Timed exercise stabilizes behavioral rhythms but not molecular programs in the brain's suprachiasmatic clock.

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.

Keeping time in the lamina terminalis: Novel oscillator properties of forebrain sensory circumventricular organs.

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.

Positive and Negative Affect Mediate the Influences of a Maladaptive Emotion Regulation Strategy on Sleep Quality.

Positive affect, negative affect, and emotion regulation strategies are related to sleep quality. Emotion regulation can also act as either a protective factor against the development of psychopathologies, or as a risk factor for their development, and therefore may be one mechanism linking mental health and sleep. However, currently it is not known whether affect can mediate the impact of emotion regulation strategy use on sleep quality. An opportunity sample in a healthy population completed the Positive and Negative Affect Schedule providing measures of positive and negative affect, the Pittsburgh Sleep Quality Index providing a measure of sleep quality, and the Emotion Regulation Questionnaire to record habitual use of emotion regulation strategies. Data were analysed using regression and mediation analyses. Negative affect and expressive suppression were positively correlated with PSQI score suggesting that as negative affect and expressive suppression use increased, sleep quality decreased. Positive affect was negatively correlated with PSQI score suggesting that as positive affect increased sleep quality improved. Further, mediation analyses revealed that both positive affect and negative affect mediated the impact of expressive suppression on sleep quality. Moreover, this partial mediation provides the first description that the influences of affect and expressive suppression on sleep quality are at least partially distinct. Targeting improvements in negative affect and effective emotion regulation strategy use may improve the efficacy of interventions aimed at improving sleep quality and the reduction in symptomology in psychopathologies.

Circadian Disruptions in the Myshkin Mouse Model of Mania Are Independent of Deficits in Suprachiasmatic Molecular Clock Function.

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.

Delayed Cryptochrome Degradation Asymmetrically Alters the Daily Rhythm in Suprachiasmatic Clock Neuron Excitability.

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 Fbxl3Afh 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 Fbxl3Afh/Afh mice. At night, vSCN cells from Fbxl3Afh/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, GABAA receptor blockade desynchronized the Fbxl3+/+ but not the Fbxl3Afh/Afh vSCN neuronal network. Further, a neurochemical mimic of the light input pathway evoked larger shifts in molecular clock rhythms in Fbxl3Afh/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.

Constant light enhances synchrony among circadian clock cells and promotes behavioral rhythms in VPAC2-signaling deficient mice.

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.

Disruption of daily rhythms in gene expression: the importance of being synchronised.

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.

Acute suppressive and long-term phase modulation actions of orexin on the mammalian circadian clock.

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.

Feedback actions of locomotor activity to the circadian clock.

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.

Neuropeptide signaling differentially affects phase maintenance and rhythm generation in SCN and extra-SCN circadian oscillators.

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.

A riot of rhythms: neuronal and glial circadian oscillators in the mediobasal hypothalamus.

BACKGROUND: In mammals, the synchronized activity of cell autonomous clocks in the suprachiasmatic nuclei (SCN) enables this structure to function as the master circadian clock, coordinating daily rhythms in physiology and behavior. However, the dominance of this clock has been challenged by the observations that metabolic duress can over-ride SCN controlled rhythms, and that clock genes are expressed in many brain areas, including those implicated in the regulation of appetite and feeding. The recent development of mice in which clock gene/protein activity is reported by bioluminescent constructs (luciferase or luc) now enables us to track molecular oscillations in numerous tissues ex vivo. Consequently we determined both clock activities and responsiveness to metabolic perturbations of cells and tissues within the mediobasal hypothalamus (MBH), a site pivotal for optimal internal homeostatic regulation. RESULTS: Here we demonstrate endogenous circadian rhythms of PER2::LUC expression in discrete subdivisions of the arcuate (Arc) and dorsomedial nuclei (DMH). Rhythms resolved to single cells did not maintain long-term synchrony with one-another, leading to a damping of oscillations at both cell and tissue levels. Complementary electrophysiology recordings revealed rhythms in neuronal activity in the Arc and DMH. Further, PER2::LUC rhythms were detected in the ependymal layer of the third ventricle and in the median eminence/pars tuberalis (ME/PT). A high-fat diet had no effect on the molecular oscillations in the MBH, whereas food deprivation resulted in an altered phase in the ME/PT. CONCLUSION: Our results provide the first single cell resolution of endogenous circadian rhythms in clock gene expression in any intact tissue outside the SCN, reveal the cellular basis for tissue level damping in extra-SCN oscillators and demonstrate that an oscillator in the ME/PT is responsive to changes in metabolism.

Live imaging of altered period1 expression in the suprachiasmatic nuclei of Vipr2-/- mice.

Vasoactive intestinal polypeptide and its receptor, VPAC(2), play important roles in the functioning of the brain's circadian clock in the suprachiasmatic nuclei (SCN). Mice lacking VPAC(2) receptors (Vipr2(-/-)) show altered circadian rhythms in locomotor behavior, neuronal firing rate, and clock gene expression, however, the nature of molecular oscillations in individual cells is unclear. Here, we used real-time confocal imaging of a destabilized green fluorescent protein (GFP) reporter to track the expression of the core clock gene Per1 in live SCN-containing brain slices from wild-type (WT) and Vipr2(-/-) mice. Rhythms in Per1-driven GFP were detected in WT and Vipr2(-/-) cells, though a significantly lower number and proportion of cells in Vipr2(-/-) slices expressed detectable rhythms. Further, Vipr2(-/-) cells expressed significantly lower amplitude oscillations than WT cells. Within each slice, the phases of WT cells were synchronized whereas cells in Vipr2(-/-) slices were poorly synchronized. Most GFP-expressing cells, from both genotypes, expressed neither vasopressin nor vasoactive intestinal polypeptide. Pharmacological blockade of VPAC(2) receptors in WT SCN slices partially mimicked the Vipr2(-/-) phenotype. These data demonstrate that intercellular communication via the VPAC(2) receptor is important for SCN neurons to sustain robust, synchronous oscillations in clock gene expression.

Spatio-temporal distribution of microglia/macrophages during regeneration in the cerebellum of adult teleost fish, Apteronotus leptorhynchus: a quantitative analysis.

In contrast to mammals, adult teleost fish exhibit an enormous capacity to replace damaged neurons with newly generated ones after injuries in the central nervous system. In the present study, the role of microglia/macrophages, identified by tomato lectin binding, was examined in this process of neuronal regeneration in the corpus cerebelli of the teleost fish Apteronotus leptorhynchus. In the intact corpus cerebelli, or after short survival times following application of a mechanical lesion to this cerebellar subdivision, microglia/macrophages were virtually absent. Conversely, approximately 3 days after application of the lesion, the areal density of microglia/macrophages started to increase at and near the lesion site in the ipsilateral hemisphere, as well as in the contralateral hemisphere, and reached maximum levels at approximately 10 days post lesion. The density remained elevated until it reached background levels approximately one month after the injury. By comparing the time course of the appearance of microglia/macrophages with that of other regenerative events occurring within the first few weeks of wound healing in this model system, we hypothesize that one possible function of microglia/macrophages might be to remove debris of cells that have undergone apoptotic cell death at the lesion site.