Rescue of Comorbid Behavioral and Metabolic Phenotypes of Arrhythmic Mice by Restoring Circadian Cry1/2 Expression in the Suprachiasmatic Nucleus.

Background: Psychiatric and metabolic disorders occur disproportionately often comorbidly, which poses particular hurdles for patients and therapists. However, the mechanisms that promote such comorbidities are largely unknown and therefore cannot yet be therapeutically targeted for the simultaneous treatment of both conditions. Because circadian clocks regulate most physiological processes and their disruption is a risk factor for both psychiatric and metabolic disorders, they may be considered as a potential mechanism for the development of comorbidities and a therapeutic target. In the current study, we investigated the latter assumption in Cry1/2-/- mice, which exhibit substantially disrupted endogenous circadian rhythms and marked metabolic and behavioral deficits. Methods: By targeted virus-induced restoration of circadian rhythms in their suprachiasmatic nucleus, we can restore behavioral as well as several metabolic processes of these animals to near-normal circadian rhythmicity. Results: Importantly, by rescuing suprachiasmatic nucleus rhythms, several of their anxiety-like behavioral as well as diabetes- and energy homeostasis-related deficits were significantly improved. Interestingly, however, this did not affect all deficits typical of Cry1/2-/- mice; for example, restlessness and body weight remained unaffected. Conclusions: Taken together, the results of this study demonstrate, on the one hand, that restoration of disturbed circadian rhythms can be used to simultaneously treat psychiatric and metabolic deficits. On the other hand, the results also allow us to distinguish processes that depend more on local canonical clocks from those that depend more on suprachiasmatic nucleus rhythms.

Biobanking in everyday clinical practice in psychiatry-The Munich Mental Health Biobank.

Translational research on complex, multifactorial mental health disorders, such as bipolar disorder, major depressive disorder, schizophrenia, and substance use disorders requires databases with large-scale, harmonized, and integrated real-world and research data. The Munich Mental Health Biobank (MMHB) is a mental health-specific biobank that was established in 2019 to collect, store, connect, and supply such high-quality phenotypic data and biosamples from patients and study participants, including healthy controls, recruited at the Department of Psychiatry and Psychotherapy (DPP) and the Institute of Psychiatric Phenomics and Genomics (IPPG), University Hospital of the Ludwig-Maximilians-University (LMU), Munich, Germany. Participants are asked to complete a questionnaire that assesses sociodemographic and cross-diagnostic clinical information, provide blood samples, and grant access to their existing medical records. The generated data and biosamples are available to both academic and industry researchers. In this manuscript, we outline the workflow and infrastructure of the MMHB, describe the clinical characteristics and representativeness of the sample collected so far, and reveal future plans for expansion and application. As of 31 October 2021, the MMHB contains a continuously growing set of data from 578 patients and 104 healthy controls (46.37% women; median age, 38.31 years). The five most common mental health diagnoses in the MMHB are recurrent depressive disorder (38.78%; ICD-10: F33), alcohol-related disorders (19.88%; ICD-10: F10), schizophrenia (19.69%; ICD-10: F20), depressive episode (15.94%; ICD-10: F32), and personality disorders (13.78%; ICD-10: F60). Compared with the average patient treated at the recruiting hospitals, MMHB participants have significantly more mental health-related contacts, less severe symptoms, and a higher level of functioning. The distribution of diagnoses is also markedly different in MMHB participants compared with individuals who did not participate in the biobank. After establishing the necessary infrastructure and initiating recruitment, the major tasks for the next phase of the MMHB project are to improve the pace of participant enrollment, diversify the sociodemographic and diagnostic characteristics of the sample, and improve the utilization of real-world data generated in routine clinical practice.

Neurobiological and behavioral mechanisms of circadian rhythm disruption in bipolar disorder: A critical multi-disciplinary literature review and agenda for future research from the ISBD task force on chronobiology.

