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Individual neurons of the hypothalamic suprachiasmatic nuclei (SCN) contain an intracellular molecular clock that drives these neurons to exhibit day-night variation in excitability. The neuropeptide vasoactive intestinal polypeptide (VIP) and its cognate receptor, VPAC 2 , are synthesized by SCN neurons and this intercellular VIP-VPAC 2 receptor signal facilitates coordination of SCN neuronal activity and timekeeping. How the loss of VPAC 2 receptor signalling affects the electrophysiological properties and states of SCN neurons as well as their responses to excitatory inputs is unclear. Here we used patch-clamp electrophysiology and made recordings of SCN neurons in brain slices prepared from transgenic animals that do not express VPAC 2 receptors (Vipr2 -/- mice) as well as animals that do (Vipr2 +/+ mice). We report that while Vipr2 +/+ neurons exhibit coordinated day-night variation in their electrical state, Vipr2 -/- neurons lack this and instead manifest a range of states during both day and night. Further, at the population level, Vipr2 +/+ neurons vary the membrane threshold potential at which they start to fire action potentials from day to night, while Vipr2 -/- neurons do not. We provide evidence that Vipr2 -/- neurons lack a component of voltage-gated sodium currents that contribute to SCN neuronal excitability. Moreover, we determine that this aberrant temporal control of neuronal state and excitability alters neuronal responses to a neurochemical mimic of the light-input pathway to the SCN. These results highlight the critical role VIP-VPAC 2 receptor signalling plays in the temporal expression of individual neuronal states as well as appropriate ensemble activity and input gating of the SCN neural network., (© 2024 The Author(s). European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Sleep is fundamental to health. The aim of this study was to analyse and determine factors predicting sleep quality during and after national lockdowns due to severe acute respiratory syndrome coronavirus 2 (COVID-19) in the UK. A longitudinal online survey-based study (SleepQuest) involving UK adults was administered in Spring 2020, Winter 2020, and Winter 2022 including questionnaires probing sleep quality, depression, anxiety, beliefs about sleep, demographics, COVID-19 status, and exercise. The primary outcome was sleep quality (Pittsburgh Sleep Quality Index). A linear mixed-effects model evaluated factors associated with baseline and longitudinal sleep quality. Complete data were provided by 3306 participants in Spring 2020, 2196 participants in Winter 2020, and 1193 in Winter 2022. Participants were mostly female (73.8%), white (97.4%), and aged over 50 years (81.0%). On average, participants reported poor sleep quality in Spring 2020 (mean [SD] Pittsburgh Sleep Quality Index score = 6.59 [3.6]) and Winter 2020 (mean [SD] Pittsburgh Sleep Quality Index score = 6.44 [3.6]), with improved but still poor sleep quality in Winter 2022 (mean [SD] Pittsburgh Sleep Quality Index score = 6.17 [3.5]). Improved sleep quality was driven by better subjective sleep and reduced daytime dysfunction and sleep latency. Being female, older, having caring responsibilities, working nightshifts, and reporting higher levels of depression, anxiety, and unhelpful beliefs about sleep were associated with worse baseline PSQI scores. Better sleep quality was associated with more days exercising per week at baseline. Interventions focusing on improving mental health, exercise, and attitudes towards sleep, particularly in at-risk groups, may improve sleep-related outcomes in future pandemics., (© 2024 The Authors. Journal of Sleep Research published by John Wiley & Sons Ltd on behalf of European Sleep Research Society.)

In mammals, intrinsic 24 h or circadian rhythms are primarily generated by the suprachiasmatic nuclei (SCN). Rhythmic daily changes in the transcriptome and proteome of SCN cells are controlled by interlocking transcription-translation feedback loops (TTFLs) of core clock genes and their proteins. SCN cells function as autonomous circadian oscillators, which synchronize through intercellular neuropeptide signalling. Physiological and behavioural rhythms can be severely disrupted by genetic modification of a diverse range of genes and proteins in the SCN. With the advent of next generation sequencing, there is unprecedented information on the molecular profile of the SCN and how it is affected by genetically targeted alteration. However, whether the expression of some genes is more readily affected by genetic alteration of the SCN is unclear. Here, using publicly available datasets from recent RNA-seq assessments of the SCN from genetically altered and control mice, we evaluated whether there are commonalities in transcriptome dysregulation. This was completed for four different phases across the 24 h cycle and was augmented by Gene Ontology Molecular Function (GO:MF) and promoter analysis. Common differentially expressed genes (DEGs) and/or enriched GO:MF terms included signalling molecules, their receptors, and core clock components. Finally, examination of the JASPAR database indicated that E-box and CRE elements in the promoter regions of several commonly dysregulated genes. From this analysis, we identify differential expression of genes coding for molecules involved in SCN intra- and intercellular signalling as a potential cause of abnormal circadian rhythms., (© 2024 The Author(s). European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

The orexinergic system of the lateral hypothalamus plays crucial roles in arousal, feeding behavior, and reward modulation. Most research has focused on adult rodents, overlooking orexins' potential role in the nervous system development. This study, using electrophysiological and molecular tools, highlights importance of orexinergic signaling in the postnatal development of the rodent dorsolateral geniculate nucleus (DLG), a primary visual thalamic center. Orexin activation of DLG thalamocortical neurons occurs in a brief seven-day window around eye-opening, concurrent to transient OX 2 receptor expression. Blocking OX 2 receptors during this period reduces sensitivity of DLG neurons to green and blue light and lowers spontaneous firing rates in adulthood. This research reveals critical and temporally confined role of orexin signaling in postnatal brain development, emphasizing its contribution to experience-dependent refinement in the DLG and its long-term impact on visual function., Competing Interests: Authors declare no conflict of interests., (© 2024 The Authors.)

Analysis of ex vivo Per2 bioluminescent rhythm previously recorded in the mouse dorsal vagal complex reveals a characteristic phase relationship between three distinct circadian oscillators. These signals represent core clock gene expression in the area postrema (AP), the nucleus of the solitary tract (NTS) and the ependymal cells surrounding the 4th ventricle (4Vep). Initially, the data suggests a consistent phasing in which the AP peaks first, followed shortly by the NTS, with the 4Vep peaking 8-9 h later. Wavelet analysis reveals that this pattern is not consistently maintained throughout a recording, however, the phase dynamics strongly imply that oscillator interactions are present. A simple phase model of the three oscillators is developed and it suggests that realistic phase dynamics occur between three model oscillators with coupling close to a synchronisation transition. The coupling topology suggests that the AP bidirectionally communicates phase information to the NTS and the 4Vep to synchronise the three structures. A comparison of the model with previous experimental manipulations demonstrates its feasibility to explain DVC circadian phasing. Finally, we show that simulating steadily decaying coupling improves the model's ability to capture experimental phase dynamics., (© 2023. The Author(s).)

