Photic Input Pathway Research Articles

Phosphatase of Regenerating Liver-1 Selectively Times Circadian Behavior in Darkness via Function in PDF Neurons and Dephosphorylation of TIMELESS

The timing of behavior under natural light-dark conditions is a function of circadian clocks and photic input pathways, but a mechanistic understanding of how these pathways collaborate in animals is lacking. Here we demonstrate in Drosophila that the Phosphatase of Regenerating Liver-1 (PRL-1) sets period length and behavioral phase gated by photic signals. PRL-1 knockdown in PDF clock neurons dramatically lengthens circadian period. PRL-1 mutants exhibit allele-specific interactions with the light- and clock-regulated gene timeless (tim). Moreover, we show that PRL-1 promotes TIM accumulation and dephosphorylation. Interestingly, the PRL-1 mutant period lengthening is suppressed in constant light, and PRL-1 mutants display a delayed phase under short, but not long, photoperiod conditions. Thus, our studies reveal that PRL-1-dependent dephosphorylation of TIM is a core mechanism of the clock that sets period length and phase in darkness, enabling the behavioral adjustment to change day-night cycles.

Clock Gene Expression in the Eye Exhibits Circadian Oscillation and Light Responsiveness but is Not Necessary for Nocturnal Locomotor Activity of Japanese Loach, Misgurnus anguillicaudatus.

There are few model fish that are both edible and suitable for use in the laboratory. The Japanese loach (Misgurnus anguillicaudatus) is a traditional food in Japan, but is highly neglected despite its great nutritional value. To understand its circadian system and photic input pathway for synchronization of physiological activities to environmental light-dark cycles, we measured locomotor activity under light-dark and constant dark (DD) conditions. Locomotor activity was found to be higher in the nighttime than daytime, and its rhythmicity was weakened under DD conditions. The nocturnal activity of the Japanese loach is mainly controlled by environmental light, rather than the circadian clock. We explored the circadian regulation and light-responsiveness of clock gene expression in the eyes of loaches. The daily expression profiles of its mRNA revealed that most of the examined Cry and Per genes were likely regulated by internal circadian and/or environmental light signals. Among the Opsin genes transcribed in the eye, we detected the retinal photopigment porphyropsin at the protein level, which was lower than in mice. This property of loach eyes prompted us to analyze the locomotor activities of eye-enucleated fish. As a result, they still showed nocturnal circadian activity. Thus, it is likely that extraocular photoreceptive tissue(s) also contribute to the photic input pathway, although loach eyes are a circadian photosensitive tissue. This suggests that the loach mainly uses not its vision but other stimuli, such as mechanical or chemical stimuli, detected by barbels, to coordinate its nocturnal behavior.

A photoperiodic time measurement served by the biphasic expression of Cryptochrome1ab in the zebrafish eye

The zebrafish (Danio rerio) is a model species that is used to study the circadian clock. It possesses light-entrainable circadian clocks in both central and peripheral tissues, and its core circadian factor cryptochromes (CRYs) have diverged significantly during evolution. In order to elucidate the functional diversity and involvement of CRYs in photoperiodic mechanisms, we investigated the daily expression profiles of six Cry transcripts in central (brain and eye) and peripheral (fin, skin and muscle) tissues. The zCry genes exhibited gene-specific diurnal conserved variations, and were divided into morning and evening groups. Notably, zCry1ab exhibited biphasic expression profiles in the eye, with peaks in the morning and evening. Comparing ocular zCry1ab expression in different photoperiods (18L:6D, 14L:10D, 10L:14D and 6L:18D) revealed that zCry1ab expression duration changed depending on the photoperiod: it increased at midnight and peaked before lights off. zCry1ab expression in constant light or dark after entrainment under long- or short-day conditions suggested that the evening clock and photic input pathway are involved in photoperiod-dependent zCry1ab expression. Laser microdissection followed by qRT-PCR analysis showed that the evening peak of zCry1ab was likely ascribed to visual photoreceptors. These results suggest the presence of an eye-specific photoperiodic time measurement served by zCry1ab.

Circadian Alterations in a Murine Model of Hypothalamic Glioma.

