Fos expression within vasopressin-containing neurons in the suprachiasmatic nucleus of diurnal rodents compared to nocturnal rodents

The underlying neural causes of the differences between nocturnal and diurnal animals with respect to their patterns of rhythmicity have not yet been identified. These differences could be due to differences in some subpopulation of neurons within the suprachiasmatic nucleus (SCN) or to differences in responsiveness to signals emanating from the SCN. The experiments described in this article were designed to address the former hypothesis by examining Fos expression within vasopressin (VP) neurons in the SCN of nocturnal and diurnal rodents. Earlier work has shown that within the SCN of the diurnal rodent Arvicanthis niloticus, approximately 30% of VP-immunoreactive (IR) neurons express Fos during the day, whereas Fos rarely is expressed in VP-IR neurons in the SCN of nocturnal rats. However, in earlier studies, rats were housed in constant darkness and pulsed with light, whereas Arvicanthis were housed in a light:dark (LD) cycle. To provide data from rats that would permit comparisons with A. niloticus, the first experiment examined VP/Fos double labeling in the SCN of rats housed in a 12:12 LD cycle and perfused 4 h into the light phase or 4 h into the dark phase. Fos was significantly elevated in the SCN of animals sacrificed during the light compared to the dark phase, but virtually no Fos at either time was found in VP-IR neurons, confirming that the SCN of rats and diurnal Arvicanthis are significantly different in this regard. The authors also evaluated the relationship between this aspect of SCN function and diurnality by examining Fos-IR and VP-IR in diurnal and nocturnal forms of Arvicanthis. In this species, most individuals exhibit diurnal wheel-running rhythms, but some exhibit a distinctly different and relatively nocturnal pattern. The authors have bred their laboratory colony for this trait and used animals with both patterns in this experiment. They examined Fos expression within VP-IR neurons in the SCN of both nocturnal and diurnal A. niloticus kept on a 12:12 LD cycle and perfused 4 h into the light phase or 4 h into the dark phase, and brains were processed for immunohistochemical identification of Fos and VP. Both the total number of Fos-IR cells and the proportion of VP-IR neurons containing Fos (20%) were higher during the day than during the night. Neither of these parameters differed between nocturnal and diurnal animals. The implications of these findings are discussed.     

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 5HT_1A or 5ht₇ receptors located on SCN neurons; and 2) a presynaptic mechanism mediated via 5HT_1B 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.     

Daily rhythms in olfactory discrimination depend on clock genes but not the suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) regulates a wide range of daily behaviors and has been described as the master circadian pacemaker. The role of daily rhythmicity in other tissues, however, is unknown. We hypothesized that circadian changes in olfactory discrimination depend on a genetic circadian oscillator outside the SCN. We developed an automated assay to monitor olfactory discrimination in individual mice throughout the day. We found olfactory sensitivity increased approximately 6-fold from a minimum during the day to a peak in the early night. This circadian rhythm was maintained in SCN-lesioned mice and mice deficient for the Npas2 gene but was lost in mice lacking Bmal1 or both Per1 and Per2 genes. We conclude that daily rhythms in olfactory sensitivity depend on the expression of canonical clock genes. Olfaction is, thus, the first circadian behavior that is not based on locomotor activity and does not require the SCN.     

Photoperiodic control of circadian activity rhythms in diurnal rodents

The responses of red squirrels(Tamiasciurus hudsonicus) and eastern chipmunks(Tamias striatus) to complete and skeleton light-dark (LD) cycles were compared. The skeletons, comprised of two 1-h pulses of light per day, effectively simulated the complete photoperiods in the squirrels, but not the chipmunks. Skeleton photoperiods greater than 12-h caused the chipmunks to shift activity from the longer to the shorter of the two intervals between the pulses. To interpret the mechanism of phase control, squirrels and chipmunks were kept in continuous darkness and exposed to 1-h light pulses every 10 days. The time-course of entrainment was also quantified. Both techniques produced light-response curves. The data suggest that the parametric and non-parametric contributions to entrainment are different in these two rodent species.Presented at the Eighth International Congress of Biometeorology, 9–14 September 1979, Shefayim, Israel.     