The intergeniculate leaflet and ventral lateral geniculate nucleus (IGL/VLG) are subcortical structures involved in entrainment of the brain's circadian system to photic and non-photic (e.g. metabolic and arousal) cues. Both receive information about environmental light from photoreceptors, exhibit infra-slow oscillations (ISO) in vivo, and connect to the master circadian clock. Although current evidence demonstrates that the IGL/VLG communicate metabolic information and are crucial for entrainment of circadian rhythms to time-restricted feeding, their sensitivity to food intake-related peptides has not been investigated yet. We examined the effect of metabolically relevant peptides on the spontaneous activity of IGL/VLG neurons. Using ex vivo and in vivo electrophysiological recordings as well as in situ hybridisation, we tested potential sensitivity of the IGL/VLG to anorexigenic and orexigenic peptides, such as cholecystokinin, glucagon-like peptide 1, oxyntomodulin, peptide YY, orexin A and ghrelin. We explored neuronal responses to these drugs during day and night, and in standard vs. high-fat diet conditions. We found that IGL/VLG neurons responded to all the substances tested, except peptide YY. Moreover, more neurons responded to anorexigenic drugs at night, while a high-fat diet affected the IGL/VLG sensitivity to orexigenic peptides. Interestingly, ISO neurons responded to light and orexin A, but did not respond to the other food intake-related peptides. In contrast, non-ISO cells were activated by metabolic peptides, with only some being responsive to light. Our results show for the first time that peptides involved in the body's energy homeostasis stimulate the thalamus and suggest functional separation of the IGL/VLG cells. KEY POINTS: The intergeniculate leaflet and ventral lateral geniculate nucleus (IGL/VLG) of the rodent thalamus process various signals and participate in circadian entrainment. In both structures, cells exhibiting infra-slow oscillatory activity as well as non-rhythmically firing neurons being observed. Here, we reveal that only one of these two groups of cells responds to anorexigenic (cholecystokinin, glucagon-like peptide 1 and oxyntomodulin) and orexigenic (ghrelin and orexin A) peptides. Neuronal responses vary depending on the time of day (day vs. night) and on the diet (standard vs. high-fat diet). Additionally, we visualised receptors to the tested peptides in the IGL/VLG using in situ hybridisation. Our results suggest that two electrophysiologically different subpopulations of IGL/VLG neurons are involved in two separate functions: one related to the body's energy homeostasis and one associated with the subcortical visual system., (© 2023 The Authors. The Journal of Physiology © 2023 The Physiological Society.)

Timed daily access to a running-wheel (scheduled voluntary exercise; SVE) synchronizes rodent circadian rhythms and promotes stable, 24h rhythms in animals with genetically targeted impairment of neuropeptide signaling ( Vipr2 -/- mice). Here we used RNA-seq and/or qRT-PCR to assess how this neuropeptide signaling impairment as well as SVE shapes molecular programs in the brain clock (suprachiasmatic nuclei; SCN) and peripheral tissues (liver and lung). Compared to Vipr2 +/+ animals, the SCN transcriptome of Vipr2 -/- mice showed extensive dysregulation which included core clock components, transcription factors, and neurochemicals. Furthermore, although SVE stabilized behavioral rhythms in these animals, the SCN transcriptome remained dysregulated. The molecular programs in the lung and liver of Vipr2 -/- mice were partially intact, although their response to SVE differed to that of these peripheral tissues in the Vipr2 +/+ mice. These findings highlight that SVE can correct behavioral abnormalities in circadian rhythms without causing large scale alterations to the SCN transcriptome., Competing Interests: The authors declare no competing interests., (© 2023.)

The dorsal vagal complex (DVC) is a key hub for integrating blood-borne, central, and vagal ascending signals that convey important information on metabolic and homeostatic state. Research implicates the DVC in the termination of food intake and the transition to satiety, and consequently it is considered a brainstem satiety centre. In natural and laboratory settings, animals have distinct times of the day or circadian phases at which they prefer to eat, but if and how circadian signals affect DVC activity is not well understood. Here, we evaluate how intrinsic circadian signals regulate molecular and cellular activity in the area postrema (AP), nucleus of the solitary tract (NTS), and dorsal motor nucleus of the vagus (DMV) of the DVC. The hierarchy and potential interactions among these oscillators and their response to changes in diet are considered a simple framework in which to model these oscillators and their interactions is suggested. We propose possible functions of the DVC in the circadian control of feeding behaviour and speculate on future research directions including the translational value of knowledge of intrinsic circadian timekeeping the brainstem., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chrobok, Ahern and Piggins.)

Melatonin is a neurohormone released in a circadian manner with peak levels at night. Melatonin mediates its effects mainly through G protein-coupled MT 1 and MT 2 receptors. Drugs acting on melatonin receptors are indicated for circadian rhythm- and sleep-related disorders. Tools to study the activation of these receptors with high temporal resolution are lacking. Here, we synthesized a family of light-activatable caged compounds by attaching o -nitrobenzyl ( o- NB) or coumarin photocleavable groups to melatonin indolic nitrogen. All caged compounds showed the expected decrease in binding affinity for MT 1 and MT 2 . The o- NB derivative MCS-0382 showed the best uncaging and biological properties, with 250-fold increase in affinity and potency upon illumination. Generation of melatonin from MCS-0382 was further demonstrated by its ability to modulate the excitation of SCN neurons in rat brain slices. MCS-0382 is available to study melatonin effects in a temporally controlled manner in cellular and physiological settings.

Temporal partitioning of daily food intake is crucial for survival and involves the integration of internal circadian states and external influences such as the light-dark cycle and dietary composition. These intrinsic and extrinsic factors are interdependent with misalignment of circadian rhythms promoting body weight gain, while consumption of a calorie-dense diet elevates the risk of obesity and blunts circadian rhythms. Recently, we defined the circadian properties of the dorsal vagal complex of the brainstem, a structure implicated in the control of food intake and autonomic tone, but whether and how 24 h rhythms in this area are influenced by diet remains unresolved. Here we focused on a key structure of this complex, the nucleus of the solitary tract (NTS). We used a combination of immunohistochemical and electrophysiological approaches together with daily monitoring of body weight and food intake to interrogate how the neuronal rhythms of the NTS are affected by a high-fat diet. We report that short-term consumption of a high-fat diet increases food intake during the day and blunts NTS daily rhythms in neuronal discharge. Additionally, we found that a high-fat diet dampens NTS responsiveness to metabolic neuropeptides, and decreases orexin immunoreactive fibres in this structure. These alterations occur without prominent body weight gain, suggesting that a high-fat diet acts initially to reduce activity in the NTS to disinhibit mechanisms that suppress daytime feeding. KEY POINTS: The dorsal vagal complex of the rodent hindbrain possesses intrinsic circadian timekeeping mechanisms In particular, the nucleus of the solitary tract (NTS) is a robust circadian oscillator, independent of the master suprachiasmatic clock Here, we reveal that rat NTS neurons display timed daily rhythms in their neuronal activity and responsiveness to ingestive cues These daily rhythms are blunted or eliminated by a short-term high-fat diet, together with increased consumption of calories during the behaviourally quiescent day Our results help us better understand the circadian control of satiety by the brainstem and its malfunctioning under a high-fat diet., (© 2021 The Authors. The Journal of Physiology © 2021 The Physiological Society.)