The mammalian circadian system is controlled by a central oscillator located in the suprachiasmatic nuclei (SCN) of the hypothalamus, in which glia appears to play a prominent role. Gliomas originate from glial cells and are the primary brain tumors with the highest incidence and mortality. Optic pathway/hypothalamic gliomas account for 4–7% of all pediatric intracranial tumors. Given the anatomical location, which compromises both the circadian pacemaker and its photic input pathway, we decided to study whether the presence of gliomas in the hypothalamic region could alter circadian behavioral outputs. Athymic nude mice implanted with LN229 human glioma cells showed an increase in the endogenous period of the circadian clock, which was also less robust in terms of sustaining the free running period throughout 2 weeks of screening. We also found that implanted mice showed a slower resynchronization rate after an abrupt 6 h advance of the light-dark (LD) cycle, advanced phase angle, and a decreased direct effect of light in general activity (masking), indicating that hypothalamic tumors could also affect photic sensitivity of the circadian clock. Our work suggests that hypothalamic gliomas have a clear impact both on the endogenous pacemaking of the circadian system, as well as on the photic synchronization of the clock. These findings strongly suggest that the observation of altered circadian parameters in patients might be of relevance for glioma diagnosis.

Nocturnal Light and Nocturnal Rodents

Investigators typically study one function of the circadian visual system at a time, be it photoreception, transmission of photic information to the suprachiasmatic nucleus (SCN), light control of rhythm phase, locomotor activity, or gene expression. There are good reasons for such a focused approach, but sometimes it is advantageous to look at the broader picture, asking how all the parts and functions complete the whole. Here, several seemingly disparate functions of the circadian visual system are examined. They share common characteristics with respect to regulation by light and, to the extent known, share a common input neuroanatomy. The argument presented is that the 3 hypothalamically mediated effects of light for which there are the most data, circadian clock phase shifts, suppression of nocturnal locomotion ("negative masking"), and suppression of nocturnal pineal function, are regulated by a common photic input pathway terminating in the SCN. For each, light triggers a relatively fixed interval response that is irradiance-dependent, the effective stimulus can be very brief light exposure, and the response continues to completion in the absence of additional light. The presence of a triggered, fixed-length response interval is of particular importance to the understanding of the circuitry and mechanisms regulating circadian rhythm phase shifts because it implies that the SCN clock response to light is not instantaneous. It also may explain why certain stimuli (neuropeptide Y or novel wheel running) administered many minutes after light exposure are able to block light-induced phase shifts. The understanding of negative masking is complicated by the fact that it can be represented as a positive change, that is, light-induced sleep, not just as a reduction in locomotion. Acute nocturnal light exposure also induces adrenal hormone secretion and a rapid drop in body temperature, physiological responses that appear to be regulated similarly to the other light effects. The likelihood of a common regulatory basis for the several responses suggests that additional light-induced responses will be forthcoming and raises questions about the relationships between light, SCN cellular anatomy, the molecular clockworks of SCN neurons, and SCN throughput mechanisms for regulating disparate downstream activities.

The mammalian molecular clockwork controls rhythmic expression of its own input pathway components.

The core molecular clockwork in the suprachiasmatic nucleus (SCN) is based on autoregulatory feedback loops of transcriptional activators (CLOCK/NPAS2 and BMAL1) and inhibitors (mPER1-2 and mCRY1-2). To synchronize the phase of the molecular clockwork to the environmental day and night condition, light at dusk and dawn increases mPer expression. However, the signal transduction pathways differ remarkably between the day/night and the night/day transition. Light during early night leads to intracellular Ca(2+) release by neuronal ryanodine receptors (RyRs), resulting in phase delays. Light during late night triggers an increase in guanylyl cyclase activity, resulting in phase advances. To date, it is still unknown how the core molecular clockwork regulates the availability of the respective input pathway components. Therefore, we examined light resetting mechanisms in mice with an impaired molecular clockwork (BMAL1(-/-)) and the corresponding wild type (BMAL1(+/+)) using in situ hybridization, real-time PCR, immunohistochemistry, and a luciferase reporter system. In addition, intracellular calcium concentrations (Ca(2+)(i)) were measured in SCN slices using two-photon microscopy. In the SCN of BMAL1(-/-) mice Ryr mRNA and RyR protein levels were reduced, and light-induced mPer expression was selectively impaired during early night. Transcription assays with NIH3T3 fibroblasts showed that Ryr expression was activated by CLOCK::BMAL1 and inhibited by mCRY1. The Ca(2+)(i) response of SCN cells to the RyR agonist caffeine was reduced in BMAL1(-/-) compared with BMAL1(+/+) mice. Our findings provide the first evidence that the mammalian molecular clockwork influences Ryr expression and thus controls its own photic input pathway components.

