not completely inhibited, suggesting the involvement of both Ca2+-sensitive and Ca2+-insensitive AC isoforms in the regulation of circadian rhythmicity in these cells. Semi-quantitative RT-PCR showed circadian profile in the expression of three AC isoforms, the Ca2+-inhibitable AC5 and AC6 and the Ca2+-insensitive AC7, and the clock genes hPer1 and hPer2. Our results demonstrate for the first time circadian rhythmicity in a human cell line, identifying the isoforms involved in the circadian profile of AC activity and showing a rhythmicity of the clock gene mRNA expression in these cells. Therefore, the results reported here provide evidence for an intertwine between AC/[Ca2+]i signalling pathways and Per genes in the HRPE circadian clockwork.     

The circadian clock system in the mammalian retina

Daily rhythms are a ubiquitous feature of living systems. Generally, these rhythms are not just passive consequences of cyclic fluctuations in the environment, but instead originate within the organism. In mammals, including humans, the master pacemaker controlling 24-hour rhythms is localized in the suprachiasmatic nuclei of the hypothalamus. This circadian clock is responsible for the temporal organization of a wide variety of functions, ranging from sleep and food intake, to physiological measures such as body temperature, heart rate and hormone release. The retinal circadian clock was the first extra-SCN circadian oscillator to be discovered in mammals and several studies have now demonstrated that many of the physiological, cellular and molecular rhythms that are present within the retina are under the control of a retinal circadian clock, or more likely a network of hierarchically organized circadian clocks that are present within this tissue. BioEssays 30:624-633, 2008. (c) 2008 Wiley Periodicals, Inc.     

Dynamic proteomic analysis reveals diurnal homeostasis of key pathways in rice leaves

Diurnal physiological acclimation regulated by a circadian system is an advantage for plant fitness. The circadian system is composed of a signal input, the clock and output pathways. Understanding the regulation mechanism of the output pathways remains a major challenge. Diurnal proteomic change reflects the state of circadian organization. We found the content of glucose, fructose, sucrose and starch diurnally changed in leaves of rice seedlings grown under a 12-h light/12-h dark condition with constant temperature. Dynamic proteomics analysis revealed 140 protein spots with diurnally changed levels at six times of the light/dark cycle; 132 spots were identified by MS, and 119 spots were of a single protein each with functional annotation. These proteins are involved in regulation of carbohydrate flow, redox, protein folding, nitrogen and protein metabolism, energy conversion, photorespiration and photosynthesis. Of these proteins, 81.5% were upregulated during the light phase, overlappingly, 41.2% showed behavior of circadian anticipation to dawn. Pattern analysis showed that the diurnal regulation involved pathways of allocation of carbohydrates between temporary reserves and consumption, maintenance of redox homeostasis, diurnal protein reassembly and nitrogen assimilation. These pathways reflect biochemical phenotypes of the circadian change linking the oscillator and circadian outputs.     

Antibodies for assessing circadian clock proteins in the rodent suprachiasmatic nucleus

Research on the mechanisms underlying circadian rhythmicity and the response of brain and body clocks to environmental and physiological challenges requires assessing levels of circadian clock proteins. Too often, however, it is difficult to acquire antibodies that specifically and reliably label these proteins. Many of these antibodies also lack appropriate validation. The goal of this project was to generate and characterize antibodies against several circadian clock proteins. We examined mice and hamsters at peak and trough times of clock protein expression in the suprachiasmatic nucleus (SCN). In addition, we confirmed specificity by testing the antibodies on mice with targeted disruption of the relevant genes. Our results identify antibodies against PER1, PER2, BMAL1 and CLOCK that are useful for assessing circadian clock proteins in the SCN by immunocytochemistry.     

Circadian Regulation of Melatonin in the Retina of Xenopus laevis: Limitation by Serotonin Availability

Treatments expected to increase retinal serotonin levels were found to stimulate melatonin production by cultured eyecups from Xenopus laevis. The monoamine oxidase inhibitor pargyline (100 microM) caused a sixfold increase in melatonin release, and the serotonin precursor 5-hydroxy-L-tryptophan (100 microM) caused a 70-fold increase. Both acted synergistically with eserine, an inhibitor of melatonin deacetylation in the retina. The effect of 5-hydroxytryptophan was dose dependent, with effects increasing from 1 to 100 microM. Increasing the tryptophan level in the culture medium had no effect on melatonin release. These results indicate that the rate-limiting step in retinal melatonin synthesis is 5-hydroxylation of tryptophan. Melatonin released from individual eyecups in superfusion culture in constant darkness with and without added 5-hydroxy-L-tryptophan was monitored over a 5-day period. Control eyecups released low levels of melatonin, with circadian rhythmicity persisting for 1-3 days. With 5-hydroxy-L-tryptophan added, melatonin levels were elevated 10-20-fold at all times, and rhythmicity was apparent for as long as five cycles. This provides a model system for studies of the circadian clock in the eye.     

