The neurons that drive infradian sleep-wake and mania-like behavioral rhythms.

Infradian mood and sleep-wake rhythms with periods of 48 hr and beyond have been observed in bipolar disorder (BD) subjects that even persist in time isolation, indicating an endogenous origin. Here we show that mice exposed to methamphetamine (Meth) in drinking water develop infradian locomotor rhythms with periods of 48 hr and beyond which extend to sleep length and mania-like behaviors in support of a model for cycling in BD. This cycling capacity is abrogated upon genetic disruption of DA production in DA neurons of the ventral tegmental area (VTA) or ablation of nucleus accumbens (NAc) projecting, dopamine (DA) neurons. Chemogenetic activation of NAc-projecting DA neurons leads to locomotor period lengthening in clock deficient mice, while cytosolic calcium in DA processes of the NAc was found fluctuating synchronously with locomotor behavior. Together, our findings argue that BD cycling relies on infradian rhythm generation that depends on NAc-projecting DA neurons.

Behavioral and Transcriptomic Changes Following Brain-Specific Loss of Noradrenergic Transmission.

Noradrenaline (NE) plays an integral role in shaping behavioral outcomes including anxiety/depression, fear, learning and memory, attention and shifting behavior, sleep-wake state, pain, and addiction. However, it is unclear whether dysregulation of NE release is a cause or a consequence of maladaptive orientations of these behaviors, many of which associated with psychiatric disorders. To address this question, we used a unique genetic model in which the brain-specific vesicular monoamine transporter-2 (VMAT2) gene expression was removed in NE-positive neurons disabling NE release in the entire brain. We engineered VMAT2 gene splicing and NE depletion by crossing floxed VMAT2 mice with mice expressing the Cre-recombinase under the dopamine ß-hydroxylase (DBH) gene promotor. In this study, we performed a comprehensive behavioral and transcriptomic characterization of the VMAT2DBHcre KO mice to evaluate the role of central NE in behavioral modulations. We demonstrated that NE depletion induces anxiolytic and antidepressant-like effects, improves contextual fear memory, alters shifting behavior, decreases the locomotor response to amphetamine, and induces deeper sleep during the non-rapid eye movement (NREM) phase. In contrast, NE depletion did not affect spatial learning and memory, working memory, response to cocaine, and the architecture of the sleep-wake cycle. Finally, we used this model to identify genes that could be up- or down-regulated in the absence of NE release. We found an up-regulation of the synaptic vesicle glycoprotein 2c (SV2c) gene expression in several brain regions, including the locus coeruleus (LC), and were able to validate this up-regulation as a marker of vulnerability to chronic social defeat. The NE system is a complex and challenging system involved in many behavioral orientations given it brain wide distribution. In our study, we unraveled specific role of NE neurotransmission in multiple behavior and link it to molecular underpinning, opening future direction to understand NE role in health and disease.

Leptin receptor-expressing pericytes mediate access of hypothalamic feeding centers to circulating leptin.

Knowledge of how leptin receptor (LepR) neurons of the mediobasal hypothalamus (MBH) access circulating leptin is still rudimentary. Employing intravital microscopy, we found that almost half of the blood-vessel-enwrapping pericytes in the MBH express LepR. Selective disruption of pericytic LepR led to increased food intake, increased fat mass, and loss of leptin-dependent signaling in nearby LepR neurons. When delivered intravenously, fluorescently tagged leptin accumulated at hypothalamic LepR pericytes, which was attenuated upon pericyte-specific LepR loss. Because a paracellular tracer was also preferentially retained at LepR pericytes, we pharmacologically targeted regulators of inter-endothelial junction tightness and found that they affect LepR neuronal signaling and food intake. Optical imaging in MBH slices revealed a long-lasting, tonic calcium increase in LepR pericytes in response to leptin, suggesting pericytic contraction and vessel constriction. Together, our data indicate that LepR pericytes facilitate localized, paracellular blood-brain barrier leaks, enabling MBH LepR neurons to access circulating leptin.

Circadian hepatocyte clocks keep synchrony in the absence of a master pacemaker in the suprachiasmatic nucleus or other extrahepatic clocks.

It has been assumed that the suprachiasmatic nucleus (SCN) synchronizes peripheral circadian oscillators. However, this has never been convincingly shown, since biochemical time series experiments are not feasible in behaviorally arrhythmic animals. By using long-term bioluminescence recording in freely moving mice, we show that the SCN is indeed required for maintaining synchrony between organs. Surprisingly, however, circadian oscillations persist in the livers of mice devoid of an SCN or oscillators in cells other than hepatocytes. Hence, similar to SCN neurons, hepatocytes can maintain phase coherence in the absence of Zeitgeber signals produced by other organs or environmental cycles.

Irregular eating patterns associate with hypomanic symptoms in bipolar disorders.