Key Points: Recently, we found that the dorsal vagal complex displays autonomous circadian timekeeping properties  The dorsal motor nucleus of the vagus (DMV) is an executory part of this complex - a source of parasympathetic innervation of the gastrointestinal tract  Here, we reveal daily changes in the neuronal activities of the rat DMV, including firing rate, intrinsic excitability and synaptic input - all of these peaking in the late day  Additionally, we establish that short term high-fat diet disrupts these daily rhythms, boosting the variability in the firing rate, but blunting the DMV responsiveness to ingestive cues  These results help us better understand daily control over parasympathetic outflow and provide evidence on its dependence on the high-fat diet ABSTRACT: The suprachiasmatic nuclei (SCN) of the hypothalamus function as the brain's primary circadian clock, but circadian clock genes are also rhythmically expressed in several extra-SCN brain sites where they can exert local temporal control over physiology and behaviour. Recently, we found that the hindbrain dorsal vagal complex possesses strong daily timekeeping capabilities, with the area postrema and nucleus of the solitary tract exhibiting the most robust clock properties. The possibility that the executory part of this complex - the dorsal motor nucleus of the vagus (DMV) - also exhibits daily changes has not been extensively studied. The DMV is the source of vagal efferent motoneurons that regulate gastric motility and emptying and consequently influence meal size and energy homeostasis. We used a combination of multi-channel electrophysiology and patch clamp recordings to gain insight into effects of time of day and diet on these DMV cells. We found that DMV neurons increase their spontaneous activity, excitability and responsiveness to metabolic neuromodulators at late day and this was paralleled with an enhanced synaptic input to these neurons. A high-fat diet typically damps circadian rhythms, but we found that consumption of a high-fat diet paradoxically amplified daily variation of DMV neuronal activity, while blunting the neurons responsiveness to metabolic neuromodulators. In summary, we show for the first time that DMV neural activity changes with time of day, with this temporal variation modulated by diet. These findings have clear implications for our understanding of the daily control of vagal efferents and parasympathetic outflow., (© 2021 The Authors. The Journal of Physiology © 2021 The Physiological Society.)

Regular exercise is important for physical and mental health. An underexplored and intriguing property of exercise is its actions on the body's 24 h or circadian rhythms. Molecular clock cells in the brain's suprachiasmatic nuclei (SCN) use electrical and chemical signals to orchestrate their activity and convey time of day information to the rest of the brain and body. To date, the long-lasting effects of regular physical exercise on SCN clock cell coordination and communication remain unresolved. Utilizing mouse models in which SCN intercellular neuropeptide signaling is impaired as well as those with intact SCN neurochemical signaling, we examined how daily scheduled voluntary exercise (SVE) influenced behavioral rhythms and SCN molecular and neuronal activities. We show that in mice with disrupted neuropeptide signaling, SVE promotes SCN clock cell synchrony and robust 24 h rhythms in behavior. Interestingly, in both intact and neuropeptide signaling deficient animals, SVE reduces SCN neural activity and alters GABAergic signaling. These findings illustrate the potential utility of regular exercise as a long-lasting and effective non-invasive intervention in the elderly or mentally ill where circadian rhythms can be blunted and poorly aligned to the external world.

Background: Heart rate follows a diurnal variation, and slow heart rhythms occur primarily at night., Objective: The lower heart rate during sleep is assumed to be neural in origin, but here we tested whether a day-night difference in intrinsic pacemaking is involved., Methods: In vivo and in vitro electrocardiographic recordings, vagotomy, transgenics, quantitative polymerase chain reaction, Western blotting, immunohistochemistry, patch clamp, reporter bioluminescence recordings, and chromatin immunoprecipitation were used., Results: The day-night difference in the average heart rate of mice was independent of fluctuations in average locomotor activity and persisted under pharmacological, surgical, and transgenic interruption of autonomic input to the heart. Spontaneous beating rate of isolated (ie, denervated) sinus node (SN) preparations exhibited a day-night rhythm concomitant with rhythmic messenger RNA expression of ion channels including hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4). In vitro studies demonstrated 24-hour rhythms in the human HCN4 promoter and the corresponding funny current. The day-night heart rate difference in mice was abolished by HCN block, both in vivo and in the isolated SN. Rhythmic expression of canonical circadian clock transcription factors, for example, Brain and muscle ARNT-Like 1 (BMAL1) and Cryptochrome (CRY) was identified in the SN and disruption of the local clock (by cardiomyocyte-specific knockout of Bmal1) abolished the day-night difference in Hcn4 and intrinsic heart rate. Chromatin immunoprecipitation revealed specific BMAL1 binding sites on Hcn4, linking the local clock with intrinsic rate control., Conclusion: The circadian variation in heart rate involves SN local clock-dependent Hcn4 rhythmicity. Data reveal a novel regulator of heart rate and mechanistic insight into bradycardia during sleep., (Copyright © 2020 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Phasic pattern of neuronal activity has been previously described in detail for magnocellular vasopressin neurons in the hypothalamic paraventricular and supraoptic nuclei. This characteristic bistable pattern consists of alternating periods of electrical silence and elevated neuronal firing, implicated in neuropeptide release. Here, with the use of multi-electrode array recordings ex vivo , we aimed to study the firing pattern of neurons in the nucleus of the solitary tract (NTS) - the brainstem hub for homeostatic, cardio-vascular, and metabolic processes. Our recordings from the mouse and rat hindbrain slices reveal the phasic activity pattern to be displayed by a subset of neurons in the dorsomedial NTS subjacent to the area postrema (AP), with the inter-spike interval distribution closely resembling that reported for phasic magnocellular vasopressin cells. Additionally, we provide interspecies comparison, showing higher phasic frequency and firing rate of phasic NTS cells in mice compared to rats. Further, we describe daily changes in their firing rate and pattern, peaking at the middle of the night. Last, we reveal these phasic cells to be sensitive to α 2 adrenergic receptors activation and to respond to electrical stimulation of the AP. This study provides a comprehensive description of the phasic neuronal activity in the rodent NTS and identifies it as a potential downstream target of the AP noradrenergic system., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chrobok, Wojcik, Klich, Pradel, Lewandowski and Piggins.)