Comparative anatomy of the mammalian hypothalamic suprachiasmatic nucleus

A detailed analysis of the cytoarchitecture, retinohypothalamic tract (RHT) projections, and immunohistochemical localization of major cell and fiber types within the hypothalamic suprachiasmatic nuclei (SCN) was conducted in five mammalian species: two species of opossum, the domestic cat, the guinea pig, and the house mouse. Cytoarchitectural and immunohistochemical studies were conducted in three additional species of marsupial mammals and in the domestic pig. The SCN in this diverse transect of mammalian taxonomy bear striking similarities. First, the SCN are similar in location, lying close to the third ventricle (3V) dorsal to the optic chiasm (OC), with a cytoarchitecture characterized by small, tightly packed neurons. Second, in all groups studied, the SCN receive bilateral retinal input. Third, the SCN contain immunohistochemically similar elements. These similarities suggest that the SCN developed characteristic features early in mammalian phylogeny. Some details of SCN organization vary among the species studied. In marsupials, vasopressin-like immunoreactive (VP-LI) and vasoactive intestinal polypeptide-like immunoreactive (VIP-LI) cells codistribute primarily in the dorsomedial aspects of the SCN, while in eutherians, VP-LI and VIP-LI cells are separated into SCN subnuclei. Furthermore, the marsupial RHT projects to the periventricular dorsomedial region, whereas the eutherian RHT projects more ventrally in the SCN into the zone that typically contains VIP-LI perikarya.     

Characterization of melatonin-induced fos-like immunoreactivity in the hypothalamic suprachiasmatic nucleus of the rat.

The hypothalamic suprachiasmatic nucleus (SCN) is primarily responsible for the regulation of circadian rhythmicity. Melatonin, the pineal-derived neurohormone, modulates the rhythmic output of the SCN. Property timed exposure to melatonin is able to induce changes in rhythmic function and thereby entrain circadian rhythms of activity. c-fos is an immediate early gene that is transiently expressed in neurons in response to receptor activation. The ventrolateral portion of the SCN (vSCN) is activated in response to phase-shifting stimuli, an event which is marked by an increase in the expression of c-fos.     

Postnatal development of the suprachiasmatic hypothalamic nucleus of the rat

Summary Light and electron microscopy of newborn, four day, one, two, three and five week old rats revealed principally a progressive increase in the diversity and number of synaptic contacts in the suprachiasmatic nucleus (SCN). The major increase in synaptic diversity occurred between four days and one week of age. Correlation between this finding and the adult synaptic morphology of SCN (Güldner, 1976) on the one hand, and the ontogeny of circadian rhythms on the other were made. This suggested that the retinal afferents arriving on day four form asymmetrical contacts with dendrites. While increase in synaptic number was progressive, it was most marked between three and five weeks of age. By five weeks, most features of the adult SCN were present. No significant morphological effects were evident as a result of neonatal retinal lesions.Supported in part by grants NS-12265, NS-12267, HD04583 and HD-08658 from the National Institutes of Health, USPHS. The electron microscopic facilities of the California Regional Primate Center, supported by NIH grant RR-00169, were utilized. The technical assistance of Mrs. Viviana Wong is gratefully acknowledged. A preliminary report of a portion of this data was given at the Society for Neuroscience, November, 1974 in St. Louis, Missouri.     

Studies in diurnal rhythms ix the water-relations of some nocturnal tropical arthropods

The rate of water-loss from tropical African centipedes (Rbysida nuda and Ethmostigmus trigonopodus) and millipedes (Oxydesmus platycercus and Ophistreptus sp.) is proportional to the saturation deficiency of the atmosphere. These animals therefore resemble forms from temperate and desert regions in this aspect of their physiology. Their nocturnal habits are probably related to their high rate of transpiration. The endogenous 24-hour rhythm shows a slight retardation in constant light and an acceleration in darkness. The probable function of this is to synchronize it with diurnal environmental changes. The West African scorpion, Pandinus imperator, has a critical temperature considerably lower than those of desert species and comparable with that of the Italian Euscorpius germanus.

