Serotonin inhibits glutamate- but not PACAP-induced per gene expression in the rat suprachiasmatic nucleus at night

Circadian rhythms of physiology and behaviour generated by the brain's biological clock located in the suprachiasmatic nucleus are entrained by light via the retinohypothalamic tract. Two neurotransmitters, glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP), found in this monosynaptic pathway mediate the effects of light to the clock. It is well known that not only light entrains the clock. Nonphotic cues mediated by neurotransmitters such as serotonin reaching the suprachiasmatic nucleus from the midbrain raphe nucleus modulate light-induced phase shifts at night. Two clock genes, per1 and per2, have been attributed a role in light-induced phase shift. In the present study, using an in vitro brain slice model and quantitative in situ hybridization for per1 and per2, we have shown that serotonin induces per1 gene expression at late subjective night but not at early night. Furthermore, serotonin application before glutamate or PACAP blocked glutamate-induced per1 expression at early night and per2 gene expression at late night. In contrast, serotonin did not influence PACAP-induced per gene expression at late night. Triple antigen immunohistochemistry and confocal microscopy supported both a pre- and post-synaptic interaction of retinohypothalamic tract (PACAP-immunoreactive) and serotonin projections on vasoactive intestinal peptide- and gastrin-releasing peptide-containing cell bodies in the ventro-lateral suprachiasmatic nucleus. Our findings suggest that the per genes could be the molecular target for the modulatory effects of serotonin on light signalling to the clock.     

The rabbit pup, a natural model of nursing-anticipatory activity

Mother rabbits nurse their young once a day with circadian periodicity. Nursing bouts are brief (ca. 3 min) and occur inside the maternal burrow. Despite this limited contact mother rabbits and their pups are tuned to each other to ensure that the capacities of each party are used efficiently to ensure the weaning of a healthy litter. In this review we present behavioral, metabolic and hormonal correlates of this phenomenon in mother rabbits and their pups. Research is revealing that the circadian rhythm of locomotion shifts in parallel to the timing of nursing in both parties. In pups corticosterone has a circadian rhythm with highest levels at the time of nursing. Other metabolic and hormonal parameters follow an exogenous or endogenous rhythm which is affected by the time of nursing. In the brain, clock genes and their proteins (e.g. Per1) are differentially expressed in specific brain regions (e.g. suprachiasmatic nucleus, paraventricular nucleus) in relation to providing or ingesting milk in mothers and young, respectively. These findings suggest that circadian activities are modulated, in the mothers, by suckling stimulation and, in the young, by the ingestion of milk and/or the perception of the mammary pheromone. In conclusion, the rabbit pup is an extraordinary model for studying the entraining by a single daily food pulse with minimal manipulations. The mother offers the possibility of studying nursing as a non-photic synchronizer, also with minimal manipulation, as suckling stimulation from the litter occurs only once daily.     

GABAergic regulation of light-induced c-Fos immunoreactivity within the suprachiasmatic nucleus.

Analysis of the photic induction of c-Fos immunoreactivity (-ir) within the suprachiasmatic nucleus (SCN) has proven to be a powerful tool with which to study the neurochemical mechanisms involved in phase shifting the circadian clock. Some systemically administered GABAergic drugs inhibit light-induced phase shifts and c-Fos-ir, whereas others inhibit light-induced phase shifts without affecting c-Fos-ir. More recently, we have found that injection of GABAergic drugs directly into the SCN region can have dramatically different effects on light-induced phase shifts than following their systemic administration. The present study investigated the effects of GABA(A) and GABA(B) agonists and antagonists injected into the SCN region on c-Fos-ir within the SCN. Microinjection of either a GABA(A) agonist, muscimol, or a GABA(B) agonist, baclofen, into the SCN region significantly reduced light-induced c-Fos-ir within the SCN when administered before light exposure at circadian time (CT) 13.5 or CT 19. In contrast, microinjection of a GABA(A) antagonist, bicuculline, but not a GABA(B) antagonist, CGP-35348, into the SCN region increased light-induced c-Fos-ir within the SCN when administered before light exposure at CT 13.5 or CT 19. These data indicate that GABAergic agonists and antagonists injected directly into the SCN region alter light-induced Fos-ir in a manner similar to their effects on light-induced phase shifts. Comparison of these data with previous studies examining the effects of systemically administered GABAergic drugs suggests that GABA(B)-active drugs have similar effects whether given systemically or within the SCN, but that GABA(A)-active drugs have more complex effects on c-fos induction and have multiple sites of action.     

