Organization of the circadian system in insects

The circadian systems of different insect groups are summarized and compared. Emphasis is placed on the anatomical identification and characterization of circadian pacemakers, as well as on their entrainment, coupling, and output pathways. Cockroaches, crickets, beetles, and flies possess bilaterally organized pacemakers in the optic lobes that appear to be located in the accessory medulla, a small neuropil between the medulla and the lobula. Neurons that are immunoreactive for the peptide pigment-dispersing hormone (PDH) arborize in the accessory medulla and appear to be important components of the optic lobe pacemakers. The neuronal architecture of the accessory medulla with associated PDH-immunoreactive neurons is best characterized in cockroaches, while the molecular machinery of rhythm generation is best understood in fruit flies. One essential component of the circadian clock is the period protein (PER), which colocalizes with PDH in about half of the fruit fly's presumptive pacemaker neurons. PER is also found in the presumptive pacemaker neurons of beetles and moths, but appears to have different functions in these insects. In moths, the pacemakers are situated in the central brain and are closely associated with neuroendocrine functions. In the other insects, neurons associated with neuroendocrine functions also appear to be closely coupled to the optic lobe pacemakers. Some crickets and flies seem to possess central brain pacemakers in addition to their optic lobe pacemakers. With respect to neuronal organization, the circadian systems of insects show striking similarities to the vertebrate circadian system.     

Diurnal variations in vasoactive intestinal polypeptide-like immunoreactivity in the suprachiasmatic nucleus of congenitally anophthalmic mice

The present study has combined recording of circadian locomotor rhythms with light microscopic immunocytochemistry for vasoactive intestinal polypeptide (VIP) in the suprachiasmatic nucleus (SCN) of congenitally anophthalmic mice. These mice, which never develop retinae or optic nerves and do not perceive light, are thus in constant darkness. Our data show a circadian rhythm in expression of VIP in the SCN of anophthalmic mice--expression is maximal during late subjective night/early subjective day and minimal in late subjective day/early subjective night. These observations support the hypothesis that expression of VIP is related to regulation of circadian rhythms by the SCN.     

Neurons containing gastrin-releasing peptide and vasoactive intestinal polypeptide are involved in the reception of the photic signal in the suprachiasmatic nucleus of the Syrian hamster: an immunocytochemical ultrastructural study

In mammals, the suprachiasmatic nuclei are involved in the generation of biological rhythms and are synchronized by light input coming from the retina. The targets of retinal afferents and the involvement of neurons containing gastrin-releasing and vasoactive intestinal peptides in photic reception were investigated in the suprachiasmatic nuclei of the Syrian hamster by using light- and electron-microscopic immunocytochemistry. Cholera toxin was used to trace retinal fibers and Fos immunoreactivity to visualize cellular response to light stimulation. Ultrastructural observations were made in the intermediate third of the nuclei, the area of highest overlap for the immunoreactivities investigated. Gastrin-releasing peptide and vasoactive intestinal peptide cell bodies were localized in the ventral part of the nuclei; their dense immunoreactive fiber network often displayed synaptic contacts. Both neuropeptides were colocalized in elongated cells observed near the optic chiasm. Following a light pulse in the middle of the subjective night, Fos protein was expressed in most gastrin-releasing peptide perikarya and in some vasoactive intestinal peptide cells. Retinal terminals mostly occurred in the midline zone between the suprachiasmatic nuclei. Symmetrical or asymmetrical retinal synapses were observed on gastrin-releasing peptide-immunoreactive dendrites and somata, but never on vasoactive intestinal peptide neurons. These results are discussed in relation to the photic entrainment of the circadian clock.     