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Zusammenfassung Bie den tropischen afrikanischen Hundertfüßlern Rhysida nuda und Ethmostigmus trigonopodus, den Tausendfüßlern Oxydesmus platycercus und Ophistreptus sp. sowie bei dem Skorpion Pandinus imperator wurden die Wasserverlustraten durch Wägen der Individuen bestimmt, die über Phosphorpentoxyd in konischen Flaschen aufgehängt wurden, die in ein Wasserbad tauchten. Die Tiere wurden jeder Temperatur 20 Minuten lang ausgesetzt, bevor der durch Evaporation bedingte Betrag des Wasserverlustes bestimmt wurde. Es wird gezeigt, daß diese Methode wertvolle Vergleiche zwischen den verschiedenen Arten zu ziehen gestattet. Sowohl bei Hundertfüßlern wie auch bei Tausendfüßlern erwies sich die Transpiration als proportional zum Sättigungsdefizit der Luft. Diese Tiere ähneln deshalb in diesem Punkte ihrer Physiologie Arten aus gemäßigten und Wüsten-Gebieten.Ihre nächtliche Lebensweise, die mittels Aktographen festgestellt wurde, steht wahrscheinlich in Beziehung zu der vergleichsweise hohen Wasserverlustrate. Der endogene diurnale Rhythmus ziegt eine leichte Verzögerung bei Dauerbelechtung und eine Beschleunigung bei Dunkelheit. Auf diese Weise wird wahrscheinlich die Periodizität dieser Tiere mit dem 24-Stunden-Rhythmus der Umgebung synchronisiert.Die kritsche Temperatur des westafrikanischen Skorpions Pandinus imperator liegt beträchtlich niedriger als die der Wüsten-Arten und ähnelt der des italienischen Euscorpius germanus. Es wird vermutet, daß in diesem Falle die nächtliche Lebensweise in Beziehung steht zu der Gefährdung des großen, langsamen Tieres durch räuberische Wirbeltiere.Es wird geschlossen, daß im allgemeinen die Transpirationsrate in Beziehung zum Habitat steht und bei Wüstenbewohnern am niedrigsten ist.

     

Involvement of the suprachiasmatic nucleus in diurnal ACTH and corticosterone responsiveness to stress

We explored the contribution of the suprachiasmatic nucleus (SCN) in ACTH and corticosterone (CORT) diurnal responsiveness of the rat to restraint stress applied either in the morning (AM) or in the evening (PM). Ablation of the SCN caused the diurnal rhythmicity of the CORT response to disappear but had no effects on AM vs. PM differences in the ACTH response. Stress-response curves in SCN-lesioned rats that had prestress levels of CORT either in the AM range or in the PM range, when compared with those obtained for AM and PM controls, showed that the SCN differentially regulates the stress response depending on the underlying secretory activity of the adrenal cortex. When basal CORT secretion is at its lowest, the SCN inhibits CORT responsiveness to stress by controlling pituitary corticotrophs; but when it is at its highest, it has a permissive action that will bypass the hypophysis and reach the adrenals to adjust the response of the gland to ACTH.     

Restraint stress delays reentrainment in male and female diurnal and nocturnal rodents

A temporary loss of normal circadian entrainment, such as that associated with shift work and transmeridian travel, can result in an array of detrimental symptoms, making rapid reentrainment of rhythmicity essential. While there is a wealth of literature examining the effects of stress on the entrained circadian system, less is known about the influence of stress on circadian function following a phase shift of the light: dark (LD) cycle. The authors find that recovery of locomotor activity synchronization is altered by restraint stress in the diurnal rodent Octodon degus (degu) and the nocturnal rat. In the first experiment, degus were subjected to a 6-h phase advance of the LD cycle. Sixty minutes after the new lights-on, animals underwent 60 min of restraint stress. The number of days it took each animal to reentrain its activity rhythms to the new LD cycle was recorded and compared to the number of days it took the animal to reentrain under control conditions. When subjected to restraint stress, degus took 30% longer to reentrain their activity rhythms (p < 0.01). In a second experiment, rats underwent a similar experimental paradigm. As with the degus, stress significantly delayed the reentrainment of rats' activity rhythms (p < 0.01). There was no interaction between sex and stress on the rate of reentrainment for either rats or degus. Furthermore, there was no effect of stress on the free-running activity rhythm of degus, suggesting that the effect of stress on reentrainment rate is not secondary to alterations of period length. Together, these data point to a detrimental effect of stress on recovery of entrainment of circadian rhythms, which is independent of activity niche and sex.     

