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.     

Cyclic AMP resets the circadian clock in cultured Xenopus retinal photoreceptor layers

The Xenopus retinal photoreceptor layer contains a circadian oscillator that regulates melatonin synthesis in vitro. The phase of this oscillator can be reset by light or dopamine. The phase-response curves for light and dopamine are similar, with transitions from phase delays to phase advances in the mid-subjective night. Light and dopamine each can inhibit adenylate cyclase in retinal photoreceptors, suggesting cyclic AMP as a candidate second messenger for entrainment of the circadian oscillator. We report here that treatments that increase intracellular cyclic AMP reset the phase of the photoreceptor circadian oscillator, and that the phase-response curves for these treatments are 180 degrees out of phase with the phase-response curves for light and dopamine. Activation of adenylate cyclase by forskolin during the late subjective day or early subjective night caused phase advances. The same treatment during the late subjective night or early subjective day caused phase delays. Similar phase shifts were induced by 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) or 8-(4-chlorophenylthio)cyclic AMP. All of these treatments also acutely increased melatonin release. Forskolin and 3-isobutyl-1-methylxanthine increased the accumulation of intracellular cyclic AMP, but not cyclic GMP, in photoreceptor layers. The results indicate that cyclic AMP-dependent pathways regulate the photoreceptor circadian oscillator and suggest that a decrease in cyclic AMP may be involved in circadian entrainment by light and/or dopamine.     

Group interviewing for the purpose of selection

Relation between retinal melatonin and corneal mitotic rhythms in the Japanese quail was investigated in experiments manipulating the ocular physiology by treatments with formoguanamine hydrochloride (FG) and eye-lid suture. In experiment 1, we investigated the effects of FG, which is known to induce photoreceptor degeneration, on retinal melatonin and corneal mitotic rhythms. FG-treatment completely abolished the retinal melatonin rhythms in both LD 12:12 and constant darkness (DD), but the corneal mitotic rhythm was maintained with high mitotic rate in darkness under a LD cycle and subjective night under DD. The result suggests that 1) the photoreceptor cells in the retina are the site for melatonin production and/or for the oscillator which drives the circadian rhythm in retinal melatonin, and 2) melatonin is not involved in generation of the corneal mitotic rhythm. In experiment 2, we investigated the effects of eye-lid suture, which is known to induce eye enlargement and bulgy cornea, on the retinal melatonin and corneal mitotic rhythms. Eye-lid suture abolished the corneal mitotic rhythm in both LD and DD, with a high mitotic rate being maintained throughout 24 hr. But retinal melatonin maintained its rhythm with high levels in darkness under a LD cycle and in subjective night under DD. The result suggests that 1) bulgy cornea in the sutured eye was induced by the increase in mitotic rate in the light period, and 2) disappearance of the corneal mitotic rhythm does not have a relation to retinal melatonin. These results suggest that retinal melatonin is not involved in generation of the corneal mitotic rhythm and that there are two circadian clock systems in the eye.     

Cellular and molecular regulation of serotonin N-acetyltransferase activity in chicken retinal photoreceptors

Serotonin N-acetyltransferase (AA-NAT; arylalkylamine N-acetyltransferase; EC 2.3.1.87) is the penultimate enzyme in melatonin synthesis and large changes in the activity of this enzyme appear to regulate the rhythm in melatonin synthesis. Recent advances have made it possible to study the mRNA encoding chicken AA-NAT, which has only been detected in the retina and pineal gland. Within the retina, AA-NAT mRNA is expressed primarily in photoreceptors. The levels of chicken retinal AA-NAT mRNA and activity exhibit 24-hour rhythms with peaks at night. These rhythms appear to reflect circadian clock control of AA-NAT mRNA abundance and independent effects of light and darkness on both mRNA levels and enzyme activity. The effects of darkness and light may occur through alterations in cAMP-dependent protein phosphorylation, which increases AA-NAT activity in photoreceptor cell cultures. The cAMP-dependent increase of AA-NAT enzyme activity reflects, at least in part, increased mRNA levels and inhibition of enzyme inactivation by a posttranslational mechanism. This review discusses a hypothetical model for the cellular and molecular regulation of AA-NAT activity by circadian oscillators and light in chicken retinal photoreceptor cells.     