Cholecystokinin‐A receptors regulate photic input pathways to the circadian clock

Daily behaviors are strongly dominated by internally generated circadian rhythms, but the underlying mechanisms remain unclear. In mammals, photoentrainment of behaviors to light-dark cycles involves signaling from both intrinsically photosensitive retinal ganglion cells and classic photoreceptor pathways to the suprachiasmatic nucleus (SCN). How classic photoreceptor pathways work with the photosensitive ganglion cells, however, is not fully understood. Although cholecystokinin (CCK) peptide has been shown to be present in a variety of vertebrate retinas, its function at a systems level is also unknown. In the present study we examined a possible role of CCK-A receptors in photoentrainment using CCK-A receptor knockout mice. The lacZ reporter gene within a gene-knockout cassette revealed precise localization of CCK-A receptors in the circadian clock system. We demonstrated that CCK-A receptors were located predominately on glycinergic amacrine cells but were rarely found on SCN neurons. Moreover, Ca(2+) imaging analysis demonstrated that the CCK-A agonist, CCK-8 sulfate (CCK-8s), mobilized intracellular Ca(2+) in amacrine cells but not glutamate-receptive SCN neurons. Furthermore, light pulse-induced mPer1/mPer2 gene expression in SCN, behavioral phase shifts, and the pupillary reflex were significantly reduced in CCK-A receptor knockout mice. These data indicate a novel function of CCK-A receptors in the nonimage-forming photoreception presumably via amacrine cell-mediated signal transduction pathways.

Light Entrainment of the Mammalian Circadian Clock by a PRKCA-Dependent Posttranslational Mechanism

Light is the most potent stimulus for synchronizing endogenous circadian rhythms with external time. Photic clock resetting in mammals involves cAMP-responsive element binding protein (CREB)-mediated transcriptional activation of Period clock genes in the suprachiasmatic nuclei (SCN). Here we provide evidence for an additional photic input pathway to the mammalian circadian clock based on Protein Kinase C alpha (PRKCA). We found that Prkca-deficient mice show an impairment of light-mediated clock resetting. In the SCN of wild-type mice, light exposure evokes a transient interaction between PRKCA and PERIOD 2 (PER2) proteins that affects PER2 stability and nucleocytoplasmic distribution. These posttranslational events, together with CREB-mediated transcriptional regulation, are key factors in the molecular mechanism of photic clock resetting.

Differential effects of constant light on circadian clock resetting by photic and nonphotic stimuli in Syrian hamsters

Circadian rhythms in Syrian hamsters can be phase shifted by behavioral arousal during the usual rest phase of the circadian rest–activity cycle. Phase shifts can be greatly potentiated by exposing the animals to constant light for 1 or 2 cycles. This could reflect a change in a specific nonphotic input pathway to the suprachiasmatic nucleus (SCN) circadian pacemaker, or it could be caused by a change in the amplitude of the pacemaker. If the latter, then phase shifts to any stimulus, including those activating the photic input pathway, should be potentiated. This hypothesis was tested by measuring phase shifts induced by microinjections of NMDA (500 nl, 10 mM) into the SCN area of hamsters exposed to constant light or dark for 2 days. NMDA induced significant phase delay shifts that mimicked those induced by light exposure early in the night. The magnitude of these shifts did not differ by prior lighting condition. Shifts induced by NMDA (200 nl, 10 mM) microinjections on day 3 and 13 of LL also did not differ. Phase shifts induced by a nonphotic stimulus (3 h of running stimulated by confinement to a novel wheel) were significantly potentiated by 2 days of exposure to constant light. These results indicate that exposure to constant light for 2 circadian cycles differentially affects phase resetting responses to photic and nonphotic inputs to the circadian pacemaker, suggesting that potentiation of shifts to nonphotic stimuli reflect changes in a nonphotic input pathway rather than in an amplitude dimension of the circadian pacemaker.