The ability to record ensemble action potential (AP) discharge frequencies from large populations of neurons over extended periods of time in vitro offers clear advantages in neuroscience and circadian biology research. Here, we provide an overview of a step-by-step method to perform multisite extracellular AP activity recordings in suprachiasmatic and mediobasal hypothalamic nuclei brain slices, using a state-of-the-art perforated multielectrode array system. Further, we describe in detail a setup architecture which systematically delivers stable, high-quality recordings with excellent anatomical accuracy and consistency. We also provide some procedural, technical, and methodological troubleshooting notes and examples of good quality recordings.

Although the mammalian rest-activity cycle is controlled by a "master clock" in the suprachiasmatic nucleus (SCN) of the hypothalamus, it is unclear how firing of individual SCN neurons gates individual features of daily activity. Here, we demonstrate that a specific transcriptomically identified population of mouse VIP+ SCN neurons is active at the "wrong" time of day-nighttime-when most SCN neurons are silent. Using chemogenetic and optogenetic strategies, we show that these neurons and their cellular clocks are necessary and sufficient to gate and time nighttime sleep but have no effect upon daytime sleep. We propose that mouse nighttime sleep, analogous to the human siesta, is a "hard-wired" property gated by specific neurons of the master clock to favor subsequent alertness prior to dawn (a circadian "wake maintenance zone"). Thus, the SCN is not simply a 24-h metronome: specific populations sculpt critical features of the sleep-wake cycle., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Metabolic and cardiovascular processes controlled by the hindbrain exhibit 24 h rhythms, but the extent to which the hindbrain possesses endogenous circadian timekeeping is unresolved. Here we provide compelling evidence that genetic, neuronal, and vascular activities of the brainstem's dorsal vagal complex are subject to intrinsic circadian control with a crucial role for the connection between its components in regulating their rhythmic properties. Robust 24 h variation in clock gene expression in vivo and neuronal firing ex vivo were observed in the area postrema (AP) and nucleus of the solitary tract (NTS), together with enhanced nocturnal responsiveness to metabolic cues. Unexpectedly, we also find functional and molecular evidence for increased penetration of blood borne molecules into the NTS at night. Our findings reveal that the hindbrain houses a local network complex of neuronal and non-neuronal autonomous circadian oscillators, with clear implications for understanding local temporal control of physiology in the brainstem.

Drinking behavior and osmotic regulatory mechanisms exhibit clear daily variation which is necessary for achieving the homeostatic osmolality. In mammals, the master clock in the brain's suprachiasmatic nuclei has long been held as the main driver of circadian (24 h) rhythms in physiology and behavior. However, rhythmic clock gene expression in other brain sites raises the possibility of local circadian control of neural activity and function. The subfornical organ (SFO) and the organum vasculosum laminae terminalis (OVLT) are two sensory circumventricular organs (sCVOs) that play key roles in the central control of thirst and water homeostasis, but the extent to which they are subject to intrinsic circadian control remains undefined. Using a combination of ex vivo bioluminescence and in vivo gene expression, we report for the first time that the SFO contains an unexpectedly robust autonomous clock with unusual spatiotemporal characteristics in core and noncore clock gene expression. Furthermore, putative single-cell oscillators in the SFO and OVLT are strongly rhythmic and require action potential-dependent communication to maintain synchrony. Our results reveal that these thirst-controlling sCVOs possess intrinsic circadian timekeeping properties and raise the possibility that these contribute to daily regulation of drinking behavior., (© 2019 The Authors. The FASEB Journal published by Wiley Periodicals, Inc. on behalf of Federation of American Societies for Experimental Biology.)

Twenty-four hour rhythms of physiology and behavior are driven by the environment and an internal endogenous timing system. Daily restricted feeding (RF) in nocturnal rodents during their inactive phase initiates food anticipatory activity (FAA) and a reorganization of the typical 24-hour sleep-wake structure. Here, we investigate the effects of daytime feeding, where food access was restricted to 4 hours during the light period ZT4-8 (Zeitgeber time; ZT0 is lights on), on sleep-wake architecture and sleep homeostasis in mice. Following 10 days of RF, mice were returned to ad libitum feeding. To mimic the spontaneous wakefulness associated with FAA and daytime feeding, mice were then sleep deprived between ZT3-6. Although the amount of wake increased during FAA and subsequent feeding, total wake time over 24 hours remained stable as the loss of sleep in the light phase was compensated for by an increase in sleep in the dark phase. Interestingly, sleep that followed spontaneous wake episodes during the dark period and the extended period of wake associated with FAA, exhibited lower levels of slow-wave activity (SWA) when compared to baseline or after sleep deprivation, despite a similar duration of waking. This suggests an evolutionary mechanism of reducing sleep drive during negative energy balance to enable greater arousal for food-seeking behaviors. However, the total amount of sleep and SWA accumulated during the 24 hours was similar between baseline and RF. In summary, our study suggests that despite substantial changes in the daily distribution and quality of wake induced by RF, sleep homeostasis is maintained., (© Sleep Research Society 2019. Published by Oxford University Press [on behalf of the Sleep Research Society].)

The kidney harbors one of the strongest circadian clocks in the body. Kidney failure has long been known to cause circadian sleep disturbances. Using an adenine-induced model of chronic kidney disease (CKD) in mice, we probe the possibility that such sleep disturbances originate from aberrant circadian rhythms in kidney. Under the CKD condition, mice developed unstable behavioral circadian rhythms. When observed in isolation in vitro, the pacing of the master clock, the suprachiasmatic nucleus (SCN), remained uncompromised, while the kidney clock became a less robust circadian oscillator with a longer period. We find this analogous to the silencing of a strong slave clock in the brain, the choroid plexus, which alters the pacing of the SCN. We propose that the kidney also contributes to overall circadian timekeeping at the whole-body level, through bottom-up feedback in the hierarchical structure of the mammalian circadian clocks.