AIM: Symptoms of bipolar disorder (BD) include changes in mood, activity, energy, sleep, and appetite. Since many of these processes are regulated by circadian function, circadian rhythm disturbance has been examined as a biological feature underlying BD. The International Society for Bipolar Disorders Chronobiology Task Force (CTF) was commissioned to review evidence for neurobiological and behavioral mechanisms pertinent to BD. METHOD: Drawing upon expertise in animal models, biomarkers, physiology, and behavior, CTF analyzed the relevant cross-disciplinary literature to precisely frame the discussion around circadian rhythm disruption in BD, highlight key findings, and for the first time integrate findings across levels of analysis to develop an internally consistent, coherent theoretical framework. RESULTS: Evidence from multiple sources implicates the circadian system in mood regulation, with corresponding associations with BD diagnoses and mood-related traits reported across genetic, cellular, physiological, and behavioral domains. However, circadian disruption does not appear to be specific to BD and is present across a variety of high-risk, prodromal, and syndromic psychiatric disorders. Substantial variability and ambiguity among the definitions, concepts and assumptions underlying the research have limited replication and the emergence of consensus findings. CONCLUSIONS: Future research in circadian rhythms and its role in BD is warranted. Well-powered studies that carefully define associations between BD-related and chronobiologically-related constructs, and integrate across levels of analysis will be most illuminating.

Circadian gene × environment perturbations influence alcohol drinking in Cryptochrome-deficient mice.

Alcohol use disorder (AUD) is a widespread addiction disorder with severe consequences for health. AUD patients often suffer from sleep disturbances and irregular daily patterns. Conversely, disruptions of circadian rhythms are considered a risk factor for AUD and alcohol relapses. In this study, we investigated the extent to which circadian genetic and environmental disruptions and their interaction alter alcohol drinking behaviour in mice. As a model of genetic circadian disruption, we used Cryptochrome1/2-deficient (Cry1/2-/- ) mice with strongly suppressed circadian rhythms and found that they exhibit significantly reduced preference for alcohol but increased incentive motivation to obtain it. Similarly, we found that low circadian SCN amplitude correlates with reduced alcohol preference in WT mice. Moreover, we show that the low alcohol preference of Cry1/2-/- mice concurs with high corticosterone and low levels of the orexin precursor prepro-orexin and that WT and Cry1/2-/- mice respond differently to alcohol withdrawal. As a model of environmentally induced disruption of circadian rhythms, we exposed mice to a "shift work" light/dark regimen, which also leads to a reduction in their alcohol preference. Interestingly, this effect is even more pronounced when genetic and environmental circadian perturbations interact in Cry1/2-/- mice under "shift work" conditions. In conclusion, our study demonstrates that in mice, disturbances in circadian rhythms have pronounced effects on alcohol consumption as well as on physiological factors and other behaviours associated with AUD and that the interaction between circadian genetic and environmental disturbances further alters alcohol consumption behaviour.

The circadian clock regulates rhythmic erythropoietin expression in the murine kidney.

Generation of circadian rhythms is cell-autonomous and relies on a transcription/translation feedback loop controlled by a family of circadian clock transcription factor activators including CLOCK, BMAL1 and repressors such as CRY1 and CRY2. The aim of the present study was to examine both the molecular mechanism and the hemopoietic implication of circadian erythropoietin expression. Mutant mice with homozygous deletion of the core circadian clock genes cryptochromes 1 and 2 (Cry-null) were used to elucidate circadian erythropoietin regulation. Wild-type control mice exhibited a significant difference in kidney erythropoietin mRNA expression between circadian times 06 and 18. In parallel, a significantly higher number of erythropoietin-producing cells in the kidney (by RNAscope®) and significantly higher levels of circulating erythropoietin protein (by ELISA) were detected at circadian time 18. Such changes were abolished in Cry-null mice and were independent from oxygen tension, oxygen saturation, or expression of hypoxia-inducible factor 2 alpha, indicating that circadian erythropoietin expression is transcriptionally regulated by CRY1 and CRY2. Reporter gene assays showed that the CLOCK/BMAL1 heterodimer activated an E-box element in the 5' erythropoietin promoter. RNAscope® in situ hybridization confirmed the presence of Bmal1 in erythropoietin-producing cells of the kidney. In Cry-null mice, a significantly reduced number of reticulocytes was found while erythrocyte numbers and hematocrit were unchanged. Thus, circadian erythropoietin regulation in the normoxic adult murine kidney is transcriptionally controlled by master circadian activators CLOCK/BMAL1, and repressors CRY1/CRY2. These findings may have implications for kidney physiology and disease, laboratory diagnostics, and anemia therapy.

Genomic perspectives on the circadian clock hypothesis of psychiatric disorders.