Expression profiles of PER2 immunoreactivity within the shell and core regions of the rat suprachiasmatic nucleus

The circadian clock cells of the mammalian suprachiasmatic nucleus (SCN) generate oscillations in physiology and behavior that are synchronized (entrained) by the external light/dark (LD) cycle. The mechanisms that mediate the effect of light on the core molecular mechanism of the clock are not well understood, but evidence suggests that the Period2 gene, which encodes a key clock regulator (PER2), might be involved. We assessed the expression of PER2 immunoreactivity in the retinorecipient core and shell compartments of the SCN of rats entrained to cycles of discrete light pulses presented at the early subjective day (dawn) or night (dusk), or housed in constant light. We found that in animals entrained to a 0.5 h:23.5-h LD cycle (light falls near dawn), PER2 expression is rhythmic both in the shell and in the core regions of the SCN and indistinguishable from that seen in the SCN of control rats housed in complete darkness. Similarly, the pattern of PER2 expression in the SCN of rats entrained to a 0.5-h:25.5-h LD cycle (light falls near dusk) resembled that in dark-housed controls. We also found that presentation of a discrete light pulse in the early subjective night did not induce PER2 protein expression in the SCN, even 6 h after photic stimulation. Finally, in constant light-housed, behaviorally arrhythmic rats, PER2 expression in the SCN was low and nonrhythmic. These results show that rhythmic PER2 expression occurs both in the shell and core regions of the rat SCN. Furthermore, they show that the expression of PER2 in the SCN is not regulated by entraining light. Finally, constant light-induced behavioral arrhythmicity is associated with a disruption of rhythmic PER2 expression in the whole SCN. Together, the results are consistent with a proposed role for PER2 in the core mechanism of the circadian clock but argue against an important role for PER2 in the mechanism mediating photic entrainment.     

The dorsomedial hypothalamic nucleus is not necessary for food-anticipatory circadian rhythms of behavior, temperature or clock gene expression in mice

Circadian rhythms in mammals are regulated by a light-entrainable circadian pacemaker in the hypothalamic suprachiasmatic nucleus and food-entrainable oscillators located elsewhere in the brain and body. The dorsomedial hypothalamic nucleus (DMH) has been proposed to be the site of oscillators driving food-anticipatory circadian rhythms, but this is controversial. To further evaluate this hypothesis, we measured clock gene, temperature and activity rhythms in intact and DMH-ablated mice. A single 4-h midday feeding after an overnight fast induced mPer1 and mPer2 mRNA expression in the DMH, arcuate nucleus, nucleus of the solitary tract and area postrema, and reset daily rhythms of mPer1 , mPer2 and mBMAL1 in the DMH, arcuate and neocortex. These rhythms persisted during 2 days of food deprivation after 12 days of scheduled daytime feeding. Acute induction of DMH mPer1 and mPer2 was N -methyl- d -aspartate (NMDA) receptor-dependent, whereas rhythmic expression after 6 days of restricted feeding was not. Thermal DMH lesions did not affect acute induction or rhythmic expression of clock genes in other brain regions in response to scheduled daytime feeding. DMH lesions attenuated mean daily activity levels and nocturnality but did not affect food-anticipatory rhythms of activity and body temperature in either light–dark or constant darkness. These results confirm that the DMH and other brain regions express circadian clock gene rhythms sensitive to daytime feeding schedules, but do not support the hypothesis that DMH oscillations drive food-anticipatory behavioral or temperature rhythms.     