Photic input pathways that mediate the Drosophila larval response to light and circadian rhythmicity are developmentally related but functionally distinct

The Drosophila melanogaster larval photosensory organ that mediates the response to light consists of bilaterally symmetrical clusters of 12 photoreceptors. These are distinguished on the basis of expression of the rhodopsins Rh5 and Rh6. The Rh6-expressing cells correspond to the Hofbauer-Buchner (H-B) eyelet found later in the posterior margin of the adult compound eye and recently shown to function as an input pathway in the entrainment of circadian rhythmicity in adult Drosophila. In addition, the axons of the larval photoreceptors are found in intimate association with a subset of the main circadian pacemaker neurons located in the developing accessory medulla, the small ventral lateral neurons (LNv). The observed spatial overlap between components of the circadian circuitry, input pathway, and pacemaker neurons-and the larval visual organ-suggest a functional relationship between these two photosensory input pathways. In this study we determined the requirement of specific rhodopsin-expressing photoreceptors including the presumptive H-B eyelet and pacemaker neurons in the larval locomotory response to visual stimuli. Our results demonstrate that two of the most important components of the neuronal circuitry underlying circadian rhythmicity in Drosophila, namely, the extraretinal H-B cluster and the circadian pacemakers, while in intimate association with the larval visual system are not required for the larval motor response to light.

Chicktacking pineal clock.

Many tissues in non-mammalian vertebrates contain both photoreceptors and circadian clock systems. Among these photosensitive clock structures, the chick pineal gland has been characterized in detail from cellular and molecular aspects of the clock oscillation and entrainment. Analyses of the pineal photic-input pathway revealed a phase-shifting mechanism mediated by activation of G11, one of the Gq-type G-proteins. A major photoreceptive molecule, pinopsin, likely triggers this pathway by transmitting the light signal to the circadian oscillator. In the chick pineal oscillator, the transcription/translation-based autoregulatory feedback loop is composed of positive and negative elements (clock gene products) that are homologous to those identified in mammals. In the molecular cycling, a CACGTG E-box located in the promoter region of the negative element genes plays a central role in the transcriptional regulation. The phase of the molecular cycling is modulated by many regulatory components, among which E4BP4 and extracellular signal-regulated kinase (ERK) are closely associated with the photic entrainment. A light-responsive element was found in the promoter region of the Pinopsin gene, and the element included a CACGTG E-box, suggesting a novel role of the E-box as a point of convergence of light and circadian signals. These observations together point to general and unique features of the chick pineal circadian system among animal clocks.

Circadian Signaling in the Chick Pineal Organ

The chick pineal organ is recognized to contain an endogenous circadian oscillator as well as having direct photic input pathways and the capability of synthesizing melatonin. Despite its interesting circadian cell biology, far less is known about the chick pineal as compared to mammalian pineal glands. The goals of our research were to identify and characterize novel components of the circadian system in this photoneuroendocrine organ. Using a subtractive screening strategy of a nocturnal chick pineal cDNA library, we identified numerous genes whose expression in the chick pineal has never been reported. Among these, we focused our attention on a homologue to the regulatory subunit of the mammalian serine/threonine protein phosphatase (STPP) 2A. The expression of this gene in the chick pineal is highly circadian both in vivo and in vitro. Analysis of the PP2A enzyme in this tissue revealed that it is predominantly cytosolic in localization, sensitive to classical PP2A inhibitors, and far more active during the subjective night. Interestingly, the acute pharmacological inhibition of PP2A leads to elevated phosphoCREB levels and concomitant melatonin secretion, indicating that this enzyme participates at some level in the control of nocturnal pineal melatonin synthesis. In a second aspect of our research, we examined the mechanisms underlying the circadian rhythmicity of cyclic GMP in the chick pineal. This signaling molecule is poorly understood, despite its well‐known, high‐amplitude circadian rhythms and the presence of many cGMP‐dependent targets in this tissue. Our work has shown that although both soluble (sGC) and membrane‐bound (mGC) forms of guanylyl cyclase are present, the primary contributor to the circadian rhythms of cGMP is the mGC‐B enzyme, which is activated only by the natriuretic peptide CNP. As pharmacological blockade of mGC‐B (but not sGC) suppresses nocturnal cGMP levels, we conclude that CNP‐dependent mechanisms are involved. Hence, the circadian clock in the chick pineal appears to drive either CNP secretion or mGC‐B expression (or synthetic efficiency) in order to elevate nocturnal cGMP. Conversely, light may inhibit cGMP by uncoupling this drive. These data provide new strategies for understanding both photic input pathways (presumed to depend on cGMP) and cGMP‐dependent cellular function in the chick pineal organ.