Circadian rhythm disturbances are frequently described in psychiatric disorders such as major depressive disorder, bipolar disorder, and schizophrenia. Growing evidence suggests a biological connection between mental health and circadian rhythmicity, including the circadian influence on brain function and mood and the requirement for circadian entrainment by external factors, which is often impaired in mental illness. Mental (as well as physical) health is also adversely affected by circadian misalignment. The marked interindividual differences in this combined susceptibility, in addition to the phenotypic spectrum in traits related both to circadian rhythms and mental health, suggested the possibility of a shared genetic background and that circadian clock genes may also be candidate genes for psychiatric disorders. This hypothesis was further strengthened by observations in animal models where clock genes had been knocked out or mutated. The introduction of genome-wide association studies (GWAS) enabled hypothesis-free testing. GWAS analysis of chronotype confirmed the prominent role of circadian genes in these phenotypes and their extensive polygenicity. However, in GWAS on psychiatric traits, only one clock gene, ARNTL (BMAL1) was identified as one of the few loci differentiating bipolar disorder from schizophrenia, and macaque monkeys where the ARNTL gene has been knocked out display symptoms similar to schizophrenia. Another lesson from genomic analyses is that chronotype has an important genetic correlation with several psychiatric disorders and that this effect is unidirectional. We conclude that the effect of circadian disturbances on psychiatric disorders probably relates to modulation of rhythm parameters and extend beyond the core clock genes themselves.

An in-depth neurobehavioral characterization shows anxiety-like traits, impaired habituation behavior, and restlessness in male Cryptochrome-deficient mice.

Many psychiatric disorders, for example, anxiety, are accompanied by disturbances of circadian rhythms, including disturbed sleep/wake cycles, changes in locomotor activity, and abnormal endocrine function. Conversely, alternations of circadian rhythms are a risk factor for the development of psychiatric disorders. This assumption is supported by animals with clock gene mutations which often display behaviors that resemble human psychiatric disorders. In this study, we performed an in-depth behavioral analysis with male mice lacking the central clock genes Cryptochrome 1 and 2 (Cry1/2-/- ), which are thus unable to express endogenous circadian rhythms. With wild-type and Cry1/2-/- mice, we performed an extensive behavioral analysis to study their cognitive abilities, social behavior, and their expression of depression-like and anxiety-like behavior. While Cry1/2-/- mice showed only mild abnormalities at cognitive and social behavioral levels, they were consistently more anxious than wildtype mice. Anxiety-like behavior was particularly evident in reduced mobility in new environments, altered ability to habituate, compensatory behavior, and consistent restless behavior across many behavioral tests. In line with their anxiety-like behavioral phenotype, Cry1/2-/- mice have higher c-Fos activity in the amygdala after exposure to an anxiogenic stressor than wild-type mice. In our study, we identified Cry1/2-/- mice as animals that qualify as a translational mouse model for anxiety disorder in humans because of its consistent behavior of restlessness, increased immobility, and dysfunctional habituation in new environments.

DAILY-A Personalized Circadian Zeitgeber Therapy as an Adjunctive Treatment for Alcohol Use Disorder Patients: Study Protocol for a Randomized Controlled Trial.

Background: Hallmarks of alcohol use disorder (AUD) are disturbances of circadian rhythms and everyday structures. While circadian rhythms dictate the timing of daily recurring activities such as sleep, activity, and meals, conversely, these activities represent time cues, so called Zeitgebers, that the circadian system uses to synchronize with the environment. Here we present a study protocol for our newly developed therapy approach for AUD patients, in which we take advantage of this mutual influence and stabilize and strengthen their circadian system by creating strict daily schedules for daily Zeitgeber activities. Since every person has a circadian system with its own characteristics and is subject to social obligations, the daily plans are personalized for each test person. Our hypothesis is that a regular exposure to Zeitgebers stabilizes behavioral and physiological circadian rhythms and thereby reduces the risk of alcohol relapses and depressive symptoms and facilitates physical recovery in AUD patients during the 1st weeks of their addiction therapy. Methods/design: The study is a 6-weeks single site trial with a controlled, randomized, single-blinded, parallel-group design including patients with a diagnosis of AUD. The study runs parallel to the standard addiction therapy of the clinic. Patients are randomly assigned to either an intervention group (DAILY) or a sham control group (placebo treatment). Questionnaires and physiological assessments of both groups are conducted before and immediately after the intervention or control treatment. According to our hypothesis, the primary outcomes of this study are improvements of regularity, alcohol consumption, and relapse rate in AUD patients compared to AUD patients receiving control treatment. Secondary outcomes are reduced depressive symptoms and increased physical recovery. Discussion: This study is a randomized controlled trial to investigate the efficacy of a personalized circadian Zeitgeber therapy as an adjunctive treatment for alcohol use disorder patients. The overall goal of this and more extended future studies is the development of an adjunctive therapy for AUD patients that is uncomplicated in its use and easy to implement in the clinical and everyday routine. Trial registration: This study is registered at the German Clinical Trial Register with the trial number DRKS00019093 on November 28, 2019.