Development of the light sensitivity of the clock genes Period1 and Period2, and immediate-early gene c-fos within the rat suprachiasmatic nucleus

The molecular mechanism underlying circadian rhythmicity within the suprachiasmatic nuclei (SCN) of the hypothalamus has two light-sensitive components, namely the clock genes Per1 and Per2 . Besides, light induces the immediate-early gene c-fos . In adult rats, expression of all three genes is induced by light administered during the subjective night but not subjective day. The aim of the present study was to ascertain when and where within the SCN the photic sensitivity of Per1 , Per2 and c-fos develops during early postnatal ontogenesis. The specific aim was to find out when the circadian clock starts to gate photic sensitivity. The effect of a light pulse administered during either the subjective day or the first or second part of the subjective night on gene expression within the rat SCN was determined at postnatal days (P) 1, 3, 5 and 10. Per1 , Per2 and c-fos mRNA levels were assessed 30 min, 1 and 2 h after the start of each light pulse by in situ hybridization histochemistry. Expression of Per1 and c-fos was light responsive from P1, and the responses began to be gated by the circadian clock at P3 and P10, respectively. Expression of Per2 was only slightly light responsive at P3, and the response was not fully gated until P5. These data demonstrate that the light sensitivity of the circadian clock develops gradually during postnatal ontogenesis before the circadian clock starts to control the response. The photoinduction of the clock gene Per2 develops later than that of Per1 .     

Perspectives in affective disorders: Clocks and sleep

Mood disorders are often characterised by alterations in circadian rhythms, sleep disturbances and seasonal exacerbation. Conversely, chronobiological treatments utilise zeitgebers for circadian rhythms such as light to improve mood and stabilise sleep, and manipulations of sleep timing and duration as rapid antidepressant modalities. Although sleep deprivation (“wake therapy”) can act within hours, and its mood‐elevating effects be maintained by regular morning light administration/medication/earlier sleep, it has not entered the regular guidelines for treating affective disorders as a first‐line treatment. The hindrances to using chronotherapeutics may lie in their lack of patentability, few sponsors to carry out large multi‐centre trials, non‐reimbursement by medical insurance and their perceived difficulty or exotic “alternative” nature. Future use can be promoted by new technology (single‐sample phase measurements, phone apps, movement and sleep trackers) that provides ambulatory documentation over long periods and feedback to therapist and patient. Light combinations with cognitive behavioural therapy and sleep hygiene practice may speed up and also maintain response. The urgent need for new antidepressants should hopefully lead to reconsideration and implementation of these non‐pharmacological methods, as well as further clinical trials. We review the putative neurochemical mechanisms underlying the antidepressant effect of sleep deprivation and light therapy, and current knowledge linking clocks and sleep with affective disorders: neurotransmitter switching, stress and cortico‐limbic reactivity, clock genes, cortical neuroplasticity, connectomics and neuroinflammation. Despite the complexity of multi‐system mechanisms, more insight will lead to fine tuning and better application of circadian and sleep‐related treatments of depression.     

Scheduled meal accelerates entrainment to a 6‐h phase advance by shifting central and peripheral oscillations in rats

Travelling across several time zones requires a fast adjustment of the circadian system and the differential adjustment speeds of organs and systems results in what is commonly referred as jet lag. During this transitory state of circadian disruption, individuals feel discomfort, appetite loss, fatigue, disturbed sleep and deficient performance of multiple tasks. We have demonstrated that after a 6‐h phase advance of the light–dark cycle (LD) scheduled food in phase with the new night onset can speed up re‐entrainment. In this study, we explored the possible mechanisms underlying the fast re‐entrainment due to the feeding schedule. We focused on first‐ and second‐order structures that provide metabolic information to the suprachiasmatic nucleus (SCN). We compared (i) control rats without change in LD cycle; (ii) rats exposed to a 6‐h phase advance of the LD cycle with food ad libitum; and (iii) rats exposed to the 6‐h phase advance combined with food access in phase with the new night. We found an immediate synchronizing effect of food on stomach distention and on c‐Fos expression in the nucleus of the solitary tract, arcuate nucleus of the hypothalamus, dorsomedial hypothalamic nucleus and paraventricular nucleus. These observations indicate that in a model of jet lag, scheduled feeding can favour an immediate shift in first‐ and second‐order relays to the SCN and that by keeping feeding schedules coupled to the new night, a fast re‐entrainment may be achieved by shifting peripheral and extra‐SCN oscillations.     