Background: Alterations in environmental light and intrinsic circadian function have strong associations with mood disorders. The neural origins underpinning these changes remain unclear, although genetic deficits in the molecular clock regularly render mice with altered mood-associated phenotypes., Methods: A detailed circadian and light-associated behavioral characterization of the Na + /K + -ATPase α3 Myshkin (Myk/+) mouse model of mania was performed. Na + /K + -ATPase α3 does not reside within the core circadian molecular clockwork, but Myk/+ mice exhibit concomitant disruption in circadian rhythms and mood. The neural basis of this phenotype was investigated through molecular and electrophysiological dissection of the master circadian pacemaker, the suprachiasmatic nuclei (SCN). Light input and glutamatergic signaling to the SCN were concomitantly assessed through behavioral assays and calcium imaging., Results: In vivo assays revealed several circadian abnormalities including lengthened period and instability of behavioral rhythms, and elevated metabolic rate. Grossly aberrant responses to light included accentuated resetting, accelerated re-entrainment, and an absence of locomotor suppression. Bioluminescent recording of circadian clock protein (PERIOD2) output from ex vivo SCN revealed no deficits in Myk/+ molecular clock function. Optic nerve crush rescued the circadian period of Myk/+ behavior, highlighting that afferent inputs are critical upstream mediators. Electrophysiological and calcium imaging SCN recordings demonstrated changes in the response to glutamatergic stimulation as well as the electrical output indicative of altered retinal input processing., Conclusions: The Myshkin model demonstrates profound circadian and light-responsive behavioral alterations independent of molecular clock disruption. Afferent light signaling drives behavioral changes and raises new mechanistic implications for circadian disruption in affective disorders., (Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Background: Glucose-sensing neurons are located in several parts of the brain, but are concentrated in the ventromedial nucleus of the hypothalamus (VMH). The importance of these VMH neurons in glucose homeostasis is well-established, however, little is known about their individual identity. In the present study, we identified a distinct glucose-sensing population in the VMH and explored its place in the glucose-regulatory network. Methods: Using patch-clamp electrophysiology on Pacap -cre::EYFP cells, we explored the glucose-sensing ability of the pituitary adenylate cyclase-activating peptide (PACAP) neurons both inside and outside the VMH. We also mapped the efferent projections of these neurons using anterograde and retrograde tracing techniques. Finally, to test the functionality of PACAP VMH in vivo , we used DREADD technology and measured systemic responses. Results: We demonstrate that PACAP neurons inside (PACAP VMH ), but not outside the VMH are intrinsically glucose inhibited (GI). Anatomical tracing techniques show that PACAP VMH neurons project to several areas that can influence autonomic output. In vivo , chemogenetic stimulation of these neurons inhibits insulin secretion leading to reduced glucose tolerance, implicating their role in systemic glucose regulation. Conclusion: These findings are important as they identify, for the first time, a specific VMH neuronal population involved in glucose homeostasis. Identifying the different glucose-sensing populations in the VMH will help piece together the different arms of glucose regulation providing vital information regarding central responses to glucose metabolic disorders including hypoglycaemia.

Over the past 20years, substantive research has firmly implicated the lateral habenula in myriad neural processes including addiction, depression, and sleep. More recently, evidence has emerged suggesting that the lateral habenula is a component of the brain's intrinsic daily or circadian timekeeping system. This system centers on the master circadian pacemaker in the suprachiasmatic nuclei of the hypothalamus that is synchronized to the external world through environmental light information received directly from the eye. Rhythmic clock gene expression in suprachiasmatic neurons drives variation in their electrical activity enabling communication of temporal information, and the organization of circadian rhythms in downstream targets. Here, we review the evidence implicating the lateral habenula as part of an extended neural circadian system. We consider findings suggesting that the lateral habenula is a recipient of circadian signals from the suprachiasmatic nuclei as well as light information from the eye. Further we examine the proposition that the lateral habenula itself expresses intrinsic clock gene and neuronal rhythms. We then speculate on how circadian information communicated from the lateral habenula could influence activity and function in downstream targets such as the ventral tegmental area and raphe nuclei., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Suprachiasmatic nuclei (SCN) neurons contain an intracellular molecular circadian clock and the Cryptochromes (CRY1/2), key transcriptional repressors of this molecular apparatus, are subject to post-translational modification through ubiquitination and targeting for proteosomal degradation by the ubiquitin E3 ligase complex. Loss-of-function point mutations in a component of this ligase complex, Fbxl3, delay CRY1/2 degradation, reduce circadian rhythm strength, and lengthen the circadian period by ∼2.5 h. The molecular clock drives circadian changes in the membrane properties of SCN neurons, but it is unclear how alterations in CRY1/2 stability affect SCN neurophysiology. Here we use male and female Afterhours mice which carry the circadian period lengthening loss-of-function Fbxl3 Afh mutation and perform patch-clamp recordings from SCN brain slices across the projected day/night cycle. We find that the daily rhythm in membrane excitability in the ventral SCN (vSCN) was enhanced in amplitude and delayed in timing in Fbxl3 Afh/Afh mice. At night, vSCN cells from Fbxl3 Afh/Afh mice were more hyperpolarized, receiving more GABAergic input than their Fbxl3 +/+ counterparts. Unexpectedly, the progression to daytime hyperexcited states was slowed by Afh mutation, whereas the decline to hypoexcited states was accelerated. In long-term bioluminescence recordings, GABA A receptor blockade desynchronized the Fbxl3 +/+ but not the Fbxl3 Afh/Afh vSCN neuronal network. Further, a neurochemical mimic of the light input pathway evoked larger shifts in molecular clock rhythms in Fbxl3 Afh/Afh compared with Fbxl3 +/+ SCN slices. These results reveal unanticipated consequences of delaying CRY degradation, indicating that the Afh mutation prolongs nighttime hyperpolarized states of vSCN cells through increased GABAergic synaptic transmission. SIGNIFICANCE STATEMENT The intracellular molecular clock drives changes in SCN neuronal excitability, but it is unclear how mutations affecting post-translational modification of molecular clock proteins influence the temporal expression of SCN neuronal state or intercellular communication within the SCN network. Here we show for the first time, that a mutation that prolongs the stability of key components of the intracellular clock, the cryptochrome proteins, unexpectedly increases in the expression of hypoexcited neuronal state in the ventral SCN at night and enhances hyperpolarization of ventral SCN neurons at this time. This is accompanied by increased GABAergic signaling and by enhanced responsiveness to a neurochemical mimic of the light input pathway to the SCN. Therefore, post-translational modification shapes SCN neuronal state and network properties., (Copyright © 2017 Wegner et al.)