Atherosclerosis in the human brachial artery

The hypothalamic suprachiasmatic nucleus (SCN) of the mammal is the circadian pacemaker responsible for generation of circadian rhythms. Several immediate-early genes are expressed in the SCN by light stimuli which induce phase shifts of animal activity rhythms. In the present study, we investigated whether Homer, a PDZ-like protein which is rapidly induced following synaptic activation, mRNA expression is regulated by light in rat SCN. Homer mRNA expression in the SCN of rat killed at 4 h after onset of the light and dark phases was very low. One hour light stimuli during the subjective night dramatically induced Homer mRNA expression in the ventrolateral portion of the SCN, whereas light stimuli during the subjective light phase did not. This finding implies that Homer may be involved in the photic entrainment of the circadian clock.     

Pigment mutations associated with altered circadian rhythms in mice

Mammalian albinism is known to alter neural pathways, reduce retinal pigment, and affect diverse behaviors. Mammalian circadian rhythms have been shown to depend on visual pathways, respond to light intensity and regulate many behaviors. Here we show that mice homozygous for the recessive albino or pinkeye-dilute mutations display shorter circadian rhythms than pigmented controls. We conclude that these pigment loci, or closely linked loci, influence the expression of circadian rhythms in mice.     

Circadian patterns of locomotor activity and body temperature in blind mole-rats, Spalax ehrenbergi

A wide variety of organisms exhibit circadian rhythms, regulated by internal clocks that are entrained primarily by the alternating cycle of light and darkness. There have been few studies of circadian rhythms in fossorial species that inhabit a microenvironment where day-night variations in most environmental parameters are minimized and where exposure to light occurs only infrequently. In this study, daily patterns of locomotor activity and body temperature (Tb) were examined in adult blind mole-rats (Spalax ehrenbergi). These fossorial rodents lack external eyes but possess rudimentary ocular structures that are embedded in the Harderian glands and covered by skin and fur. Most individual mole-rats exhibited circadian rhythms of locomotor activity, but some animals were arrhythmic. Individuals that did exhibit robust rhythms of locomotor activity also showed rhythms of Tb. In most cases, Tb was highest during the phase of intense locomotor activity. Locomotor activity rhythms could be entrained to light:dark cycles, and several mole-rats exhibited entrainment to non-24-h light cycles (T-cycles) with period lengths ranging from T = 23 h to T = 25 h. Some individuals also showed entrainment to daily cycles of ambient temperature. There was considerable interindividual variation in the daily patterns of locomotor activity among mole-rats in virtually all the conditions of environmental lighting and temperature employed in this study. Thus, whereas it appears likely that photic cues have a significant role in the entrainment of circadian rhythms in mole-rats, the amount of variability in rhythm patterns among individuals appears to be much greater than for most species that have been studied.     

Circadian clock function in isolated eyestalk tissue of crayfish

Electrical mass response of crayfish photoreceptors (electroretinogram) was recorded continuously for up to seven days in isolated preparations that consisted of the retina and lamina ganglionaris. Electroretinogram amplitude varied in a circadian manner with a nocturnal acrophase and a period of 22-23 h in preparations kept in darkness. Acclimatization of animals to reversed light/dark cycles resulted in a phase reversal of the rhythm in vitro. The per (period) gene of Drosophila has been implicated in the genesis of rhythms in insects and in vertebrates. Immunocytochemical staining with an antibody against the PER gene product revealed immunoreactivity in the retinal photoreceptors, as well as in cell bodies in the lamina ganglionaris. Labelled axons run distally towards the photoreceptors and proximally to other areas of the lamina.     

PSD-95 promotes Fyn-mediated tyrosine phosphorylation of the N-methyl-D-aspartate receptor subunit NR2A