Experimental jetlag disrupts circadian clock genes but improves performance in racehorses after light-dependent rapid resetting of neuroendocrine systems and the rest-activity cycle

Abrupt alterations in the 24-h light : dark cycle, such as those resulting from transmeridian air travel, disrupt circadian biological rhythms in humans with detrimental consequences on cognitive and physical performance. In the present study, a jetlag-simulated phase shift in photoperiod temporally impaired circadian peaks of peripheral clock gene expression in racehorses but acutely enhanced athletic performance without causing stress. Indices of aerobic and anaerobic capacities were significantly increased by a phase-advance, enabling prolonged physical activity before fatigue occurred. This was accompanied by rapid re-entrainment of the molecular clockwork and the circadian pattern of melatonin, with no disturbance of the adrenal cortical axis, but a timely rise in prolactin, which is a hormone known to target organs critical for physical performance. Subsequent studies showed that, unlike the circadian pattern of melatonin, and in contrast to other species, the daily rhythm of locomotor activity was completely eliminated under constant darkness, but it was restored immediately upon the reintroduction of a light : dark cycle. Resetting of the rhythm of locomotion was remarkably fast, revealing a rapid mechanism of adaptation and a species dependency on light exposure for the expression of daily diurnal activity. These results show that horses are exquisitely sensitive to sudden changes in photoperiod and that, unlike humans, can benefit from them; this appears to arise from powerful effects of light underlying a fast and advantageous process of adjustment to the phase shift.     

Targeted mutation of the calbindin D 28k gene selectively alters nonvisual photosensitivity

Light intensity is an important determinant of diverse physiological and behavioral responses within the non-image-forming visual system. Thresholds differ among various photic responses, namely control of circadian rhythms, vigilance state, activity level and pupil constriction, but the mechanisms that regulate photosensitivity are not known. Calbindin D(28k) (CalB) is a calcium-binding protein associated with light processing in the mammalian circadian clock. Loss-of-function studies indicate that CalB-deficient mice (CalB(-/-)) have deficits in their ability to entrain to light-dark cycles. To explore the role of CalB in modulating photosensitivity, thresholds for three behaviors mediated by the non-image-forming visual system (entrainment, masking and pupillary light reflex; PLR) were compared in CalB(-/-) and wildtype mice, and the localization of CalB protein in these circuits was examined in adult and juvenile mice. The results reveal a divergence in how CalB affects thresholds to photic cues among these responses. Entrainment and masking were 40- to 60-fold less sensitive in CalB(-/-) than in wildtype mice. On the other hand, the PLR in CalB(-/-) mice was 80- to 200-fold more sensitive. Though CalB is expressed in the retina and in brain circuits regulating entrainment we found no CalB expression in any component of the PLR pathway, namely the olivary pretectal nucleus, Edinger-Westphal nucleus and ciliary ganglion. The behavioral and anatomical data together suggest that, in normal animals, the retinal response to light is blunted in the presence of CalB, but responsiveness of the higher order processes that transduce afferent retinal input is enhanced.     

Unpredictable feeding schedules unmask a system for daily resetting of behavioural and metabolic food entrainment

Restricted feeding schedules (RFS) are a potent Zeitgeber that uncouples daily metabolic and clock gene oscillations in peripheral tissues from the suprachiasmatic nucleus (SCN), which remains entrained to the light-dark cycle. Under RFS, animals develop food anticipatory activity (FAA), characterized by arousal and increased locomotion. Food availability in nature is not precise, which suggests that animals need to adjust their food-associated activity on a daily basis. This study explored the capacity of rats to adjust to variable and unpredictable feeding schedules. Rats were exposed either to RFS with fixed daily meal (RF) or to a variable meal time (VAR) during the light phase. RF and VAR rats exhibited daily metabolic oscillations driven by the last meal event; however, VAR rats were not able to show a robust adjustment in the anticipating corticosterone peak. VAR rats were unable to exhibit FAA but exhibited a daily activation pattern in phase with the previous meal. In both groups the dorsomedial nucleus of the hypothalamus and arcuate nucleus, involved in energy balance, exhibited increased c-Fos expression 24 h after the last meal, while only RF rats exhibited low c-Fos expression in the SCN. Data show that metabolic and behavioural food-entrained rhythms can be reset on a daily basis; the two conditions elicit a similar hypothalamic response, while only the SCN is inhibited in rats exhibiting anticipatory activity. The variable feeding strategy uncovered a rapid (24-h basis) resetting mechanism for metabolism and general behaviour.     

