Hypothalamic regulation of circadian noradrenergic input to the chick pineal gland

Summary While the avian pineal gland contains circadian oscillators and photoreceptors capable of producing circadian rhythms of the hormone melatonin, it is extensively innervated by post-ganglionic fibers of the superior cervical ganglia which release norepinephrine (NE) rhythmically. Norepinephrine turnover is higher during subjective day than during subjective night. In mammals, this rhythmic input, which is higher in subjective night than subjective day, derives from the hypothalamic suprachiasmatic nuclei (SCN) and is essential for rhythmic melatonin production. The present study was designed to determine whether one of two candidates for the avian homologue of the mammalian SCN is necessary for rhythmic NE turnover in the chick pineal gland. Either electrolytic lesions or sham lesions were delivered to the periventricular preoptic nuclei (PPN) or to the visual suprachiasmatic nucleus (vSCN). After recovery, the rates of decline in [NE] were determined following pretreatment with -methyl-p-tyrosine, a tyrosine hydroxylase inhibitor, at mid-subjective day or at mid-subjective night. Birds receiving sham surgeries in either PPN or vSCN and birds receiving lesions of the PPN exhibited rhythmicity in NE turnover. No rhythm of NE turnover could be determined in birds with ablated vSCN.Abbreviations AMPT -methyl-p-tyrosine - DS supraoptic decussation - EBZ ear bar zero (see Methods) - GLv ventral lateral geniculate body - NE norepinephrine - PPN periventricular preoptic nuclei - RH retinohypothalamic projection - SCN suprachiasmatic nuclei - vSCN visual suprachiasmatic nucleus     

Phase response curve and light-induced fos expression in the suprachiasmatic nucleus and adjacent hypothalamus of Arvicanthis niloticus

This article describes the phase response curve (PRC), the effect of light on Fos immunoreactivity (Fos-IR) in the suprachiasmatic nucleus (SCN), and the effect of SCN lesions on circadian rhythms in the murid rodent, Arvicanthis niloticus. In this species, all individuals are diurnal when housed without a running wheel, but running in a wheel induces a nocturnal pattern in some individuals. First, the authors characterized the PRC in animals with either the nocturnal or diurnal pattern. Both groups of animals were less affected by light during the middle of the subjective day than during the night and were phase delayed and phase advanced by pulses in the early and late subjective night, respectively. Second, the authors characterized the Fos response to light at circadian times 5, 14, or 22. Light induced an increase in Fos-IR within the SCN during the subjective night but not subjective day; this effect was especially pronounced in the ventral SCN, where retinal inputs are most concentrated, but was also evident in other regions. Both light and time influenced Fos-IR within the lower subparaventricular area. Third, SCN lesions caused animals to become arrhythmic when housed in a light-dark cycle as well as constant darkness. In summary, Arvicanthis appear to be very similar to nocturnal rodents with respect to their PRC, temporal patterns of light-induced Fos expression in the SCN, and the effects of SCN lesions on activity rhythms.     

Circadian expression of clock genes in the rat eye and brain

The light sensing system in the eye directly affects the circadian oscillator in the mammalian suprachiasmatic nucleus (SCN). To investigate this relationship in the rat, we examined the circadian expression of clock genes in the SCN and eye tissue during a 24 h day/night cycle. In the SCN, rPer1 and rPer2 mRNAs were expressed in a clear circadian rhythm like rCry1 and rCry2 mRNAs, whereas the level of BMAL1 and CLOCK mRNAs decreased during the day and increased during the night with a relatively low amplitude. It seems that the clock genes of the SCN may function in response to a master clock oscillation in the rat. In the eye, the rCry1 and rCry2 were expressed in a circadian rhythm with an increase during subjective day and a decrease during subjective night. However, the expression of Opn4 mRNA did not exhibit a clear circadian pattern, although its expression was higher in daytime than at night. This suggests that cryptochromes located in the eye, rather than melanopsin, are the major photoreceptive system for synchronizing the circadian rhythm of the SCN in the rat.     