Serotonergic innervation of the hypothalamic suprachiasmatic nucleus and photic regulation of circadian rhythms

Converging lines of evidence have firmly established that the hypothalamic suprachiasmatic nucleus (SCN) is a light-entrainable circadian oscillator in mammals, critically important for the expression of behavioral and physiological circadian rhythms. Photic information essential for the daily phase resetting of the SCN circadian clock is conveyed directly to the SCN from retinal ganglion cells via the retinohypothalamic tract. The SCN also receives a dense serotonergic innervation arising from the mesencephalic raphe. The terminal fields of retinal and serotonergic afferents within the SCN are co-extensive, and serotonergic agonists can modify the response of the SCN circadian oscillator to light. However, the functional organization and subcellular localization of 5HT receptor subtypes in the SCN are just beginning to be clarified. This information is necessary to understand the role 5HT afferents play in modulating photic input to the SCN. In this paper, we review evidence suggesting that the serotonergic modulation of retinohypothalamic neurotransmission may be achieved via at least two different cellular mechanisms: 1) a postsynaptic mechanism mediated via 5HT1A or 5ht7 receptors located on SCN neurons; and 2) a presynaptic mechanism mediated via 5HT1B receptors located on retinal axon terminals in the SCN. Activation of either of these 5HT receptor mechanisms in the SCN by specific 5HT agonists inhibits the effects of light on circadian function. We hypothesize that 5HT modulation of photic input to the SCN may serve to set the gain of the SCN circadian system to light.     

Neuropeptidergic organization of the suprachiasmatic nucleus in the blind mole rat (Spalax ehrenbergi)

The blind mole rat, Spalax, is a subterranean rodent with atrophied, subcutaneous eyes. Whereas most of the visual system is highly degenerated, the retino-hypothalamic pathway in this species has remained intact. Although Spalax is considered to be visually blind, circadian locomotor rhythms are entrained by the light/dark cycle. In the present study we used anterograde tracing techniques to demonstrate retinal afferents to the suprachiasmatic nucleus (SCN) and immunohistochemistry to examine the distribution of neuropeptides that are known to be involved in the regulation or expression of circadian rhythmicity. Based on the localization of retinal afferents and neuropeptides, the SCN can be divided into two subdivisions. The ventral region, which receives retinal afferents, also contains vasoactive intestinal polypeptide (VIP)-containing neurons, and fibers that are immunopositive to neuropeptide Y (NPY) and serotonin (5-HT). The dorsal region contains vasopressinergic neurons, but this latter cell population is extremely sparse compared to that described in other rodents. The dorsal region is also characterized by numerous VIP-immunoreactive fibers. The presence of NPY and 5-HT fibers suggests that the SCN receives afferent projections from the intergeniculate leaflet and from the raphe nuclei, respectively. These neuroanatomical results, together with previous studies of behavior, visual tract tracing, and immediate early gene expression, confirm that an endogenous clock and the capacity for light entrainment of circadian rhythms are conserved in the blind mole rat.     

Subclavian venogram as a guide to lead implantation

Melatonin synthesis in retinal photoreceptors is stimulated at night by a circadian oscillator and suppressed acutely by light. To identify photoreceptor mechanisms involved in the acute suppression of melatonin synthesis, an action spectrum was measured for dark-adapted Xenopus laevis eyecups at night. Intensity-response curves at six wavelengths from 400 to 650 nm were parallel, suggesting that a single photopigment predominates in melatonin suppression. Half-saturating intensities at 400, 440, 480, and 533 nm were not significantly different from one another, at 1-2 x 10(8) quanta cm(-2) s(-1). Significantly higher intensities of 580- and 650-nm light were required for melatonin suppression. These results indicate a predominant role for the principal green-absorbing rods in acute regulation of retinal melatonin synthesis in response to light, and argue against an important role for the red-absorbing cones. Higher than expected sensitivity at short wavelengths suggests that photoreceptors sensitive to blue and/or violet light may also contribute to melatonin suppression.     

