Diverse genetic alteration dysregulates neuropeptide and intracellular signalling in the suprachiasmatic nuclei.

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.

Sleep quality during and after severe acute respiratory syndrome coronavirus 2 (COVID-19) lockdowns in the UK: Results from the SleepQuest study.

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.

Metabolic cues impact non-oscillatory intergeniculate leaflet and ventral lateral geniculate nucleus: standard versus high-fat diet comparative study.

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.

Daily changes in neuronal activities of the dorsal motor nucleus of the vagus under standard and high-fat diet.

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.

Rhythmic neuronal activities of the rat nucleus of the solitary tract are impaired by high-fat diet - implications for daily control of satiety.

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.

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.

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.

Distinct roles for GABA across multiple timescales in mammalian circadian timekeeping.

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.

Circadian regulation of mouse suprachiasmatic nuclei neuronal states shapes responses to orexin.

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-GABAA 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.

Dietary fat and corticosterone levels are contributing factors to meal anticipation.

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.

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.

Daily variation in the electrophysiological activity of mouse medial habenula neurones.

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.

Intrinsic and extrinsic cues regulate the daily profile of mouse lateral habenula neuronal activity.

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.

Causes and consequences of hyperexcitation in central clock neurons.

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.

The circadian clock in murine chondrocytes regulates genes controlling key aspects of cartilage homeostasis.

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.

Suppressed cellular oscillations in after-hours mutant mice are associated with enhanced circadian phase-resetting.

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.

Melanopsin contributions to irradiance coding in the thalamo-cortical visual system.

Photoreception in the mammalian retina is not restricted to rods and cones but extends to a subset of retinal ganglion cells expressing the photopigment melanopsin (mRGCs). These mRGCs are known to drive such reflex light responses as circadian photoentrainment and pupillomotor movements. By contrast, until now there has been no direct assessment of their contribution to conventional visual pathways. Here, we address this deficit. Using new reporter lines, we show that mRGC projections are much more extensive than previously thought and extend across the dorsal lateral geniculate nucleus (dLGN), origin of thalamo-cortical projection neurons. We continue to show that this input supports extensive physiological light responses in the dLGN and visual cortex in mice lacking rods+cones (a model of advanced retinal degeneration). Moreover, using chromatic stimuli to isolate melanopsin-derived responses in mice with an intact visual system, we reveal strong melanopsin input to the ∼40% of neurons in the LGN that show sustained activation to a light step. We demonstrate that this melanopsin input supports irradiance-dependent increases in the firing rate of these neurons. The implication that melanopsin is required to accurately encode stimulus irradiance is confirmed using melanopsin knockout mice. Our data establish melanopsin-based photoreception as a significant source of sensory input to the thalamo-cortical visual system, providing unique irradiance information and allowing visual responses to be retained even in the absence of rods+cones. These findings identify mRGCs as a potential origin for aspects of visual perception and indicate that they may support vision in people suffering retinal degeneration.

A new phase model of the spatiotemporal relationships between three circadian oscillators in the brainstem.

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.

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.