Identification of the suprachiasmatic nucleus in birds

Circadian rhythms are generated by an internal biological clock. The suprachiasmatic nucleus (SCN) in the hypothalamus is known to be the dominant biological clock regulating circadian rhythms in mammals. In birds, two nuclei, the so-called medial SCN (mSCN) and the visual SCN (vSCN), have both been proposed to be the avian SCN. However, it remains an unsettled question which nuclei are homologous to the mammalian SCN. We have identified circadian clock genes in Japanese quail and demonstrated that these genes are expressed in known circadian oscillators, the pineal and the retina. Here, we report that these clock genes are expressed in the mSCN but not in the vSCN in Japanese quail, Java sparrow, chicken, and pigeon. In addition, mSCN lesions eliminated or disorganized circadian rhythms of locomotor activity under constant dim light, but did not eliminate entrainment under light-dark (LD) cycles in pigeon. However, the lesioned birds became completely arrhythmic even under LD after the pineal and the eye were removed. These results indicate that the mSCN is a circadian oscillator in birds.     

Patterns of wheel running are related to Fos expression in neuropeptide-Y-containing neurons in the intergeniculate leaflet of Arvicanthis niloticus

A variety of nonphotic influences on circadian rhythms have been documented in mammals. In hamsters, one such influence, running in a novel wheel, is mediated in part by the pathway extending from neuropeptide-Y (NPY)-containing cells within the intergeniculate leaflet (IGL) of the thalamus to the hypothalamic suprachiasmatic nucleus (SCN). Arvicanthis niloticus is a species in which all individuals are diurnal with respect to general activity and body temperature when they are housed without a running wheel, but access to a running wheel induces a subset of individuals to become nocturnal. In the first study, the authors evaluated the possibility that nocturnal and diurnal patterns of wheel running in Arvicanthis are correlated with differences in IGL function. Adult male Arvicanthis housed in a 12:12 light-dark (LD) cycle were monitored in wheels, classified as nocturnal or diurnal, and then perfused either 4 h after lights-on or 4 h after lights-off. Sections through the intergeniculate leaflet were processed for immunohistochemical labeling of Fos and NPY. The percentage of NPY cells that expressed Fos was significantly influenced by an interaction between time of day and phenotype such that it rose from night to day in diurnal animals, and from day to night in nocturnal animals. In the second experiment, the authors established that running in a wheel actually induces Fos in the IGL of Arvicanthis. Specifically, the proportion of NPY cells expressing Fos was increased by access to wheels in nocturnal animals at night and in diurnal animals during the day. In the third experiment, the authors established that lesions of the IGL eliminate NPY fibers within the SCN, suggesting that these IGL cells project to the SCN in this species as has been established in other rodents. Together, these data demonstrate a clear difference in NPY cell function in nocturnal and diurnal Arvicanthis that appears to be caused, at least in part, by the differences in their wheel-running patterns, and that NPY cells within the IGL project to the SCN in Arvicanthis.     

Glucocorticoid hormones inhibit food-induced phase-shifting of peripheral circadian oscillators.

The circadian timing system in mammals is composed of a master pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus and slave clocks in most peripheral cell types. The phase of peripheral clocks can be completely uncoupled from the SCN pacemaker by restricted feeding. Thus, feeding time, while not affecting the phase of the SCN pacemaker, is a dominant Zeitgeber for peripheral circadian oscillators. Here we show that the phase resetting in peripheral clocks of nocturnal mice is slow when feeding time is changed from night to day and rapid when switched back from day to night. Unexpectedly, the inertia in daytime feeding-induced phase resetting of circadian gene expression in liver and kidney is not an intrinsic property of peripheral oscillators, but is caused by glucocorticoid signaling. Thus, glucocorticoid hormones inhibit the uncoupling of peripheral and central circadian oscillators by altered feeding time.     

c-Fos expression in the brains of behaviorally "split" hamsters in constant light: calling attention to a dorsolateral region of the suprachiasmatic nucleus and the medial division of the lateral habenula

"Splitting" of circadian activity rhythms in Syrian hamsters maintained in constant light appears to be the consequence of a reorganized SCN, with left and right halves oscillating in antiphase; in split hamsters, high mRNA levels characteristic of day and night are simultaneously expressed on opposite sides of the paired SCN. To visualize the splitting phenomenon at a cellular level, immunohistochemical c-Fos protein expression in the SCN and brains of split hamsters was analyzed. One side of the split SCN exhibited relatively high c-Fos levels, in a pattern resembling that seen in normal, unsplit hamsters during subjective day in constant darkness; the opposite side was labeled only within a central-dorsolateral area of the caudal SCN, in a region that likely coincides with a photo-responsive, glutamate receptor antagonist-insensitive, pERK-expressing cluster of cells previously identified by other laboratories. Outside the SCN, visual inspection revealed an obvious left-right asymmetry of c-Fos expression in the medial preoptic nucleus and subparaventricular zone of split hamsters killed during the inactive phase and in the medial division of the lateral habenula during the active phase (when the hamsters were running in their wheels). Roles for the dorsolateral SCN and the mediolateral habenula in circadian timekeeping are not yet understood.     