Our previous study has shown that the phases of circadian rhythms of ocular melatonin and dopamine are always opposite and intraocular melatonin injection suppresses dopamine release. Therefore, it is possible that dopamine rhythms result from inhibitory action of melatonin. We have examined this possibility in the following experiments. In the first experiment effects of continuous light on melatonin and dopamine release were examined. The data indicated that continuous light exposure resulted in loss of circadian rhythmicity of melatonin and dopamine by suppressing melatonin and enhancing dopamine levels throughout the day. To further examine the effects of light in the second experiment, 2 h light pulse was applied during the night, then temporal changes of melatonin and dopamine release were studied. The light pulse rapidly suppressed melatonin release, whereas it rapidly increased dopamine release. These changes occurred within 30 min in both melatonin and dopamine. However, the recovery after the cessation of the light stimulus was slower in melatonin than dopamine. In the third experiment it was tested if dopamine release was increased by lowering melatonin release with an intraocular injection of the D2 agonist, quinpirol. Although quinpirol strongly inhibited melatonin release independently of the time of injection, dopamine did not always increase by the inhibition of melatonin. These results indicate that ocular dopamine rhythms are not simply produced by melatonin inhibitory action.     

Differences in tryptophan binding to hepatic nuclei of NZBWF1 and Swiss mice: insight into mechanism of tryptophan''s effects

Catecholamine receptors of multiple classes have been shown to influence pineal melatonin synthesis in a species-specific manner. In these experiments, the effects of catecholamine receptor agonists on circadian melatonin rhythms of zebrafish (Danio rerio) pineal in vitro were examined. Cyclic application of adrenergic receptor agonists (norepinephrine, phenylephrine, clonidine, and isoproterenol) had no effect on zebrafish pineal melatonin release, nor on the circadian oscillator that regulates melatonin rhythms. Pineal melatonin release was partially suppressed by quinpirole, a D2 dopamine receptor agonist, but cyclic application of quinpirole did not reset the pineal circadian oscillator. Pineal melatonin release was unaffected by either dopamine or SKF38393, a D1 receptor agonist, suggesting that the effects of quinpirole were not mediated by dopamine receptors. The regulatory mechanisms underlying pineal melatonin rhythms appear to differ among teleosts.     

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.     

Photic and circadian regulations of melatonin rhythms in fishes

Photic and circadian regulations of melatonin rhythms in the pineal organ and the retina of several teleosts were studied to investigate the regulatory mechanisms of melatonin rhythms in fishes. In the eyecup preparations of the goldfish, Carassius auratus, both time of day and lighting conditions affected melatonin production, with high melatonin production observed only in the dark-treated group incubated during the 'subjective' night. Thus, in the goldfish retina, local photoreceptors and an ocular circadian clock seem to regulate melatonin production, as in the zebrafish retina and in the pineal organ of a number of teleosts, including the goldfish. However, this circadian regulation of melatonin rhythms is not universal among fishes. Although the superfused pineal organ of the masu salmon Oncorhynchus masou secreted melatonin in a rhythmic fashion under light-dark (LD) cycles, the rhythm disappeared under constant darkness (DD), as in the rainbow trout, with a large amount of melatonin released both during the subjective day and the subjective night. These results suggest that all salmonids lack circadian regulation of melatonin rhythms. Furthermore, when ocular melatonin rhythms were compared in two cyprinids, the ugui Tribolodon hakonensis and the oikawa Zacco platypus occupying different ecological niches, ocular melatonin contents exhibited daily variations, with higher values during the dark phase of LD cycles in both species. The rhythmic changes persisted in the ugui under DD, with higher levels at subjective midnight than at subjective midday; however, ocular melatonin levels in the oikawa were consistently high under DD. Thus, the circadian regulation of melatonin rhythms in fishes is influenced not only by phylogeny, but also by the ecological niches of the animals. These results suggest that the physiological functions of melatonin in the circadian and photoperiodic systems differ among fishes.     