Light-induced phase-delay of the chicken pineal circadian clock is associated with the induction of cE4bp4, a potential transcriptional repressor of cPer2 gene.

The chicken pineal gland contains the autonomous circadian oscillator together with the photic-input pathway. We searched for chicken pineal genes that are induced by light in a time-of-day-dependent manner, and isolated chicken homolog of bZIP transcription factor E4bp4 (cE4bp4) showing high similarity to vrille, one of the Drosophila clock genes. cE4bp4 was expressed rhythmically in the pineal gland with a peak at very early (subjective) night under both 12-h light/12-h dark cycle and constant dark conditions, and the phase was nearly opposite to the expression rhythm of cPer2, a chicken pineal clock gene. Luciferase reporter gene assays showed that cE4BP4 represses cPer2 promoter through a E4BP4-recognition sequence present in the 5'-flanking region, indicating that cE4BP4 can down-regulate the chick pineal cPer2 expression. In vivo light-perturbation studies showed that the prolongation of the light period to early subjective night maintained the high level expression of the pineal cE4bp4, and presumably as a consequence delayed the onset of the induction of the pineal cPer2 expression in the next morning. These light-dependent changes in the mRNA levels of the pineal cE4bp4 and cPer2 were followed by a phase-delay of the subsequent cycles of cE4bp4/cPer2 expression, suggesting that cE4BP4 plays an important role in the phase-delaying process as a light-dependent suppressor of cPer2 gene.

Photoreception and circadian clock system of the chicken pineal gland.

Chicken pinealocytes contain three major components of the circadian clock system: 1) a self-sustained oscillator, 2) a photic-input pathway to the oscillator, and 3) an overt output represented by the rhythmic production of melatonin. Even under cultured conditions of isolated pineal gland or dissociated pinealocytes, the input-oscillator-output functions are well maintained. Because of these experimental advantages, chicken pineal gland has been one of the best models for the study of the circadian clock system. Since the finding of a pineal-specific photoreceptive molecule, pinopsin, we have characterized the endogenous phototransduction pathway in the pinealocytes. On the other hand, despite the long history of chick pineal research, the molecular mechanism underlying the pineal clock oscillation has been largely unknown. Our recent characterization of the chick pineal clock genes strongly suggests that they constitute a transcription/translation-based autoregulatory feedback loop, which is very similar to that generating circadian rhythmicity in mammalian SCN.

A Noradrenergic Mechanism Influences the Circadian Timing System in Rats and Hamsters