An improved sample packing device for rapid freeze-trap electron paramagnetic resonance spectroscopy kinetic measurements

Circadian rhythms are generated by the suprachiasmatic nuclei (SCN) and synchronized (entrained) to environmental light-dark cycles by the retinohypothalamic tract (RHT), a direct pathway from the retina to the suprachiasmatic nuclei. In anophthalmic mice, the optic primordia are resorbed between embryonic days 11.5 and 13, before retinal ganglion cells emerge. Thus the retinohypothalamic tract, which is the primary "zeitgeber" for circadian rhythms in sighted animals, never forms, and there is no retinal or photic input to the circadian system. We have used wheel running activity, a highly consistent and reliable measure of circadian rhythmicity in rodents, to establish the properties of endogenous locomotor rhythms of anophthalmic mice. We have identified three subpopulations of anophthalmic mice: a) rhythmic with strong stable circadian period but significantly increased period length; b) rhythmic with unstable circadian period; and c) arrhythmic. Future correlation of locomotor rhythms with properties of the suprachiasmatic nuclei in these mice will clarify the relationship between generation and properties of circadian rhythms and the neuroanatomical, neurochemical, and molecular organization of the circadian clock.     

Circadian rhythms in cultured mammalian retina

Many retinal functions are circadian, but in most instances the location of the clock that drives the rhythm is not known. Cultured neural retinas of the golden hamster (Mesocricetus auratus) exhibited circadian rhythms of melatonin synthesis for at least 5 days at 27 degrees celsius. The rhythms were entrained by light cycles applied in vitro and were free-running in constant darkness. Retinas from hamsters homozygous for the circadian mutation tau, which shortens the free-running period of the circadian activity rhythm by 4 hours, showed a shortened free-running period of melatonin synthesis. The mammalian retina contains a genetically programmed circadian oscillator that regulates its synthesis of melatonin.     

Rapid resetting of the mammalian circadian clock

The suprachiasmatic nuclei (SCN) contain the principal circadian clock governing overt daily rhythms of physiology and behavior. The endogenous circadian cycle is entrained to the light/dark via direct glutamatergic retinal afferents to the SCN. To understand the molecular basis of entrainment, it is first necessary to define how rapidly the clock is reset by a light pulse. We used a two-pulse paradigm, in combination with cellular and behavioral analyses of SCN function, to explore the speed of resetting of the circadian oscillator in Syrian hamster and mouse. Analysis of c-fos induction and cAMP response element-binding protein phosphorylation in the retinorecipient SCN demonstrated that the SCN are able to resolve and respond to light pulses presented 1 or 2 hr apart. Analysis of the phase shifts of the circadian wheel-running activity rhythm of hamsters presented with single or double pulses demonstrated that resetting of the oscillator occurred within 2 hr. This was the case for both delaying and advancing phase shifts. Examination of delaying shifts in the mouse showed resetting within 2 hr and in addition showed that resetting is not completed within 1 hr of a light pulse. These results establish the temporal window within which to define the primary molecular mechanisms of circadian resetting in the mammal.     

What makes a mother? Interviews with women involved in egg donation and surrogacy

The fish pineal organ contains typical and, in some species, modified photoreceptor cells involved in the photoperiodic control of melatonin production. In the majority of species studied, the rhythm in melatonin production is driven by an intra-pineal circadian oscillator synchronized by the light:dark cycle. In the present study, it is shown that the endogenous rhythm in melatonin release of superfused pike pineals maintained under constant darkness is expressed at temperatures of 19 degrees C, 20 degrees C, 25 degrees C, and 30 degrees C (period > 24 hr), but not at temperatures of 10 degrees C and 15 degrees C. Under constant darkness, pineal fractions containing either typical photoreceptors, modified photoreceptors, or both behaved like total organs. Dissociated pike pineal cells, cultured statically at 20 degrees C, expressed a high amplitude rhythm in melatonin secretion under a light:dark cycle. Under constant darkness, circadian oscillations, which appeared better sustained than in organ culture, were also observed. This study provides the first evidence that the rhythmic production of melatonin, by a fish pineal, is driven by a population of circadian oscillators or clocks. It is hypothesized that each typical and modified photoreceptor might be the locus of a circadian clock. Damping of the overall rhythm under constant darkness might reflect the desynchronization (uncoupling) between these clocks and/or damping of individual oscillators.     