Prospects for circadian treatment of mood disorders.

Disruption of circadian clocks is strongly associated with mood disorders. Chronotherapies targeting circadian rhythms have been shown to be very effective treatments of mood disorders, but still are not widely used in clinical practice. The mechanisms by which circadian disruption leads to mood disorders are poorly characterized and, therefore, may not convince clinicians to apply chronotherapies. Hence, in this review, we describe specific potential mechanisms, in order to make this connection more credible to clinicians. We believe that four major features of disrupted clocks may contribute to the development of mood disorders: (1) loss of synchronization to environmental 24-h rhythms, (2) internal desynchronization among body clocks, (3) low rhythm amplitude, and (4) changes in sleep architecture. Discussing these attributes and giving plausible examples, we will discuss prospects for relatively simple chronotherapies addressing these features that are easy to implement in clinical practice. Key messages In this review, we describe specific potential mechanisms by which disrupted clocks may contribute to the development of mood disorders: (1) loss of synchronization to environmental 24-h rhythms, (2) internal desynchronization among body clocks, (3) low rhythm amplitude, and (4) changes in sleep architecture. We provide prospects for relatively simple chronotherapies addressing these features that are easy to implement in clinical practice.

Inositol polyphosphates contribute to cellular circadian rhythms: Implications for understanding lithium's molecular mechanism.

Most living organisms maintain cell autonomous circadian clocks that synchronize critical biological functions with daily environmental cycles. In mammals, the circadian clock is regulated by inputs from signaling pathways including glycogen synthase kinase 3 (GSK3). The drug lithium has actions on GSK3, and also on inositol metabolism. While it is suspected that lithium's inhibition of GSK3 causes rhythm changes, it is not known if inositol polyphosphates can also affect the circadian clock. We examined whether the signaling molecule inositol hexaphosphate (IP6) has effects on circadian rhythms. Using a bioluminescent reporter (Per2::luc) to measure circadian rhythms, we determined that IP6 increased rhythm amplitude and shortened period in NIH3T3 cells. The IP6 effect on amplitude was attenuated by selective siRNA knockdown of GSK3B and pharmacological blockade of AKT kinase. However, unlike lithium, IP6 did not induce serine-9 phosphorylation of GSK3B. The synthesis of IP6 involves the enzymes inositol polyphosphate multikinase (IPMK) and inositol pentakisphosphate 2-kinase (IPPK). Knockdown of Ippk had effects opposite to those of IP6, decreasing rhythm amplitude and lengthening period. Ipmk knockdown had few effects on rhythm alone, but attenuated the effects of lithium on rhythms. However, lithium did not change the intracellular content of IP6 in NIH3T3 cells or neurons. Pharmacological inhibition of the IP6 kinases (IP6K) increased rhythm amplitude and shortened period, suggesting secondary effects of inositol pyrophosphates may underlie the period shortening effect, but not the amplitude increasing effect of IP6. Overall, we conclude that inositol phosphates, in particular IP6 have effects on circadian rhythms. Manipulations affecting IP6 and related inositol phosphates may offer a novel means through which circadian rhythms can be regulated.

Circadian clock-gastrointestinal peptide interaction in peripheral tissues and the brain.

Food intake and sleep are two mutually exclusive behaviors and both are normally confined to opposing phases of the diurnal cycle. The temporal coordination of behavior and physiology along the 24-h day-night cycle is organized by a network of circadian clocks that orchestrate transcriptional programs controlling cellular physiology. Many of the peptide hormones of the gastrointestinal tract are not only secreted in a circadian fashion, they can also affect circadian clock function in peripheral metabolic tissues and the brain, thus providing metabolic feedback to metabolic and neurobehavioral circuits. In this review, we summarize the current knowledge on this gastrointestinal peptide crosstalk and its potential role in the coordination of nutrition and the maintenance of metabolic homeostasis.