Differential contribution of rod and cone circadian clocks in driving retinal melatonin rhythms in Xenopus

Background

Although an endogenous circadian clock located in the retinal photoreceptor layer governs various physiological events including melatonin rhythms in Xenopus laevis, it remains unknown which of the photoreceptors, rod and/or cone, is responsible for the circadian regulation of melatonin release.

Methodology/Principal Findings

We selectively disrupted circadian clock function in either the rod or cone photoreceptor cells by generating transgenic Xenopus tadpoles expressing a dominant-negative CLOCK (XCLΔQ) under the control of a rod or cone-specific promoter. Eyecup culture and continuous melatonin measurement revealed that circadian rhythms of melatonin release were abolished in a majority of the rod-specific XCLΔQ transgenic tadpoles, although the percentage of arrhythmia was lower than that of transgenic tadpole eyes expressing XCLΔQ in both rods and cones. In contrast, whereas a higher percentage of arrhythmia was observed in the eyes of the cone-specific XCLΔQ transgenic tadpoles compare to wild-type counterparts, the rate was significantly lower than in rod-specific transgenics. The levels of the transgene expression were comparable between these two different types of transgenics. In addition, the average overall melatonin levels were not changed in the arrhythmic eyes, suggesting that CLOCK does not affect absolute levels of melatonin, only its temporal expression pattern.

Conclusions/Significance

These results suggest that although the Xenopus retina is made up of approximately equal numbers of rods and cones, the circadian clocks in the rod cells play a dominant role in driving circadian melatonin rhythmicity in the Xenopus retina, although some contribution of the clock in cone cells cannot be excluded.     

Circadian activation of bullfrog retinal mitogen-activated protein kinase associates with oscillator function

The vertebrate retina retains a circadian oscillator, and its oscillation is self-sustained with a period close to 24 h under constant environmental conditions. Here we show that bullfrog retinal mitogen-activated protein kinase (MAPK) exhibits an in vivo circadian rhythm in phosphorylation with a peak at night in a light/dark cycle. The phosphorylation rhythm of MAPK persists in constant darkness with a peak at subjective night, and this self-sustained rhythm is also observed in cultured retinas, indicating its close interaction with the retinal oscillator. The rhythmically phosphorylated MAPK is detected only in a discrete subset of amacrine cells despite ubiquitous distribution of MAPK throughout the retinal layers. Treatment of the cultured retinas with MAPK kinase (MEK) inhibitor PD98059 suppresses MAPK phosphorylation during the subjective night, and this pulse perturbation of MEK activity induces a significant phase delay (4-8 h) of the retinal circadian rhythm in MAPK and MEK phosphorylation. These observations strongly suggest that the site-specific and time-of-day-specific activation of MAPK contributes to the circadian time-keeping mechanism of the retinal clock system.     

Early development of circadian rhythmicity in the suprachiamatic nuclei and pineal gland of teleost,flounder (Paralichthys olivaeus), embryos

Circadian rhythms enable organisms to coordinate multiple physiological processes and behaviors with the earth's rotation. In mammals, the suprachiasmatic nuclei (SCN), the sole master circadian pacemaker, has entrainment mechanisms that set the circadian rhythm to a 24‐h cycle with photic signals from retina. In contrast, the zebrafish SCN is not a circadian pacemaker, instead the pineal gland (PG) houses the major circadian oscillator. The SCN of flounder larvae, unlike that of zebrafish, however, expresses per2 with a rhythmicity of daytime/ON and nighttime/OFF. Here, we examined whether the rhythm of per2 expression in the flounder SCN represents the molecular clock. We also examined early development of the circadian rhythmicity in the SCN and PG. Our three major findings were as follows. First, rhythmic per2 expression in the SCN was maintained under 24 h dark (DD) conditions, indicating that a molecular clock exists in the flounder SCN. Second, onset of circadian rhythmicity in the SCN preceded that in the PG. Third, both 24 h light (LL) and DD conditions deeply affected the development of circadian rhythmicity in the SCN and PG. This is the first report dealing with the early development of circadian rhythmicity in the SCN in fish.