Hypothalamic circadian organization in birds. I. Anatomy,functional morphology,and terminology of the suprachiasmatic region

In mammals, the “master clock” controlling circadian rhythmicity is located in the hypothalamic suprachiasmatic nuclei (SCN). Until now, no comparable structure has been unambiguously described in the brain of any nonmammalian vertebrate. In birds, early anatomical and lesioning studies described a SCN located in the anterior hypothalamus, but whether birds possess a nucleus equivalent to the mammalian SCN remained controversial. By reviewing the existing literature it became evident that confusion in delineation and nomenclature of hypothalamic cell groups may be one of the major reasons that no coherent picture of the avian hypothalamus exists. In this review, we attempt to clarify certain aspects of the organization of the avian hypothalamus by summarizing anatomical and functional studies and comparing them to immunocytochemical results from our laboratory. There is no single cell group in the avian hypothalamus that combines the morphological and neurochemical features of the mammalian SCN. Instead, certain aspects of anatomy and morphology suggest that at least two anatomically distinct cell groups, the SCN and the lateral hypothalamic nucleus (LHN), bear some of the characteristics of the mammalian SCN.     

Primary optic projections in the rabbit. Study using the horseradish peroxidase anterograde labeling technic

The primary visual pathways, in particular those to the lateral geniculate body, of 11 albino and 7 pigmented rabbits, were studied using the method of anterograde labelling with horseradish peroxidase following injection of the tracer into the vitreous body of one eye. A heavy projection to the contralateral dorsal lateral geniculate nucleus was seen in all animals. In both albino and pigmented animals a region devoid of label was present in the medial part of the alpha sector of the nucleus. This region corresponded to a compact, oval or wedge-shaped field of terminal label in the ipsilateral nucleus, which was much heavier in pigmented than in albino rabbits. In the ventral lateral geniculate nucleus, contralateral retinal input was almost entirely confined to the caudal half of the lateral sector of the nucleus, where two laminae of dense terminal label, separated by a less densely labelled area, were oriented parallel to one another and to the optic tract. This bilaminar distribution of retinal afferents to the ventral lateral geniculate nucleus has not been described in previous studies. The ipsilateral projection was to the dorsal part of the lateral sector and was most prominent in pigmented animals. The "intergeniculate leaflet" received a prominent contralateral input in all animals, and a clear ipsilateral input in pigmented animals, which overlapped with the contralateral input. Projections to other primary visual centres (pretectal nuclei, superior colliculus, nuclei of the accessory optic tract) are also described.     

A reexamination of the role of GABA in the mammalian suprachiasmatic nucleus

Three independent electrophysiological approaches in hypothalamic slices were used to test the hypothesis that gamma-amino butyric acid (GABA)A receptor activation excites suprachiasmatic nucleus (SCN) neurons during the subjective day, consistent with a recent report. First, multiple-unit recordings during either the subjective day or night showed that GABA or muscimol inhibited firing activity of the SCN population in a dose-dependent manner. Second, cell-attached recordings during the subjective day demonstrated an inhibitory effect of bath- or microapplied GABA on action currents of single SCN neurons. Third, gramicidin perforated-patch recordings showed that bicuculline increased the spontaneous firing rate during the subjective day. Therefore, electrophysiological data obtained by three different experimental methods provide evidence that GABA is inhibitory rather than excitatory during the subjective day.     

Loss of dexras1 Alters Nonphotic Circadian Phase Shifts and Reveals a Role for the Intergeniculate Leaflet (IGL) in Gene-Targeted Mice

