Gregory M. Cahill

Circadian clock functions localized in xenopus retinal photoreceptors.

A circadian oscillator that regulates visual function is located somewhere within the vertebrate eye. To determine whether circadian rhythmicity is generated by retinal photoreceptors, we isolated and cultured photoreceptor layers from Xenopus retina. On average, 94% of the viable cells in these preparations were rod or cone photoreceptors. Photoreceptor layers produced melatonin rhythmically, with an average period of 24.3 hr, in constant darkness. The phase of the melatonin rhythm was reset by in vitro exposure of the photoreceptor layers to cycles of either light or quinpirole, a D2 dopamine receptor agonist. These data indicate that other parts of the eye are not necessary for generation or entrainment of retinal circadian melatonin rhythms and suggest that rod and/or cone photoreceptors are circadian clock cells.

Clock mechanisms in zebrafish.

Abstract. Recent research on the circadian system of the zebrafish is reviewed. This teleost has become an attractive model system because of its advantages for genetic analyses. Circadian rhythms of zebrafish behavior, visual system function, and pineal melatonin synthesis have been described, and behavioral and pineal rhythms are being used to identify and characterize clock mutants. Zebrafish heart, kidney, and embryonic cell lines contain circadian oscillators and phototransduction mechanisms for entrainment, suggesting that circadian pacemaking functions may be distributed throughout the animal. Studies of circadian system development in zebrafish have found that a molecular circadian oscillation in unfertilized oocytes persists through embryonic development with its phase intact, but that the pacemakers that drive rhythms of melatonin synthesis and behavior require environmental entraining signals late in development for initial synchronization. Zebrafish homologs of several of the core clock genes identified in other animals have been cloned. Transcripts for most of these are rhythmically expressed in multiple tissues. The interactions of clock gene products are for the most part similar to their interactions in mammals, although there are some potentially interesting differences.

Circadian rhythmicity in vertebrate retinas: Regulation by a photoreceptor oscillator

Abstract Circadian regulation of retinal function has now been shown in all classes of vertebrates and attempts are now under way to understand the mechanisms underlying this regulation. The functions and organization of retinal rhythm mechanisms seem to vary among species and types of rhythms. To some extent this reflects the experimental advantages of different systems, but it also seems to reflect real variation in control mechanisms among species. Many aspects of retinal function, from gene transcription to complex intercellular interactions, are regulated by circadian oscillators (Fig. 8). Circadian clock regulation of the visual system ranges from dramatic, spontaneous rhythmicity in melatonin synthesis and disc shedding in some species to subtle rhythmic regulation of responsiveness to light and dark signals in other species. Most of the known retinal rhythms, as well as a circadian oscillator, are localized in photoreceptors. This suggests that circadian rhythmicity is fundamental for normal photoreceptor function. Some of the intracellular and paracrine components of the pathways linking different retinal rhythms have been identified. However, because of the complex feedback interactions present in the system, it is still difficult to draw strong conclusions about the causal relationships among rhythms. For example, melatonin is rhythmic and regulates dopamine release, but it is not clear that the rhythms in dopamine are driven directly by melatonin rhythms. Dopamine can modulate melatonin rhythms, but it is not necessary for generation of those rhythms (Cahill and Besharse, 1993). The recently discovered circadian regulation of photoreceptor gene expression (Pierce et al. , 1993; Yoshida et al. , 1993; Green and Besharse, 1994) may underlie rhythmicity at higher levels of organization. It will be important to determine which other photoreceptor genes are also regulated by circadian mechanisms and whether common factors are involved. Mechanisms of the photoreceptor oscillator itself are now approachable in a few experimental preparations, including Xenopus eyecups and photoreceptor layers and chick retinal cell cultures (Cahill and Besharse, 1991, 1993; Pierce et al. , 1993). These in vitro preparations eliminate systemic influences from the interpretation of experimental results. Furthermore, by measuring the timing of rhythmicity, it is possible to distinguish oscillator responses to experimental manipulations from acute changes in rhythmic variables. This is the key to unraveling the mechanisms underlying retinal circadian rhythmicity.

Circadian regulation of melatonin production in cultured zebrafish pineal and retina

Melatonin release was measured from zebrafish pineal organs and retinas maintained in flow-through culture. Pineal organs released melatonin in a strong circadian rhythm through 5 days in constant darkness, and the phase of this rhythm was reset by in vitro exposure to phase-shifted light cycles. In contrast, the retinal melatonin rhythm rapidly damped out in constant darkness, even in the presence of (phase-shifted) light cycles. The zebrafish pineal should be useful for in vitro studies of vertebrate circadian clock mechanisms.

Rhythmic regulation of retinal melatonin: Metabolic pathways, neurochemical mechanisms, and the ocular circadian clock

Summary1.Current knowledge of the mechanisms of circadian and photic regulation of retinal melatonin in vertebrates is reviewed, with a focus on recent progress and unanswered questions.2.Retinal melatonin synthesis is elevated at night, as a result of acute suppression by light and rhythmic regulation by a circadian oscillator, or clock, which has been localized to the eye in some species.3.The development of suitablein vitro retinal preparations, particularly the eyecup from the African clawed frog,Xenopus laevis, has enabled identification of neural, cellular, and molecular mechanisms of retinal melatonin regulation.4.Recent findings indicate that retinal melatonin levels can be regulated at multiple points in indoleamine metabolic pathways, including synthesis and availability of the precursor serotonin, activity of the enzyme serotoninN-acetyltransferase, and a novel pathway for degradation of melatonin within the retina.5.Retinal dopamine appears to act through D2 receptors as a signal for light in this system, both in the acute suppression of melatonin synthesis and in the entrainment of the ocular circadian oscillator.6.A recently developedin vitro system that enables high-resolution measurement of retinal circadian rhythmicity for mechanistic analysis of the circadian oscillator is described, along with preliminary results that suggest its potential for elucidating general circadian mechanisms.7.A model describing hypothesized interactions among circadian, neurochemical, and cellular mechanisms in regulation of retinal melatonin is presented.