Cellular circadian oscillators in the suprachiasmatic nucleus remain coupled in the absence of connexin-36.

In mammals, the master circadian clock resides in the suprachiasmatic nucleus (SCN). The SCN is characterized by robust circadian oscillations of clock gene expression and neuronal firing. The synchronization of circadian oscillations among individual cells in the SCN is attributed to intercellular coupling. Previous studies have shown that gap junctions, specifically those composed of connexin-36 (Cx36) subunits, are required for coupling of electrical firing among SCN neurons at a time scale of milliseconds. However, it remains unknown whether Cx36 gap junctions also contribute to coupling of circadian (∼24h) rhythms of clock gene expression. Here, we investigated circadian expression patterns of the clock gene Period 2 (Per2) in the SCN of Cx36-deficient mice using luminometry and single-cell bioluminescence imaging. Surprisingly, we found that synchronization of circadian PER2 expression rhythms is maintained in SCN explants from Cx36-deficient mice. Since Cx36 expression levels change with age, we also tested circadian running-wheel behavior of juvenile (3-4weeks old) and adult (9-30weeks old) Cx36-deficient mice. We found that impact of connexin-36 expression on circadian behavior changes greatly during postnatal development. However, consistent with the intact synchrony among SCN cells in cultured explants, Cx36-deficient mice had intact locomotor circadian rhythms, although adults displayed a lengthened period in constant darkness. Our data indicate that even though Cx36 may be required for electrical coupling of SCN cells, it does not affect coupling of molecular clock gene rhythms. Thus, electrical coupling of neurons and coupling of circadian clock gene oscillations can be regulated independently in the SCN.

Enhancing circadian clock function in cancer cells inhibits tumor growth.

BACKGROUND: Circadian clocks control cell cycle factors, and circadian disruption promotes cancer. To address whether enhancing circadian rhythmicity in tumor cells affects cell cycle progression and reduces proliferation, we compared growth and cell cycle events of B16 melanoma cells and tumors with either a functional or dysfunctional clock. RESULTS: We found that clock genes were suppressed in B16 cells and tumors, but treatments inducing circadian rhythmicity, such as dexamethasone, forskolin and heat shock, triggered rhythmic clock and cell cycle gene expression, which resulted in fewer cells in S phase and more in G1 phase. Accordingly, B16 proliferation in vitro and tumor growth in vivo was slowed down. Similar effects were observed in human colon carcinoma HCT-116 cells. Notably, the effects of dexamethasone were not due to an increase in apoptosis nor to an enhancement of immune cell recruitment to the tumor. Knocking down the essential clock gene Bmal1 in B16 tumors prevented the effects of dexamethasone on tumor growth and cell cycle events. CONCLUSIONS: Here we demonstrated that the effects of dexamethasone on cell cycle and tumor growth are mediated by the tumor-intrinsic circadian clock. Thus, our work reveals that enhancing circadian clock function might represent a novel strategy to control cancer progression.

Disinhibition of the extracellular-signal-regulated kinase restores the amplification of circadian rhythms by lithium in cells from bipolar disorder patients.

UNLABELLED: Bipolar disorder (BD) is characterized by depression, mania, and circadian rhythm abnormalities. Lithium, a treatment for BD stabilizes mood and increases circadian rhythm amplitude. However, in fibroblasts grown from BD patients, lithium has weak effects on rhythm amplitude compared to healthy controls. To understand the mechanism by which lithium differentially affects rhythm amplitude in BD cells, we investigated the extracellular-signal-regulated kinase (ERK) and related signaling molecules linked to BD and circadian rhythms. In fibroblasts from BD patients, controls and mice, we assessed the contribution of the ERK pathway to lithium-induced circadian rhythm amplification. Protein analyses revealed low phospho-ERK1/2 (p-ERK) content in fibroblasts from BD patients vs. CONTROLS: Pharmacological inhibition of ERK1/2 by PD98059 attenuated the rhythm amplification effect of lithium, while inhibition of two related kinases, c-Jun N-terminal kinase (JNK), and P38 did not. Knockdown of the transcription factors CREB and EGR-1, downstream effectors of ERK1/2, reduced baseline rhythm amplitude, but did not alter rhythm amplification by lithium. In contrast, ELK-1 knockdown amplified rhythms, an effect that was not increased further by the addition of lithium, suggesting this transcription factor may regulate the effect of lithium on amplitude. Augmentation of ERK1/2 signaling through DUSP6 knockdown sensitized NIH3T3 cells to rhythm amplification by lithium. In BD fibroblasts, DUSP6 knockdown reversed the BD rhythm phenotype, restoring the ability of lithium to increase amplitude in these cells. We conclude that the inability of lithium to regulate circadian rhythms in BD may reflect reduced ERK activity, and signaling through ELK-1.