Our knowledge of how circadian and homeostatic brain circuits interact to temporally organize physiology and behavior is limited. Progress has been made with the determination that lateral hypothalamic orexin (OXA) neurons control arousal and appetitive states, while suprachiasmatic nuclei (SCN) neurons function as the master circadian clock. During the day, SCN neurons exhibit heterogeneity in spontaneous resting membrane potential (RMP), with some neurons becoming severely depolarized (hyperexcited) and ceasing to fire action potentials (APs), while other neurons rest at moderate RMP and fire APs. Intriguingly, the day phase is when the SCN clock is most readily influenced by arousal, but it is unclear if and how heterogeneity in the excitability state of SCN neurons shapes their response to arousal signals, such as OXA. In whole-cell recordings we show that during the day OXA recruits GABA-GABA A receptor signaling to suppress the RMP of hyperexcited silent as well as moderately hyperpolarized AP-firing SCN neurons. In the AP-firing neurons, OXA hyperpolarized and silenced these SCN cells, while in the hyperexcited silent neurons OXA suppressed the RMP of these cells and evoked either AP-firing, depolarized low-amplitude membrane oscillations, or continued silence at a reduced RMP. These results demonstrate how the resting state of SCN neurons determines their response to OXA, and illustrate that the inhibitory action of this neurochemical correlate of arousal can trigger paradoxical AP firing., (© 2016 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Daily restricted access to food leads to the development of food anticipatory activity and metabolism, which depends upon an as yet unidentified food-entrainable oscillator(s). A premeal anticipatory peak in circulating hormones, including corticosterone is also elicited by daily restricted feeding. High-fat feeding is associated with elevated levels of corticosterone with disrupted circadian rhythms and a failure to develop robust meal anticipation. It is not clear whether the disrupted corticosterone rhythm, resulting from high-fat feeding contributes to attenuated meal anticipation in high-fat fed rats. Our aim was to better characterize meal anticipation in rats fed a low- or high-fat diet, and to better understand the role of corticosterone in this process. To this end, we utilized behavioral observations, hypothalamic c-Fos expression, and indirect calorimetry to assess meal entrainment. We also used the glucocorticoid receptor antagonist, RU486, to dissect out the role of corticosterone in meal anticipation in rats given daily access to a meal with different fat content. Restricted access to a low-fat diet led to robust meal anticipation, as well as entrainment of hypothalamic c-Fos expression, metabolism, and circulating corticosterone. These measures were significantly attenuated in response to a high-fat diet, and animals on this diet exhibited a postanticipatory rise in corticosterone. Interestingly, antagonism of glucocorticoid activity using RU486 attenuated meal anticipation in low-fat fed rats, but promoted meal anticipation in high-fat-fed rats. These findings suggest an important role for corticosterone in the regulation of meal anticipation in a manner dependent upon dietary fat content., (Copyright © 2016 the American Physiological Society.)

Individual neurons in the suprachiasmatic nuclei (SCN) contain an intracellular molecular clock and use intercellular signaling to synchronize their timekeeping activities so that the SCN can coordinate brain physiology and behavior. The neuropeptide vasoactive intestinal polypeptide (VIP) and its VPAC2 receptor form a key component of intercellular signaling systems in the SCN and critically control cellular coupling. Targeted mutations in either the intracellular clock or intercellular neuropeptide signaling mechanisms, such as VIP-VPAC2 signaling, can lead to desynchronization of SCN neuronal clocks and loss of behavioral rhythms. An important goal in chronobiology is to develop interventions to correct deficiencies in circadian timekeeping. Here we show that extended exposure to constant light promotes synchrony among SCN clock cells and the expression of ~24 h rhythms in behavior in mice in which intercellular signaling is disrupted through loss of VIP-VPAC2 signaling. This study highlights the importance of SCN synchrony for the expression of rhythms in behavior and reveals how non-invasive manipulations in the external environment can be used to overcome neurochemical communication deficits in this important brain system.

The suprachiasmatic nuclei (SCN), the central circadian pacemakers in mammals, comprise a multiscale neuronal system that times daily events. We use recent advances in graphics processing unit computing to generate a multiscale model for the SCN that resolves cellular electrical activity down to the timescale of individual action potentials and the intracellular molecular events that generate circadian rhythms. We use the model to study the role of the neurotransmitter GABA in synchronizing circadian rhythms among individual SCN neurons, a topic of much debate in the circadian community. The model predicts that GABA signaling has two components: phasic (fast) and tonic (slow). Phasic GABA postsynaptic currents are released after action potentials, and can both increase or decrease firing rate, depending on their timing in the interspike interval, a modeling hypothesis we experimentally validate; this allows flexibility in the timing of circadian output signals. Phasic GABA, however, does not significantly affect molecular timekeeping. The tonic GABA signal is released when cells become very excited and depolarized; it changes the excitability of neurons in the network, can shift molecular rhythms, and affects SCN synchrony. We measure which neurons are excited or inhibited by GABA across the day and find GABA-excited neurons are synchronized by-and GABA-inhibited neurons repelled from-this tonic GABA signal, which modulates the synchrony in the SCN provided by other signaling molecules. Our mathematical model also provides an important tool for circadian research, and a model computational system for the many multiscale projects currently studying brain function.

A hormone released from the gut after a meal can reset clock gene activity in the liver.

The vasoactive intestinal peptide receptor 2 (VPAC2) is widely distributed throughout the body and is also overexpressed in a variety of human neoplastic tissues. However, little is known about its precise tissue distribution, regulation and function, which is in part be due to the lack of specific monoclonal anti-VPAC2 antibodies. In this study, we extensively characterised the novel rabbit monoclonal anti-VPAC2 antibody (clone SP235) using transfected cells and mouse, rat and human tissues. SP235 was then subjected to a comparative immunohistochemical study on a series of 167 histological specimens from formalin-fixed, paraffin-embedded human tumours and adjacent normal tissues. SP235 detected a broad band migrating at a molecular weight of 50-70 kDa in western blotting analyses of various mouse tissues as well as VPAC2- but not VPAC1-transfected human embryonic kidney 293 cells. SP235 yielded an efficient immunostaining of distinct cell populations in human tissue samples with a predominance of plasma membrane staining, which was completely abolished by preadsorption with its immunising peptide. SP235 immunohistochemistry detected VPAC2 receptors in lymphocytes present in spleen, tonsils, lymph nodes and Peyer's patches, chief cells of gastric mucosa, exocrine and endocrine pancreas, kidney tubules and blood vessels. In addition, VPAC2 was observed in thyroid, gastric and lung carcinomas, pancreatic adenocarcinomas, sarcomas and neuroendocrine tumours. SP235 may prove of great value in the identification of VPAC2 receptors during routine histopathological examination. VPAC2 visualisation with this simple and rapid immunohistochemical method will facilitate identification of candidate tumours for vasoactive intestinal peptide (VIP)-based diagnostics or therapeutic interventions., (© 2015 The authors.)