Circadian expression of tryptophan hydroxylase mRNA in the chicken retina

Many aspects of retinal physiology are controlled by a circadian clock located within the eye. This clock controls the rhythmic synthesis of melatonin, which results in elevated levels during the night and low levels during the day. The rate-limiting enzyme in melatonin biosynthesis in retina appears to be tryptophan hydroxylase (TPH)[G.M. Cahill and J.C. Besharse, Circadian regulation of melatonin in the retina of Xenopus laevis: Limitation by serotonin availability, J. Neurochem. 54 (1990) 716-719]. In this report, we found that TPH mRNA is strongly expressed in the photoreceptor layer and the vitread portion of the inner nuclear layer; the message is also expressed, but to a lesser extent, in the ganglion cell layer. The abundance of retinal TPH mRNA exhibits a circadian rhythm which persists in constant light or constant darkness. The phase of the rhythm can be reversed by reversing the light:dark cycle. In parallel experiments we found a similar pattern of expression in the chicken pineal gland. However, whereas a pulse of light at midnight suppressed retinal TPH mRNA by 25%, it did not alter pineal TPH mRNA, suggesting that there are tissue-specific differences in photic regulation of TPH mRNA. In retinas treated with kainic acid to destroy serotonin-containing amacrine and bipolar cells, a high amplitude rhythm of TPH mRNA was observed indicating that melatonin-synthesizing photoreceptors are the primary source of the rhythmic message. These observations provide the first evidence that chick retinal TPH mRNA is under control of a circadian clock.     

Tripartite hemolysin BL from Bacillus cereus. Hemolytic analysis of component interactions and a model for its characteristic paradoxical zone phenomenon

This study describes the use of the microdialysis technique to elucidate specific properties of the circadian pacemaking system in the hypothalamus, by measurement of melatonin production in the pineal gland. Melatonin has appeared to be a reliable marker of the pacemaker activity, which is influenced by the light/dark cycle. A phase shift in the light/dark cycle was applied to perturb the rhythm generating system. An 8-h phase advance resulted in the disappearance of melatonin production over two days, with basal levels comparable to normal daytime levels. In the subsequent return of rhythmic melatonin production, new clock characteristics could be revealed, due to the high time-resolution measurements of microdialysis. While half of the animals still did not show any rhythmicity, the other half of the animals regained rhythmicity with entrained onset of melatonin production, while the offset was variable and not stably entrained to lights on. Ten days after the shift, the system had completely recovered and all animals regained normal rhythmicity, in phase with the new light/dark cycle. The results are interpreted in terms of the two-oscillator model, with one oscillator reacting with a phase advance and the other with a phase delay to adapt to the phase shift.     

A chronobiological approach to circulating levels of renin, angiotensin-converting enzyme, aldosterone, ACTH, and cortisol in Addison's disease

This study deals with a chronobiological approach to the circadian rhythm of the renin-angiotensin-aldosterone system (RAAS) and the ACTH-cortisol axis (ACA) in patients with Addison's disease (PAD). The aim is to explore the mechanism(s) for which the circadian rhythmicity of the RAAS and ACA takes place. The study has shown that both the RAAS and ACA are devoid of a circadian rhythm in PAD. The lack of rhythmicity for renin and ACTH provides indirect evidence that their rhythmic secretion is in some way related to the circadian oscillation of aldosterone and cortisol. This implies a new concept: a positive feedback may be included among the mechanisms which chronoregulate the RAAS and ACA.     

Intraocular gene transfer of ciliary neurotrophic factor prevents death and increases responsiveness of rod photoreceptors in the retinal degeneration slow mouse

Several mutations causing both photoreceptor degeneration and malfunction have been identified in humans and animals. Although intraocular injection of trophic factors has been shown to reduce photoreceptor death in a few conditions of rapid photoreceptor loss, it is unclear whether long-term beneficial changes in functional properties of affected photoreceptors can be obtained by treatment with these factors. The rds/rds mouse is a spontaneous mutant bearing a null mutation in the rds/peripherin gene, which is linked to many forms of dominant retinal degenerations in humans. Here, we report that intraocular adenovirus-mediated gene transfer of ciliary neurotrophic factor (CNTF) in this mutant reduces photoreceptor loss, causes a significant increase in the length of photoreceptor segments, and results in a redistribution and an increase in the retinal content of the photopigment rhodopsin. These effects are accompanied by a significant increase in the amplitude of the a- and b-waves of the scotopic electroretinogram. These results suggest that continuous administration of CNTF could potentially be useful for the treatment of some forms of retinal degeneration.     