Genetic Disruption of Circadian Rhythms in the Suprachiasmatic Nucleus Causes Helplessness, Behavioral Despair, and Anxiety-like Behavior in Mice.

BACKGROUND: Major depressive disorder is associated with disturbed circadian rhythms. To investigate the causal relationship between mood disorders and circadian clock disruption, previous studies in animal models have employed light/dark manipulations, global mutations of clock genes, or brain area lesions. However, light can impact mood by noncircadian mechanisms; clock genes have pleiotropic, clock-independent functions; and brain lesions not only disrupt cellular circadian rhythms but also destroy cells and eliminate important neuronal connections, including light reception pathways. Thus, a definitive causal role for functioning circadian clocks in mood regulation has not been established. METHODS: We stereotactically injected viral vectors encoding short hairpin RNA to knock down expression of the essential clock gene Bmal1 into the brain's master circadian pacemaker, the suprachiasmatic nucleus (SCN). RESULTS: In these SCN-specific Bmal1-knockdown (SCN-Bmal1-KD) mice, circadian rhythms were greatly attenuated in the SCN, while the mice were maintained in a standard light/dark cycle, SCN neurons remained intact, and neuronal connections were undisturbed, including photic inputs. In the learned helplessness paradigm, the SCN-Bmal1-KD mice were slower to escape, even before exposure to inescapable stress. They also spent more time immobile in the tail suspension test and less time in the lighted section of a light/dark box. The SCN-Bmal1-KD mice also showed greater weight gain, an abnormal circadian pattern of corticosterone, and an attenuated increase of corticosterone in response to stress. CONCLUSIONS: Disrupting SCN circadian rhythms is sufficient to cause helplessness, behavioral despair, and anxiety-like behavior in mice, establishing SCN-Bmal1-KD mice as a new animal model of depression.

The mood stabilizer valproic acid opposes the effects of dopamine on circadian rhythms.

Endogenous circadian (∼24 h) clocks regulate key physiological and cognitive processes via rhythmic expression of clock genes. The main circadian pacemaker is the hypothalamic suprachiasmatic nucleus (SCN). Mood disorders, including bipolar disorder (BD), are commonly associated with disturbed circadian rhythms. Dopamine (DA) contributes to mania in BD and has direct impact on clock gene expression. Therefore, we hypothesized that high levels of DA during episodes of mania contribute to disturbed circadian rhythms in BD. The mood stabilizer valproic acid (VPA) also affects circadian rhythms. Thus, we further hypothesized that VPA normalizes circadian disturbances caused by elevated levels of DA. To test these hypotheses, we examined locomotor rhythms and circadian gene cycling in mice with reduced expression of the dopamine transporter (DAT-KD mice), which results in elevated DA levels and mania-like behavior. We found that elevated DA signaling lengthened the circadian period of behavioral rhythms in DAT-KD mice and clock gene expression rhythms in SCN explants. In contrast, we found that VPA shortened circadian period of behavioral rhythms in DAT-KD mice and clock gene expression rhythms in SCN explants, hippocampal cell lines, and human fibroblasts from BD patients. Thus, DA and VPA have opposing effects on circadian period. To test whether the impact of VPA on circadian rhythms contributes to its behavioral effects, we fed VPA to DAT-deficient Drosophila with and without functioning circadian clocks. Consistent with our hypothesis, we found that VPA had potent activity-suppressing effects in hyperactive DAT-deficient flies with intact circadian clocks. However, these effects were attenuated in DAT-deficient flies in which circadian clocks were disrupted, suggesting that VPA functions partly through the circadian clock to suppress activity. Here, we provide in vivo and in vitro evidence across species that elevated DA signaling lengthens the circadian period, an effect remediated by VPA treatment. Hence, VPA may exert beneficial effects on mood by normalizing lengthened circadian rhythm period in subjects with elevated DA resulting from reduced DAT.

NPAS2 Compensates for Loss of CLOCK in Peripheral Circadian Oscillators.