The suprachiasmatic nucleus exhibits diurnal variations in spontaneous excitatory postsynaptic activity

A most prominent feature of neurons in the suprachiasmatic nucleus (SCN) is the circadian rhythm in spontaneous firing frequency. To disclose synaptic mechanisms associated with the rhythmic activity, the spontaneous postsynaptic activity was studied using whole-cell, patch clamp recordings in the ventral region of the SCN in slice preparations from rats. The synaptic events were compared between two time intervals corresponding to the highest and lowest electrical activity within the SCN during subjective daytime and nighttime, respectively. The gamma-aminobutyric acid (GABA)-mediated spontaneous inhibitory activity showed no diurnal variations, but the excitatory activity was markedly higher in frequency, without differences in amplitude, during the subjective day compared to the subjective night. Spontaneous and evoked inhibitory synaptic events were blocked by the GABA(A) receptor antagonist bicuculline. The alpha-amino-hydroxy-5-methylisoxazole-4-propionic acid (AMPA/kainate) receptor antagonist 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) blocked most of the excitatory activity. In addition, CNQX reduced the spontaneous inhibitory activity. The N-methyl-D-aspartate antagonist D-2-amino-5-phosphonopentanoic acid reduced the inhibitory activity to a lesser degree, and there was no significant difference in amplitude or frequency of synaptic events in control and Mg2+-free solutions, indicating that the AMPA receptor plays an important role in regulating the inhibitory release of GABA within the SCN. Ipsi- and contralateral stimulation of the SCN consistently evoked excitatory synaptic responses. Inhibitory synaptic responses occurred in some neurons upon increasing stimulus strength. In conclusion, this study shows that there is a substantial influence from spontaneous glutamatergic synapses on the ventral part of the SCN and that these exhibit daily variations in activity. Diurnal fluctuations in spontaneous excitatory postsynaptic activity within this network may contribute to the mechanisms for synchronization of rhythms between individual SCN neurons and may underlie the daily variations in the spontaneous firing frequency of SCN neurons.     

c-fos expression in the putative avian suprachiasmatic nucleus

c-fos induction was investigated as a potential component in the avian photic entrainment pathway and as a possible means of locating the central pacemaker in birds. In both quail (Coturnix coturnix japonica) and starlings (Sturnus vulgaris) exposure to 1 h of light induced Fos-lir in the visual suprachiasmatic nucleus but not in the medial suprachiasmatic nucleus. However, the degree of c-fos induction in the visual suprachiasmatic nucleus was similar at different circadian times despite the fact that the light pulses caused differential phase shifts in the locomotor rhythm. For golden hamsters the same experiment resulted in significantly different levels of Fos-lir in the suprachiasmatic nucleus, as well as different phase shifts. Starlings and hamsters were also entrained to T-cycles that caused a large daily phase shift (T = 21.5 h in starlings, T = 22.67 hours in hamsters), or no daily phase shift (T = free running period). No difference in the induced levels of Fos-lir in the visual suprachiasmatic nucleus region was observed between the two groups of starlings, but in hamsters there were significantly different levels of Fos-lir in the suprachiasmatic nucleus between the two groups. Accepted: 15 November 1996     

Circadian activity rhythms and phase-shifting of cultured neurons of the rat suprachiasmatic nucleus

The mammalian suprachiasmatic nucleus (SCN) is the major endogenous pacemaker that coordinates various daily rhythms including locomotor activity and autonomous and endocrine responses, through a neuronal and humoral influence. In the present study we examined the behavior of dispersed individual SCN neurons obtained from 1- to 3-day-old rats cultured on multi-microelectrode arrays (MEAs). SCN neurons were identified by immunolabeling for the neuropeptides arginine-vasopressin (AVP) and vasoactive intestinal polypeptide (VIP). Single SCN neurons cultured at low density onto an MEA can express firing rate patterns with different circadian phases. In these cultures we observed rarely synchronized firing patterns on adjacent electrodes. This suggests that, in cultures of low cell densities, SCN neurons function as independent pacemakers. To investigate whether individual pacemakers can be influenced independently by phase-shifting stimuli, we applied melatonin (10 pM to 100 nM) for 30 min at different circadian phases and continuously monitored the firing rate rhythms. Melatonin could elicit phase-shifting responses in individual clock cells which had no measurable input from other neurons. In several neurons, phase-shifts occurred with a long delay in the second or third cycle after melatonin treatment, but not in the first cycle. Phase-shifts of isolated SCN neurons were also observed at times when the SCN showed no sensitivity to these phase-shifting stimuli in recordings from brain slices. This finding suggests that the neuronal network plays an essential role in the control of phase-shifts.     