The epithalamic lateral habenula (LHb) is implicated as part of the mammalian brain's circadian system. Anatomical evidence suggests that the LHb receives extrinsic circadian timing cues from retinal ganglion cells and the master clock in the suprachiasmatic nuclei (SCN). Intriguingly, some LHb neurones contain the molecular circadian clock, but it is unclear if and how intrinsic and extrinsic circadian processes influence neuronal activity in the mouse LHb. Here, using an in vitro brain slice preparation isolating the LHb from the SCN, we show through whole-cell patch-clamp recordings that LHb neurones exhibit heterogeneity in their resting state, but the majority spontaneously fire action potentials (APs). Discharge rate of APs varied from low firing in the early day to higher firing later in the day and was absent in LHb brain slices prepared from Cry1(-/-)Cry2(-/-) mice that lack a functional molecular clock. Low amplitude circadian oscillations in the molecular circadian clock were also monitored in LHb brain slices, but were absent in Cry1(-/-)Cry2(-/-) LHb brain tissue. A putative neurochemical output signal of the SCN, prokineticin 2 (PK2), inhibited some LHb neurones by elevating the frequency of GABA release in the LHb. Using multi-electrode recordings in vivo, we found that LHb neurones sluggishly respond to retinal illumination, suggesting that they receive such information through polysynaptic processes. In summary, our results show for the first time that intrinsic circadian signals are important for regulating LHb neuronal state, while the SCN-derived signal PK2 is less influential. Moreover, we demonstrate that mouse LHb neurones have access to and can respond to visual input, but such signals are unlikely to be directly communicated to the LHb. Broadly, these findings raise the possibility that intrinsic circadian signals are likely to be influential in shaping LHb contributions to cognition and emotionality., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

Extending a normal 24 hours day by four hours is unexpectedly highly disruptive to daily rhythms in gene expression in the blood. Using a paradigm in which human subjects were exposed to a 28 hours day, Archer and colleagues show how this sleep-altering forced desynchrony protocol caused complex disruption to daily rhythms in distinct groups of genes. Such perturbations in the temporal organisation of the blood transcriptome arise quickly, and point to the fragile nature of coordinated genomic activity. Chronic disruption of the daily and circadian rhythms in sleep compromise health and well-being and this study reveals potential new molecular targets to combat the disruptive effects of shift work and jetlag., (© 2014 The Authors. Bioessays published by WILEY Periodicals, Inc.)

Circadian and homeostatic neural circuits organize the temporal architecture of physiology and behavior, but knowledge of their interactions is imperfect. For example, neurons containing the neuropeptide orexin homeostatically control arousal and appetitive states, while neurons in the suprachiasmatic nuclei (SCN) function as the brain's master circadian clock. The SCN regulates orexin neurons so that they are much more active during the circadian night than the circadian day, but it is unclear whether the orexin neurons reciprocally regulate the SCN clock. Here we show both orexinergic innervation and expression of genes encoding orexin receptors (OX1 and OX2) in the mouse SCN, with OX1 being upregulated at dusk. Remarkably, we find through in vitro physiological recordings that orexin predominantly suppresses mouse SCN Period1 (Per1)-EGFP-expressing clock cells. The mechanisms underpinning these suppressions vary across the circadian cycle, from presynaptic modulation of inhibitory GABAergic signaling during the day to directly activating leak K(+) currents at night. Orexin also augments the SCN clock-resetting effects of neuropeptide Y (NPY), another neurochemical correlate of arousal, and potentiates NPY's inhibition of SCN Per1-EGFP cells. These results build on emerging literature that challenge the widely held view that orexin signaling is exclusively excitatory and suggest new mechanisms for avoiding conflicts between circadian clock signals and homeostatic cues in the brain.

Intrinsic daily or circadian rhythms arise through the outputs of the master circadian clock in the brain's suprachiasmatic nuclei (SCN) as well as circadian oscillators in other brain sites and peripheral tissues. SCN neurones contain an intracellular molecular clock that drives these neurones to exhibit pronounced day-night differences in their electrical properties. The epithalamic medial habenula (MHb) expresses clock genes, but little is known about the bioelectric properties of mouse MHb neurones and their potential circadian characteristics. Therefore, in this study we used a brain slice preparation containing the MHb to determine the basic electrical properties of mouse MHb neurones with whole-cell patch clamp electrophysiology, and investigated whether these vary across the day-night cycle. MHb neurones (n = 230) showed heterogeneity in electrophysiological state, ranging from highly depolarised cells (∼ -25 to -30 mV) that are silent with no membrane activity or display depolarised low-amplitude membrane oscillations, to neurones that were moderately hyperpolarised (∼40 mV) and spontaneously discharging action potentials. These electrical states were largely intrinsically regulated and were influenced by the activation of small-conductance calcium-activated potassium channels. When considered as one population, MHb neurones showed significant circadian variation in their spontaneous firing rate and resting membrane potential. However, in recordings of MHb neurones from mice lacking the core molecular circadian clock, these temporal differences in MHb activity were absent, indicating that circadian clock signals actively regulate the timing of MHb neuronal states. These observations add to the extracellularly recorded rhythms seen in other brain areas and establish that circadian mechanisms can influence the membrane properties of neurones in extra-SCN sites. Collectively, the results of this study indicate that the MHb may function as an intrinsic secondary circadian oscillator in the brain, which can shape daily information flow in key brain processes, such as reward and addiction.

Objective: To characterize the circadian clock in murine cartilage tissue and identify tissue-specific clock target genes, and to investigate whether the circadian clock changes during aging or during cartilage degeneration using an experimental mouse model of osteoarthritis (OA)., Methods: Cartilage explants were obtained from aged and young adult mice after transduction with the circadian clock fusion protein reporter PER2::luc, and real-time bioluminescence recordings were used to characterize the properties of the clock. Time-series microarrays were performed on mouse cartilage tissue to identify genes expressed in a circadian manner. Rhythmic genes were confirmed by quantitative reverse transcription-polymerase chain reaction using mouse tissue, primary chondrocytes, and a human chondrocyte cell line. Experimental OA was induced in mice by destabilization of the medial meniscus (DMM), and articular cartilage samples were microdissected and subjected to microarray analysis., Results: Mouse cartilage tissue and a human chondrocyte cell line were found to contain intrinsic molecular circadian clocks. The cartilage clock could be reset by temperature signals, while the circadian period was temperature compensated. PER2::luc bioluminescence demonstrated that circadian oscillations were significantly lower in amplitude in cartilage from aged mice. Time-series microarray analyses of the mouse tissue identified the first circadian transcriptome in cartilage, revealing that 615 genes (∼3.9% of the expressed genes) displayed a circadian pattern of expression. This included genes involved in cartilage homeostasis and survival, as well as genes with potential importance in the pathogenesis of OA. Several clock genes were disrupted in the early stages of cartilage degeneration in the DMM mouse model of OA., Conclusion: These results reveal an autonomous circadian clock in chondrocytes that can be implicated in key aspects of cartilage biology and pathology. Consequently, circadian disruption (e.g., during aging) may compromise tissue homeostasis and increase susceptibility to joint damage or disease., (© 2013 The Authors. Arthritis & Rheumatism is published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.)