Heterodimers of CLOCK and BMAL1 are the major transcriptional activators of the mammalian circadian clock. Because the paralog NPAS2 can substitute for CLOCK in the suprachiasmatic nucleus (SCN), the master circadian pacemaker, CLOCK-deficient mice maintain circadian rhythms in behavior and in tissues in vivo. However, when isolated from the SCN, CLOCK-deficient peripheral tissues are reportedly arrhythmic, suggesting a fundamental difference in circadian clock function between SCN and peripheral tissues. Surprisingly, however, using luminometry and single-cell bioluminescence imaging of PER2 expression, we now find that CLOCK-deficient dispersed SCN neurons and peripheral cells exhibit similarly stable, autonomous circadian rhythms in vitro. In CLOCK-deficient fibroblasts, knockdown of Npas2 leads to arrhythmicity, suggesting that NPAS2 can compensate for loss of CLOCK in peripheral cells as well as in SCN. Our data overturn the notion of an SCN-specific role for NPAS2 in the molecular circadian clock, and instead indicate that, at the cellular level, the core loops of SCN neuron and peripheral cell circadian clocks are fundamentally similar.

Depression-like behaviour in mice is associated with disrupted circadian rhythms in nucleus accumbens and periaqueductal grey.

An association between circadian rhythms and mood regulation is well established, and disturbed circadian clocks are believed to contribute to the development of mood disorders, including major depressive disorder. The circadian system is coordinated by the suprachiasmatic nucleus (SCN), the master pacemaker in the hypothalamus that receives light input from the retina and synchronizes circadian oscillators in other brain regions and peripheral tissues. Lacking the tight neuronal network that couples single-cell oscillators in the SCN, circadian clocks outside the SCN may be less stable and more susceptible to disturbances, for example by clock gene mutations or uncontrollable stress. However, non-SCN circadian clocks have not been studied extensively in rodent models of mood disorders. In the present study, it was hypothesized that disturbances of local circadian clocks in mood-regulating brain areas are associated with depression-like behaviour in mice. Using the learned helplessness procedure, depression-like behaviour was evoked in mice bearing the PER2::LUC circadian reporter, and then circadian rhythms of PER2 expression were examined in brain slices from these mice using luminometry and bioluminescence imaging. It was found that helplessness is associated with absence of circadian rhythms in the nucleus accumbens and the periaqueductal grey, two of the most critical brain regions within the reward circuit. The current study provides evidence that susceptibility of mice to depression-like behaviour is associated with disturbed local circadian clocks in a subset of mood-regulating brain areas, but the direction of causality remains to be determined.

Circadian Clocks as Modulators of Metabolic Comorbidity in Psychiatric Disorders.

Psychiatric disorders such as schizophrenia, bipolar disorder, and major depressive disorder are often accompanied by metabolic dysfunction symptoms, including obesity and diabetes. Since the circadian system controls important brain systems that regulate affective, cognitive, and metabolic functions, and neuropsychiatric and metabolic diseases are often correlated with disturbances of circadian rhythms, we hypothesize that dysregulation of circadian clocks plays a central role in metabolic comorbidity in psychiatric disorders. In this review paper, we highlight the role of circadian clocks in glucocorticoid, dopamine, and orexin/melanin-concentrating hormone systems and describe how a dysfunction of these clocks may contribute to the simultaneous development of psychiatric and metabolic symptoms.

Dissociation of learned helplessness and fear conditioning in mice: a mouse model of depression.

The state of being helpless is regarded as a central aspect of depression, and therefore the learned helplessness paradigm in rodents is commonly used as an animal model of depression. The term 'learned helplessness' refers to a deficit in escaping from an aversive situation after an animal is exposed to uncontrollable stress specifically, with a control/comparison group having been exposed to an equivalent amount of controllable stress. A key feature of learned helplessness is the transferability of helplessness to different situations, a phenomenon called 'trans-situationality'. However, most studies in mice use learned helplessness protocols in which training and testing occur in the same environment and with the same type of stressor. Consequently, failures to escape may reflect conditioned fear of a particular environment, not a general change of the helpless state of an animal. For mice, there is no established learned helplessness protocol that includes the trans-situationality feature. Here we describe a simple and reliable learned helplessness protocol for mice, in which training and testing are carried out in different environments and with different types of stressors. We show that with our protocol approximately 50% of mice develop learned helplessness that is not attributable to fear conditioning.