Circuit development in the master clock network of mammals

Daily rhythms are generated by the circadian timekeeping system, which is orchestrated by the master circadian clock in the suprachiasmatic nucleus (SCN) of mammals. Circadian timekeeping is endogenous and does not require exposure to external cues during development. Nevertheless, the circadian system is not fully formed at birth in many mammalian species and it is important to understand how SCN development can affect the function of the circadian system in adulthood. The purpose of the current review is to discuss the ontogeny of cellular and circuit function in the SCN, with a focus on work performed in model rodent species (i.e., mouse, rat, and hamster). Particular emphasis is placed on the spatial and temporal patterns of SCN development that may contribute to the function of the master clock during adulthood. Additional work aimed at decoding the mechanisms that guide circadian development is expected to provide a solid foundation upon which to better understand the sources and factors contributing to aberrant maturation of clock function.     

Flies and fish: birds of a feather

The identification of specific clock-containing structures has been a major endeavour of the circadian field for many years. This has lead to the identification of many key components of the circadian system, including the suprachiasmatic nucleus in mammals, and the eyes and pineal glands in lower vertebrates. However, the idea that these structures represent the only clocks in animals has been challenged by the discovery of peripheral pacemakers in most organs and tissues, and even a number of cell lines. In Drosophila, and vertebrates such as the zebrafish, these peripheral clocks appear to be highly autonomous, being set directly by the environmental light/dark cycle. However, a hierarchy of clocks may still exist in mammals. In this review, we examine some of the current views regarding peripheral clocks, their organization and how they are entrained.     

The photoperiod entrains the molecular clock of the rat pineal

The suprachiasmatic nucleus-pineal system acts as a neuroendocrine transducer of seasonal changes in the photoperiod by regulating melatonin formation. In the present study, we have investigated the extent to which the photoperiod entrains the nonself-cycling oscillator in the Sprague-Dawley rat pineal. For this purpose, the 24-h expression of nine clock genes (bmal1, clock, per1, per2, per3, cry1, cry2, dec1 and dec2) and the aa-nat gene was monitored under light-dark 8 : 16 and light-dark 16 : 8 in the rat pineal by using real-time RT-PCR. The 24-h pattern of the expression of only per1, dec2 and aa-nat genes was affected by photoperiod. In comparison with the short photoperiod, the duration of elevated expression under the long photoperiod was elongated for per1 and shortened for dec2 and aa-nat. For each of the genes, photoperiod-dependent variations partly persisted under constant darkness. Therefore, the pineal clockwork appears to memorize the photoperiod of prior entrained cycles. The findings of the present study indicate that the nonself-cycling oscillator of the rat pineal is entrained by photoperiodic information and therefore that it participates in seasonal timekeeping.     

Aging and the clock: Perspective from flies to humans

Endogenous circadian oscillators regulate molecular, cellular and physiological rhythms, synchronizing tissues and organ function to coordinate activity and metabolism with environmental cycles. The technological nature of modern society with round‐the‐clock work schedules and heavy reliance on personal electronics has precipitated a striking increase in the incidence of circadian and sleep disorders. Circadian dysfunction contributes to an increased risk for many diseases and appears to have adverse effects on aging and longevity in animal models. From invertebrate organisms to humans, the function and synchronization of the circadian system weakens with age aggravating the age‐related disorders and pathologies. In this review, we highlight the impacts of circadian dysfunction on aging and longevity and the reciprocal effects of aging on circadian function with examples from Drosophila to humans underscoring the highly conserved nature of these interactions. Additionally, we review the potential for using reinforcement of the circadian system to promote healthy aging and mitigate age‐related pathologies. Advancements in medicine and public health have significantly increased human life span in the past century. With the demographics of countries worldwide shifting to an older population, there is a critical need to understand the factors that shape healthy aging. Drosophila melanogaster, as a model for aging and circadian interactions, has the capacity to facilitate the rapid advancement of research in this area and provide mechanistic insights for targeted investigations in mammals.