Brainstem monoaminergic projections to the suprachiasmatic nucleus (SCN), and to the intergeniculate leaflet (IGL), appear to modulate both photic and non-photic effects on the circadian system. Recent work in this laboratory has concentrated on the role of noradrenaline in the regulation of circadian period and phase. Previously, this lab has shown that chronic administration of the alpha2 adrenergic agonist, clonidine, to rats maintained in constant light (LL) shortens free-running circadian period and promotes dissociation of rhythmicity, while acute clonidine administration to hamsters produces phase shifts similar to those observed with photic stimuli. These results suggest an interaction between clonidine and photic input on circadian rhythmicity, and so the present study was designed to examine systematically the relationship between chronic clonidine administration and photic input in both rats and hamsters. In DD and low intensity LL, clonidine did not alter free-running circadian wheel-running rhythms of rats, but under moderate to high intensity LL, clonidine significantly reduced the period-lengthening effects of LL. Chronic clonidine administration also altered several aspects of circadian phase in hamsters; phase shifts in response to light pulses of varying intensity at CT 19 were reduced; steady-state entrainment phase under a 24-h light-dark cycle (LD 14:10)was delayed; and synchronization to a 23-h light-dark cycle (LD 13:10) was impaired. Clonidine appeared to have little effect on free-running period of hamsters, but a trend towards dissociation of rhythmicity under LL was observed. These effects may reflect an action of clonidine at the photic input pathways to the circadian system, or directly at the circadian pacemaker, since alpha 2 adrenoceptors have been localized both in the suprachiasmatic nucleus (SCN) and in several of its projection areas. As both clinical and experimental studies suggest that clonidine may have depressogenic properties, chronic administration of clonidine to rodents may provide an animal model of the alterations in circadian rhythmicity seen in human depression.

Role of Circadian Activation of Mitogen-Activated Protein Kinase in Chick Pineal Clock Oscillation

A circadian pacemaker generates a rhythm with a period of approximately 24 hr even in the absence of environmental time cues. Several photosensitive neuronal tissues such as the retina and pineal gland contain the autonomous circadian pacemaker together with the photic-input pathway responsible for entrainment of the pacemaker to the daily light/dark cycle. We show here that, in constant darkness, chick pineal mitogen-activated protein kinase (MAPK) exhibited an in vivo circadian rhythm in tyrosine phosphorylation and in enzymatic activity with a peak during subjective night. Phosphorylated and hence activated MAPK was rapidly dephosphorylated after light illumination during the nighttime when light induces a phase-shift of the pacemaker. The circadian rhythmicity in MAPK phosphorylation was also observed in the cultured pineal gland, and importantly, MAPK kinase inhibitor treatment during subjective night not only shifted the time-of-peak of MAPK phosphorylation but also induced a remarkable phase-delay of the circadian pacemaker. These results indicate an important role of MAPK for time keeping in circadian clock systems.

Clonidine Shortens Circadian Period in Both Constant Light and Constant Darkness

Chronic treatment of rats with clonidine, an α-adrenoceptor agonist, alters the period and amplitude of free-running circadian activity rhythms, as well as the level of home cage locomotor activity. Agents that alter free-running period are presumed to act directly on the circadian pacemaker or on its input pathways. Because shortening of free-running period during clonidine treatment has been observed consistently under constant light but not under constant darkness, and because increasing light intensity itself lengthens free-running period, this agent may influence the circadian pacemaker by modulating light-evoked activity in the photic input pathway. The present study reexamined the possible dependence of clonidine-induced alterations of free-running circadian activity rhythms on lighting conditions. Similar effects were seen in both constant light and constant darkness, indicating that the effects of clonidine on the circadian pacemaker are not due to blockade of light-evoked activity in the photic input pathway. Instead, clonidine may act directly on the circadian pacemaker, or on other unspecified mechanisms influencing free-running period.

Clonidine shortens circadian period in both constant light and constant darkness

Chronic treatment of rats with clonidine, an α-adrenoceptor agonist, alters the period and amplitude of free-running circadian activity rhythms, as well as the level of home cage locomotor activity. Agents that alter free-running period are presumed to act directly on the circadian pacemaker or on its input pathways. Because shortening of free-running period during clonidine treatment has been observed consistently under constant light but not under constant darkness, and because increasing light intensity itself lengthens free-running period, this agent may influence the circadian pacemaker by modulating light-evoked activity in the photic input pathway. The present study reexamined the possible dependence of clonidine-induced alterations of free-running circadian activity rhythms on lighting conditions. Similar effects were seen in both constant light and constant darkness, indicating that the effects of clonidine on the circadian pacemaker are not due to blockade of light-evoked activity in the photic input pathway. Instead, clonidine may act directly on the circadian pacemaker, or on other unspecified mechanisms influencing free-running period.