Loss of Dexras1 in gene-targeted mice impairs circadian entrainment to light cycles and produces complex changes to phase-dependent resetting responses (phase shifts) to light. The authors now describe greatly enhanced and phase-specific nonphotic responses induced by arousal in dexras1^?/? mice. In constant conditions, mutant mice exhibited significant arousal-induced phase shifts throughout the subjective day. Unusual phase advances in the late subjective night were also produced when arousal has little effect in mice. Bilateral lesions of the intergeniculate leaflet (IGL) completely eliminated both the nonphotic as well as the light-induced phase shifts of circadian locomotor rhythms during the subjective day, but had no effect on nighttime phase shifts. The expression of FOS-like protein in the suprachiasmatic nucleus (SCN) was not affected by either photic or nonphotic stimulation in the subjective day in either genotype. Therefore, the loss of Dexras1 (1) enhances nonphotic phase shifts in a phase-dependent manner, and (2) demonstrates that the IGL in mice is a primary mediator of circadian phase-resetting responses to both photic and nonphotic events during the subjective day, but plays a different functional role in the subjective night. Furthermore, (3) the change in FOS level does not appear to be a critical step in the entrainment pathways for either light or arousal during the subjective day. The cumulative evidence suggests that Dexras1 regulates multiple photic and nonphotic signal-transduction pathways, thereby playing an essential role modulating species-specific characteristics of circadian entrainment. (Author correspondence: ralph@psych.utoronto.ca)     

Hormones, subjective night and season of the year

Production and release of many mammalian hormones exhibit circadian rhythms controlled by a pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Under conditions when the circadian pacemaker free-runs with a period close to, but not equal to 24 h, subjective day and night may not be identical with the environmental day and night. The present study was aimed to define the phase and state of the circadian pacemaker when the circadian system is experiencing subjective night and to ascertain whether and how such a defined subjective night depends on the photoperiod. The results indicate that the subjective night may be defined as the time interval when i) light stimuli can reset the circadian system, ii) pineal melatonin production and photic induction of the c-Fos gene in the ventrolateral SCN are high, and iii) the spontaneous c-Fos protein production in the dorsomedial SCN is low. Such a defined subjective night and, logically, the whole circadian pacemaking system depend on the photoperiod and hence on the season of the year which the animals are experiencing.     

SCOP, a novel gene product expressed in a circadian manner in rat suprachiasmatic nucleus

To elucidate the mechanism of the circadian rhythm, genes differentially expressed during subjective day and night in the rat suprachiasmatic nucleus (SCN), a circadian oscillator in mammals, were surveyed by a differential display method. We isolated a novel gene, scop (SCN circadian oscillatory protein), that was expressed in a circadian manner in the SCN. SCOP protein is predominantly expressed in the brain and has domains including a pleckstrin homology domain, leucine-rich repeats, a protein phosphatase 2C-like domain and a glutamine-rich region. The structural feature of SCOP protein suggests its role in the intracellular signaling in the SCN.     

How do the suprachiasmatic nuclei of the hypothalamus integrate photoperiodic information?

The suprachiasmatic nucleus of the hypothalamus (SCN) plays an essential role in the generation and maintenance of circadian rhythms in mammals. The SCN activity is also dependent upon the photoperiod. The duration of the SCN sensitive phase to light, in term of Fos induction, is variable and tied to the length of the night. The question is how and by which pathways can photoperiod influence SCN? It is possible following the theoretical model of evening and morning component of the clock that the SCN build itself the photoperiodic signal. That the SCN integrate the photoperiodic information through indirect neural or neuroendocrine pathways is also to consider. Data in favor of these different interpretations are presented.     

Hamsters running on time: is the lateral habenula a part of the clock?

Previous anatomical and physiological studies have implicated the lateral habenula, and especially its medial division (LHbM), as a candidate component of the circadian timing system in rodents. We assayed lateral habenula rhythmicity in rodents using c-FOS immunohistochemistry and found a robust rhythm in immunoreactive cell counts in the LHbM, with higher counts during the dark phase of a light-dark (LD) cycle and during subjective night in constant darkness. We have also observed an obvious asymmetry of c-FOS expression in the LHbM of behaviorally "split" hamsters in constant light, but only during their active phase (when they were running in wheels). Locomotor activity rhythms appear to be regulated by the suprachiasmatic nucleus (SCN) via multiple output pathways, one of which might be diffusible while the other might be neural, involving the lateral habenula.     