Differential sensitivity of membrane-associated pyrophosphatases to inhibition by diphosphonates and fluoride delineates two classes of enzyme

The participation of GABAergic mechanisms in the regulation of circadian rhythmicity by the suprachiasmatic nuclei (SCN) has been suggested from different lines of evidence. Little is known, however, whether GABA synthesis, release, uptake or content within the SCN may show a circadian pattern. The present results show that the activity of the GABAergic system within the SCN region of the rat exhibits circadian rhythmicity, which is manifested by correlative changes of the GABA content and the glutamic acid decarboxylase activity under the light/dark cycle, and by changes in the GABA content in animals kept under constant darkness.     

A myosin III from Limulus eyes is a clock-regulated phosphoprotein

The lateral eyes of the horseshoe crab Limulus polyphemus undergo dramatic daily changes in structure and function that lead to enhanced retinal sensitivity and responsiveness to light at night. These changes are controlled by a circadian neural input that alters photoreceptor and pigment cell shape, pigment migration, and phototransduction. Clock input to the eyes also regulates photomechanical movements within photoreceptors, including membrane shedding. The biochemical mechanisms underlying these diverse effects of the clock on the retina are unknown, but a major biochemical consequence of activating clock input to the eyes is a rise in the concentration of cAMP in photoreceptors and the phosphorylation of a 122 kDa visual system-specific protein. We have cloned and sequenced cDNA encoding the clock-regulated 122 kDa phosphoprotein and show here that it is a new member of the myosin III family. We report that Limulus myosin III is similar to other unconventional myosins in that it binds to calmodulin in the absence of Ca2+; it is novel in that it is phosphorylated within its myosin globular head, probably by cAMP-dependent protein kinase. The protein is present throughout the photoreceptor, including the region occupied by the photosensitive rhabdom. We propose that the phosphorylation of Limulus myosin III is involved in one or more of the structural and functional changes that occur in Limulus eyes in response to clock input.     

Phase control of ultradian feeding rhythms in the common vole (Microtus arvalis): the roles of light and the circadian system

In their ultradian (2- to 3-hr) feeding rhythm, common voles show intraindividual synchrony from day to day, as well as interindividual synchrony between members of the population, even at remote distances. This study addresses the question of how resetting of the ultradian rhythm, a prerequisite for such synchronization, is achieved. Common voles were subjected to short light-dark cycles (1 hr darkness with light varying between 0.7 and 2.5 hr); to T cycles (long light-dark cycles in the circadian range--16 hr darkness and 3-13 hr light); to light pulses (15 min) during different circadian and ultradian phases; and to addition of D2O to the drinking water (25%). Short light-dark cycles and D2O were also applied to voles without circadian rhythmicity, after lesions of the suprachiasmatic nuclei. In these experiments, four hypotheses on synchronization of ultradian rhythmicity were tested: (I) synchronization by a direct response to light; (II) synchronization via the circadian system with multiple triggers, here called "cogs," each controlling a single ultradian feeding bout; and (III and IV) synchronization via the circadian system with a single "cog," which resets an ultradian oscillator and either (III) originates directly from the circadian pacemaker, or (IV) is mediated via the overt circadian activity rhythm. Short light-dark cycles failed to entrain ultradian rhythms, either in circadian-rhythmic or in non-circadian-rhythmic voles; light pulses did not cause phase shifts; and in extreme T cycles no stable phase relationship with light could be demonstrated. Thus, Hypothesis I was rejected. Changes in the circadian period (tau) were generated as aftereffects of light pulses, by entrainment in various T cycles, and by the addition of D2O to the drinking water. These changes in tau did not lead to parallel, let alone proportional, changes in the ultradian period. This excluded Hypothesis II. Both in T-cycle experiments and in the D2O experiments with circadian-rhythmic voles, the phase of ultradian feeding bouts was locked to the end of circadian activity rather than to the most prominent marker of the pacemaker, the onset of circadian activity. This was not expected under Hypothesis III, but was consistent with entrainment via activity (Hypothesis IV). On the basis of these experiments, we conclude that the most likely mechanism of ultradian entrainment is that of a light-insensitive ultradian oscillator, reset every dawn by the termination of the activity phase controlled by the circadian pacemaker, which is itself entrained by the light-dark cycle. Neither in circadian-rhythmic nor in non-circadian-rhythmic voles was the period of the feeding rhythm lengthened by administration of D2O. This insensitivity to deuterium is exceptional among biological rhythms.