The role of the intergeniculate leaflet in entrainment of circadian rhythms to a skeleton photoperiod

Mammalian circadian rhythms are synchronized to environmental light/dark (LD) cycles via daily phase resetting of the circadian clock in the suprachiasmatic nucleus (SCN). Photic information is transmitted to the SCN directly from the retina via the retinohypothalamic tract (RHT) and indirectly from the retinorecipient intergeniculate leaflet (IGL) via the geniculohypothalamic tract (GHT). The RHT is thought to be both necessary and sufficient for photic entrainment to standard laboratory light/dark cycles. An obligatory role for the IGL-GHT in photic entrainment has not been demonstrated. Here we show that the IGL is necessary for entrainment of circadian rhythms to a skeleton photoperiod (SPP), an ecologically relevant lighting schedule congruous with light sampling behavior in nocturnal rodents. Rats with bilateral electrolytic IGL lesions entrained normally to lighting cycles consisting of 12 hr of light followed by 12 hr of darkness, but exhibited free-running rhythms when housed under an SPP consisting of two 1 hr light pulses given at times corresponding to dusk and dawn. Despite IGL lesions and other damage to the visual system, the SCN displayed normal sensitivity to the entraining light, as assessed by light-induced Fos immunoreactivity. In addition, all IGL-lesioned, free-running rats showed masking of the body temperature rhythm during the SPP light pulses. These results show that the integrity of the IGL is necessary for entrainment of circadian rhythms to a lighting schedule like that experienced by nocturnal rodents in the natural environment.     

Glutamatergic antagonists do not attenuate light-induced fos protein in rat intergeniculate leaflet

Photic information that entrains circadian rhythms is transmitted to the suprachiasmatic nucleus (SCN) from the retina and from the retinorecipient intergeniculate leaflet (IGL). Expression of light-induced Fos protein in SCN neurons is correlated with the effectiveness of such light to induce phase shifts, and is prevented by pretreatment with glutamate receptor antagonists that prevent phase shifts as well. In the present study we demonstrate that treatments with N-methyl-d-aspartate (NMDA) and non-NMDA receptor antagonists prior to light pulses during the subjective night have no effect on light-induced Fos immunoreactivity (Fos-IR) in IGL neurons despite attenuating Fos-IR in the SCN. Transmission of photic information along retinogeniculate and retinohypothalamic pathways appears to be mediated by different mechanisms.     

Hierarchically coupled ultradian oscillators generating robust circadian rhythms

Ensembles of mutually coupled ultradian cellular oscillators have been proposed by a number of authors to explain the generation of circadian rhythms in mammals. Most mathematical models using many coupled oscillators predict that the output period should vary as the square root of the number of participating units, thus being inconsistent with the well-established experimental result that ablation of substantial parts of the suprachiasmatic nuclei (SCN), the main circadian pacemaker in mammals, does not eliminate the overt circadian functions, which show no changes in the phases or periods of the rhythms. From these observations, we have developed a theoretical model that exhibits the robustness of the circadian clock to changes in the number of cells in the SCN, and that is readily adaptable to include the successful features of other known models of circadian regulation, such as the phase response curves and light resetting of the phase.     

Dynamics of spontaneous beating of cardiomyocytes in vitro depend on the number of involved cells

The hypothalamic suprachiasmatic nucleus (SCN) is the predominant pacemaker of the mammalian brain that generates and controls circadian rhythms of various endocrine and behavioral processes. Different lines of evidence suggest that stress interferes with the maintenance of such rhythms. As a first approach to investigate whether the neuropeptide arginine vasopressin (AVP), which shows circadian rhythms of synthesis and release within the SCN, might contribute to this stress-induced alterations in circadian rhythms, we monitored acute effects of swim stress on the intra-SCN release of AVP in male rats by means of the microdialysis technique. A 10-min forced swimming session triggered a marked but relatively short-lasting increase in the intranuclear release of AVP (to approx. 440%). This effect was restricted to the area containing predominantly somata and dendrites of vasopressinergic neurons, since no changes in AVP release could be measured in one of their major projection areas, the nucleus of the dorsomedial hypothalamus. Our data provide evidence that the amount of AVP released within the SCN can vary widely not only in accordance with AVP's intrinsically regulated circadian rhythm but also in response to a physiologically relevant stressor. In this way, the neuropeptide may contribute to the regulation of endocrine and behavioral rhythms particularly in challenging situations associated with resettings of the endogenous clock.     

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.