RHYTHMIC cFOS EXPRESSION IN THE VENTRAL SUBPARAVENTRICULAR ZONE INFLUENCES GENERAL ACTIVITY RHYTHMS IN THE NILE GRASS RAT,ARVICANTHIS NILOTICUS

Circadian rhythms in behavior and physiology are very different in diurnal and nocturnal rodents. A pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus is responsible for generating and maintaining circadian rhythms in mammals, and cellular and molecular rhythms within the SCN of diurnal and nocturnal rodents are very similar. The neural substrates determining whether an animal has a diurnal or nocturnal phase preference are thus likely to reside downstream of the SCN. The ventral subparaventricular zone (vSPVZ), a major target of the SCN that is important for the expression of circadian rhythmicity in nocturnal lab rats (Rattus norvegicus), exhibits different rhythms in cFos expression in diurnal Nile grass rats compared to lab rats. We examined the effects of chemotoxic lesions of the cFos-expressing cells of the vSPVZ on activity rhythms of grass rats to evaluate the hypothesis that these cells support diurnality in this species. Male grass rats housed in a 12:12 light:dark (LD) cycle were given bilateral injections of the neurotoxin n-methyl-D-L-aspartic acid (NMA) or vehicle aimed at the vSPVZ; cells in the SCN are resistant to NMA, which kills neurons in other brain regions, but leaves fibers of passage intact. vSPVZ-damaged grass rats exhibited highly unstable patterns of activity in constant darkness (DD) and in the LD cycle that followed. However, crepuscular bouts of activity could be seen in all animals with vSPVZ lesions. Damage to the vSPVZ reduced cFos expression in this area but not in the SCN. Using correlational analyses, we found that the number of cFos-ir cells in the vSPVZ was unrelated to several parameters of the activity rhythms during the initial post-surgical period, when animals were in LD. However, the number of cells expressing cFos in the vSPVZ was positively correlated with general activity during the subjective day relative to the subjective night when the animals were switched to DD, and this pattern persisted when a LD cycle was reinstated. Also, the number of cFos-ir cells in the vSPVZ was negatively correlated with the strength of rhythmicity in DD and the number of days required to re-entrain to a LD cycle following several weeks in DD. These data suggest that the vSPVZ emits signals important for the expression of stable diurnal activity patterns in grass rats, and that species differences in these signals may contribute to differences in behavioral and physiological rhythms of diurnal and nocturnal mammals. (Author correspondence: mschw009@umaryland.edu)     

Circadian modulation in photic induction of Fos-like immunoreactivity in the suprachiasmatic nucleus cells of diurnal chipmunk,Eutamias asiaticus

Photic induction of immediate early genes including c-fos in the suprachiasmatic nucleus (SCN) has been well demonstrated in the nocturnal rodents. On the other hand, in diurnal rodents, no data is available whether the light can induce c-fos or Fos in the SCN. We therefore examined whether 60 min light exposure induces Fos-like immunoreactivity (Fos-lir) in the SCN cells of diurnal chipmunks and whether the induction is phase dependent, comparing with the results in nocturnal hamsters. We also examined an effect of light on the locomotor activity rhythm under continuous darkness. Fos-lir was induced in the chipmunk SCN. The induction was clearly phase dependent. The light during the subjective night induced strong expression of Fos-lir. This phase dependency is similar to that in hamsters. However, unlike in hamsters, the Fos-lir was induced in some SCN cells of chipmunks exposed to light during the subjective day. In the locomotor rhythm, on the other hand, the light pulse failed to induce the phase shift at phases at which the Fos-lir was induced. These results suggest that the photic induction of Fos-lir in the diurnal chipmunks is gated by a circadian oscillator as well as in the nocturnal hamsters. However, the functional role of Fos protein may be different in the diurnal rodents from in the nocturnal rodents.     

Distribution of vasoactive intestinal peptide-like and neurophysin-like immunoreactive neurons and acetylcholinesterase staining in the ring dove hypothalamus with emphasis on the question of an avian suprachiasmatic nucleus

Summary Two nuclei, termed here the medial hypothalamic nucleus and the lateral hypothalamic retinorecipient nucleus, are possible homologs of the mammalian suprachiasmatic nucleus. As the mammalian suprachiasmatic nucleus is characterized by a dense concentration of vasoactive intestinal peptide (VIP)-and neurophysin (NP)-immunoreactive neurons and an absence of acetylcholinesterase (AChE) staining, we decided to examine these factors in the ring dove hypothalamus. Neither the medial hypothalamic nucleus nor the lateral hypothalamic retinorecipient nucleus contained either VIP-or NP-like immunoreactive neurons. The lateral hypothalamic retinorecipient nucleus stained darkly for AChE. Although there was some overlap in the distribution of VIP-and NP-like immunoreactive neurons, a clustering of both types into a well defined nucleus was not observed. Therefore, an avian homolog to the mammalian suprachiasmatic nucleus must differ in its chemoarchitecture from that of mammalian species described to date.