Within the core molecular clock, protein phosphorylation and degradation play a vital role in determining circadian period. The 'after-hours' (Afh) mutation in mouse slows the degradation of the core clock protein Cryptochrome, lengthening the period of the molecular clock in the suprachiasmatic nuclei (SCN) and behavioural wheel-running rhythms. However, we do not yet know how the Afh mutation affects other aspects of physiology or the activity of circadian oscillators in other brain regions. Here we report that daily rhythms of metabolism and ingestive behaviours are altered in these animals, as are PERIOD2::LUCIFERASE (PER2::LUC) rhythms in mediobasal hypothalamic nuclei, which influence these behaviours. Overall there is a trend towards period lengthening and a decrease in amplitude of PER2::LUC rhythms throughout the brain. Imaging of single cells from the arcuate and dorsomedial hypothalamic nuclei revealed this reduction in tissue oscillator amplitude to be due to a decrease in the amplitude, rather than a desynchrony, of single cells. Consistent with existing models of oscillator function, this cellular phenotype was associated with a greater susceptibility to phase-shifting stimuli in vivo and in vitro, with light evoking high-amplitude Type 0 resetting in Afh mutant mice. Together, these findings reveal unexpected consequences of the Afh mutation on the amplitude and synchrony of individual cellular oscillators in the SCN.

Hyperexcited states, including depolarization block and depolarized low amplitude membrane oscillations (DLAMOs), have been observed in neurons of the suprachiasmatic nuclei (SCN), the site of the central mammalian circadian (~24-hour) clock. The causes and consequences of this hyperexcitation have not yet been determined. Here, we explore how individual ionic currents contribute to these hyperexcited states, and how hyperexcitation can then influence molecular circadian timekeeping within SCN neurons. We developed a mathematical model of the electrical activity of SCN neurons, and experimentally verified its prediction that DLAMOs depend on post-synaptic L-type calcium current. The model predicts that hyperexcited states cause high intracellular calcium concentrations, which could trigger transcription of clock genes. The model also predicts that circadian control of certain ionic currents can induce hyperexcited states. Putting it all together into an integrative model, we show how membrane potential and calcium concentration provide a fast feedback that can enhance rhythmicity of the intracellular circadian clock. This work puts forward a novel role for electrical activity in circadian timekeeping, and suggests that hyperexcited states provide a general mechanism for linking membrane electrical dynamics to transcription activation in the nucleus.

Plasma levels of corticosterone exhibit both circadian and ultradian rhythms. The circadian component of these rhythms is regulated by the suprachiasmatic nucleus (SCN). Our studies investigate the importance of the SCN in regulating ultradian rhythmicity. Two approaches were used to dissociate the hypothalamic-pituitary-adrenal (HPA) axis from normal circadian input in rats: (i) exposure to a constant light (LL) environment and (ii) electrolytic lesioning of the SCN. Blood was sampled using an automated sampling system. As expected, both treatments resulted in a loss of the circadian pattern of corticosterone secretion. Ultradian pulsatile secretion of corticosterone however, was maintained across the 24 h in all animals. Furthermore, the loss of SCN input revealed an underlying relationship between locomotor and HPA activity. In control (LD) rats there was no clear correlation between ultradian locomotor activity and hormone secretion, whereas, in LL rats, episodes of ultradian activity were consistently followed by periods of increased pulsatile hormone secretion. These data clearly demonstrate that the ultradian rhythm of corticosterone secretion is generated through a mechanism independent of the SCN input, supporting recent evidence for a sub-hypothalamic pulse generator., (© 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

The phase of the mammalian circadian system can be entrained to a range of environmental stimuli, or zeitgebers, including food availability and light. Further, locomotor activity can act as an entraining signal and represents a mechanism for an endogenous behavior to feedback and influence subsequent circadian function. This process involves a number of nuclei distributed across the brain stem, thalamus, and hypothalamus and ultimately alters SCN electrical and molecular function to induce phase shifts in the master circadian pacemaker. Locomotor activity feedback to the circadian system is effective across both nocturnal and diurnal species, including humans, and has recently been shown to improve circadian function in a mouse model with a weakened circadian system. This raises the possibility that exercise may be useful as a noninvasive treatment in cases of human circadian dysfunction including aging, shift work, transmeridian travel, and the blind., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Humans and other mammals exhibit a remarkable array of cyclical changes in physiology and behaviour. These are often synchronized to the changing environmental light-dark cycle and persist in constant conditions. Such circadian rhythms are controlled by an endogenous clock, located in the suprachiasmatic nuclei of the hypothalamus. This structure and its cells have unique properties, and some of these are reviewed to highlight how this central clock controls and sculpts our daily activities.

Circadian rhythms in physiology and behavior are coordinated by the brain's dominant circadian pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Vasoactive intestinal polypeptide (VIP) and its receptor, VPAC(2), play important roles in the functioning of the SCN pacemaker. Mice lacking VPAC(2) receptors (Vipr2(-/-)) express disrupted behavioral and metabolic rhythms and show altered SCN neuronal activity and clock gene expression. Within the brain, the SCN is not the only site containing endogenous circadian oscillators, nor is it the only site of VPAC(2) receptor expression; both VPAC(2) receptors and rhythmic clock gene/protein expression have been noted in the arcuate (Arc) and dorsomedial (DMH) nuclei of the mediobasal hypothalamus, and in the pituitary gland. The functional role of VPAC(2) receptors in rhythm generation and maintenance in these tissues is, however, unknown. We used wild type (WT) and Vipr2(-/-) mice expressing a luciferase reporter (PER2::LUC) to investigate whether circadian rhythms in the clock gene protein PER2 in these extra-SCN tissues were compromised by the absence of the VPAC(2) receptor. Vipr2(-/-) SCN cultures expressed significantly lower amplitude PER2::LUC oscillations than WT SCN. Surprisingly, in Vipr2(-/-) Arc/ME/PT complex (Arc, median eminence and pars tuberalis), DMH and pituitary, the period, amplitude and rate of damping of rhythms were not significantly different to WT. Intriguingly, while we found WT SCN and Arc/ME/PT tissues to maintain a consistent circadian phase when cultured, the phase of corresponding Vipr2(-/-) cultures was reset by cull/culture procedure. These data demonstrate that while the main rhythm parameters of extra-SCN circadian oscillations are maintained in Vipr2(-/-) mice, the ability of these oscillators to resist phase shifts is compromised. These deficiencies may contribute towards the aberrant behavior and metabolism associated with Vipr2(-/-) animals. Further, our data indicate a link between circadian rhythm strength and the ability of tissues to resist circadian phase resetting.