Daily changes of neuropeptide Y-like immunoreactivity in the suprachiasmatic nucleus of the rat

Most of the biochemical, physiological and behavioural events in living organisms show diurnal fluctuations, normally synchronized with 24-h environmental rhythms, such as the light-dark cycle. The suprachiasmatic nucleus (SCN) of the hypothalamus is considered to be a pacemaker of the circadian rhythms in several mammals. The light-dark cycle is the primary synchronizing agent for many of the circadian rhythms which are regulated by the SCN. The photic information reaches the SCN also through a neuropeptide Y(NPY)-like immunoreactive pathway from the ventro-lateral geniculate nucleus. We found that in 12-h-dark and 12-h-light housed rats the NPY-like immunoreactive innervation of the ventro-lateral part of the SCN shows a 24 h rhythm with values rising gradually during the light phase and falling during the dark phase. Besides this rhythm, we found two peaks corresponding to the switching on and switching off of the light. The average level of NPY-like immunoreactivity, as assessed by means of semiquantitative immunocytochemistry and expressed in 'arbitrary units', is reduced in rats housed in total darkness for 2 weeks. These results confirm the physiological role of NPY in the timing of the circadian activity of the SCN.     

Development of hamster circadian rhythms: Role of the maternal suprachiasmatic nucleus

Summary During development, the circadian rhythms of rodents become entrained to rhythmicity of the mother. Rhythms in behavior and in neuroendocrine function are regulated by a circadian pacemaker thought to be located within the suprachiasmatic nucleus (SCN) of the hypothalamus. Evidence indicates that this pacemaker begins to function and to be entrained by maternal rhythms before birth. Although the maternal rhythms which mediate prenatal entrainment of the fetal circadian pacemaker have not been identified, it is likely that they are regulated by the maternal SCN.The role of the maternal SCN in entrainment of the offspring was examined in Syrian hamsters (Mesocricetus auratus) by measuring the activity/rest rhythms of pups. Using the synchrony among the rhythms of pups within a litter as an indication that the pups had been entrained, the effect on entrainment of ablating the maternal SCN was determined. Lesions of the maternal SCN which were performed early in gestation (day 7) and which destroyed at least 75% of the SCN were found to disrupt the normal within litter synchrony among pups, indicating interference with the normal mechanism of entrainment.The effect of lesions on day 7 of gestation could mean that the maternal SCN is important for entrainment of the pups before birth, after birth, or during both of these times. To determine if the maternal SCN is specifically important for prenatal entrainment, lesions were performed two days before birth on day 14 of gestation. Lesions of the maternal SCN on day 14 were not as disruptive as were lesions on day 7. This suggests that the maternal SCN is important between days 7 and 14 of gestation and that the synchrony normally observed at weaning is already established, in part, on or before day 14 of gestation. This further suggests that an entrainable circadian pacemaker is present in the fetus only two weeks after fertilization.Abbreviations SCN suprachiasmatic nucleus - L:D light:dark - LL constant light - r mean vector length - 2DG 2-deoxyglucose - NAT N-acetyltransferase     

Daily rhythms of P-glycoprotein expression in mice

Recent studies have shown the gene expression of several transporters to be circadian rhythmic. However, it remains to be elucidated whether the expression of P-glycoprotein, which is involved in the transport of many medications, undergoes 24 h rhythmicity. To address this issue, we investigated daily profiles of P-glycoprotein mRNA and protein levels in peripheral mouse tissues. In the liver and intestine, but not in the kidney, Abcb1a mRNA expression showed clear 24 h rhythmicity. On the other hand, Abcb1b and Abcb4, the other P-glycoprotein genes, did not exhibit significant rhythmic expression in the studied tissues. In the intestine, levels of whole P-glycoprotein also exhibited a daily rhythm, with a peak occurring in the latter half of the light phase and a trough at the onset of the light phase. Consistent with the day-night change of P-glycoprotein level, the ex vivo accumulation of digoxin, an Abcb1a P-glycoprotein substrate, into the intestinal segments at the onset of dark phase was significantly lower than it was at the onset of the light phase. Thus, Abcb1a P-glycoprotein expression, and apparently its function, are 24 h rhythmic at least in mouse intestine tissue. This circadian variation might be involved in various chronopharmacological phenomena.