Light-sensitive melatonin synthesis by Xenopus photoreceptors after destruction of the inner retina.

Several lines of evidence indicate that retinal photoreceptors produce melatonin. However, there are other potential melatonin sources in the retina, and melatonin synthesis can be regulated by feedback from the inner retina. To analyze cellular mechanisms of melatonin regulation in retinal photoreceptors, we have developed an in vitro method for destruction of the inner retina that preserves functional photoreceptors in contact with the pigment epithelium. Eyecups, which include the neural retina, retinal pigment epithelium, choriod, and sclera were prepared. The vitreal surface of the retina in each eyecup was washed sequentially with 1% Triton X-100, water, and culture medium. This lysed the ganglion cells and neurons and glia of the inner nuclear layer, causing the retina to split apart within the inner nuclear layer. The damaged inner retina was peeled away, leaving photoreceptors attached to the pigment epithelium. The cell density of the inner nuclear layer was reduced 94% by this method, but there was little apparent damage to the photoreceptors. Lesioned eyecups produced normal melatonin levels in darkness at night, and melatonin production was inhibited by light. These results indicate that the inner retina is not necessary for melatonin production nor for regulation of photoreceptor melatonin synthesis by light. The lesion method used in this study may be useful for other physiological and biochemical studies of photoreceptors.

Development of a circadian melatonin rhythm in embryonic zebrafish.

We investigated the time course of circadian system development in zebrafish and the role of environmental light cycles in this process, using a rhythm in melatonin content of embryos and larvae as a marker of circadian function. When zebrafish were raised in a cycle of 14 h light and 10 h dark at 28.5 degrees C, nocturnal increases in melatonin content were detectable beginning on the second night post-fertilization (PF). When embryos were transferred to constant darkness (DD) at the end of the second light period, a circadian rhythm of melatonin content persisted for at least three cycles. However, when embryos were transferred from light to DD at 14 h PF, no rhythm was detectable in the population. Phase-locked circadian melatonin rhythms were measurable after embryos were exposed to a transition from constant light (LL) to darkness at 26 or 32 h PF, but not at 20 h. These data indicate that a circadian oscillator that regulates melatonin synthesis becomes functional and responsive by light between 20 and 26 h PF. At this stage, pineal photoreceptors have begun to differentiate, but retinal photoreceptors have not, suggesting that the first circadian melatonin rhythms are of pineal origin. The absence of melatonin rhythms in populations of embryos exposed to DD beginning at earlier stages indicates that there is no timed developmental event that sets the circadian clock in the absence of environmental input. Exposure to DD starting at 14 or 20 h PF did not retard overall development as determined by gross morphological staging criteria, and did not prevent later synchronization of melatonin rhythms by light-dark (LD) cycles.

Melatonin suppresses nighttime memory formation in zebrafish.

Memory processes are modulated by the biological clock, although the mechanisms are unknown. Here, we report that in the diurnal zebrafish both learning and memory formation of an operant conditioning paradigm occur better during the day than during the night. Melatonin treatment during the day mimics the nighttime suppression of memory formation. Training in constant light improves nighttime memory formation while reducing endogenous melatonin concentrations. Treatment with melatonin receptor antagonists at night dramatically improves memory. Pinealectomy also significantly improves nighttime memory formation. We adduce that melatonin is both sufficient and necessary for poor memory formation during the night.

Light induction of a vertebrate clock gene involves signaling through blue-light receptors and MAP kinases.

The signaling pathways that couple light photoreception to entrainment of the circadian clock have yet to be deciphered. Two prominent groups of candidates for the circadian photoreceptors are opsins (e.g., melanopsin) and blue-light photoreceptors (e.g., cryptochromes). We have previously showed that the zebrafish is an ideal model organism in which to study circadian regulation and light response in peripheral tissues. Here, we used the light-responsive zebrafish cell line Z3 to dissect the response of the clock gene zPer2 to light. We show that the MAPK (mitogen-activated protein kinase) pathway is essential for this response, although other signaling pathways may also play a role. Moreover, action spectrum analyses of zPer2 transcriptional response to monochromatic light demonstrate the involvement of a blue-light photoreceptor. The Cry1b and Cry3 cryptochromes constitute attractive candidates as photoreceptors in this setting. Our results establish a link between blue-light photoreceptors, probably cryptochromes, and the MAPK pathway to elicit light-induced transcriptional activation of clock genes.

Transcripts Encoding Two Melatonin Synthesis Enzymes in the Teleost Pineal Organ: Circadian Regulation in Pike and Zebrafish, But Not in Trout1

In this report the photosensitive teleost pineal organ was studied in three teleosts, in which melatonin production is known to exhibit a daily rhythm with higher levels at night; in pike and zebrafish this increase is driven by a pineal clock, whereas in trout it occurs exclusively in response to darkness. Here we investigated the regulation of messenger RNA (mRNA) encoding serotonin N-acetyltransferase (AA-NAT), the penultimate enzyme in melatonin synthesis, which is thought to be primarily responsible for changes in melatonin production. AA-NAT mRNA was found in the pineal organ of all three species and in the zebrafish retina. A rhythm in AA-NAT mRNA occurs in vivo in the pike pineal organ in a light/dark (L/D) lighting environment, in constant lighting (L/L), or in constant darkness (D/D) and in vitro in the zebrafish pineal organ in L/D and L/L, indicating that these transcripts are regulated by a circadian clock. In contrast, trout pineal AA-NAT mRNA levels are stable in vivo and in vitro in L/D, L...