Objective: We present novel dimensional methods to describe the timing of eating in psychopathology. We focused on the relationship between current mood in bipolar disorder (BD) and the stability of the temporal pattern of daily eating events. Methods: Consenting BD patients (n = 69) from an outpatient, tertiary care clinic completed hourly charts of mood and eating for two weeks. Mood was also evaluated with Montgomery-Åsberg Depression Rating Scale (MADRS) and Young Mania Rating Scale (YMRS). Results: Illustrative displays, or eatograms, enabling visualization of all recorded eating events were used to guide assessment of the temporal structure of eating across the two week assessment period. We computed indices to quantify irregularities in timing of eating, namely IFRQ, ITIM and IINT for the variability of frequency, timing, and interval of eating events, respectively. In this cohort, irregular temporal pattern of eating correlated with hypomanic symptoms (YMRS with IFRQ, Spearman rank order rh = 0.28, p = .019, with ITIM, rh = 0.44, p < .001, and with IINT rh = 0.38, p = .001), but not depressive symptoms or anthropometric measures. Conclusions: Our data suggest a link between the instability of the temporal order of daily eating and mood. The dimensional measures for eating pattern introduced here enable future investigations of correlations with psychopathology.

Effects of antipsychotics on circadian rhythms in humans: a systematic review and meta-analysis.

Antipsychotics are widely used to treat psychiatric illness and insomnia. However, the etiology of insomnia is multifactorial, including disrupted circadian rhythms. Several studies show that antipsychotics might modulate even healthy circadian rhythms. The purpose of this systematic review is to integrate current knowledge about the effects of antipsychotics on the circadian rhythms in humans, and to conduct a meta- analysis with the available data. Nine electronic databases were searched. We followed the PRISMA guidelines and included randomized controlled trials (RCTs), non-RCTs, case-control studies, case series, and case reports. Of 7,217 articles, 70 were included. The available data was mainly from healthy individuals, or patients having schizophrenia, but the findings showed a transdiagnostic impact on circadian parameters. This was consistently seen as decreased amplitude of cortisol, melatonin, and body temperature. Particularly, a meta-analysis of 16 RCTs measuring cortisol rhythm showed that antipsychotics, especially atypical antipsychotics, decreased the cortisol area under the curve and morning cortisol level, compared to placebo. The data with melatonin or actigraphy was limited. Overall, this evidence about the circadian effect of antipsychotics showed a need for longitudinal, real-time monitoring of specific circadian markers to differentiate a change in amplitude from a shift in phasing, and for knowledge about optimal timing of administration of antipsychotics, according to individual baseline circadian parameters. Standardizing selection criteria and outcome methods could facilitate good quality intervention studies and evidence-based treatment guidelines. This is relevant considering the accumulating evidence of the high prevalence and unfavorable impact of disrupted circadian rhythms in psychiatric disorders.

The core clock transcription factor BMAL1 drives circadian ß-cell proliferation during compensatory regeneration of the endocrine pancreas.

Circadian clocks in pancreatic islets participate in the regulation of glucose homeostasis. Here we examined the role of these timekeepers in ß-cell regeneration after the massive ablation of ß cells by doxycycline-induced expression of diphtheria toxin A (DTA) in Insulin-rtTA/TET-DTA mice. Since we crossed reporter genes expressing α- and ß-cell-specific fluorescent proteins into these mice, we could follow the fate of α- and ß cells separately. As expected, DTA induction resulted in an acute hyperglycemia, which was accompanied by dramatic changes in gene expression in residual ß cells. In contrast, only temporal alterations of gene expression were observed in α cells. Interestingly, ß cells entered S phase preferentially during the nocturnal activity phase, indicating that the diurnal rhythm also plays a role in the orchestration of ß-cell regeneration. Indeed, in arrhythmic Bmal1-deficient mice, which lack circadian clocks, no compensatory ß-cell proliferation was observed, and the ß-cell ablation led to aggravated hyperglycemia, hyperglucagonemia, and fatal diabetes.

Suicidal Ideation and Insomnia in Bipolar Disorders: Idéation suicidaire et insomnie dans les troubles bipolaires.

OBJECTIVE: Bipolar disorder (BD) confers elevated suicide risk and associates with misaligned circadian rhythm. Real-time monitoring of objectively measured sleep is a novel approach to detect and prevent suicidal behavior. We aimed at understanding associations between subjective insomnia and actigraphy data with severity of suicidal ideation in BDs. METHODS: This prospective cohort study comprised 76 outpatients with a BD aged 18 to 65 inclusively. Main measures included 10 consecutive days of wrist actigraphy; the Athens Insomnia Scale (AIS); the Montgomery-Åsberg Depression Rating Scale (MADRS); the Quick Inventory of Depressive Symptoms-16, self-rating (QIDS-SR-16); and the Columbia Suicide Severity Rating Scale. Diagnoses, medications, and suicide attempts were obtained from chart review. RESULTS: Suicidal ideation correlated moderately with subjective insomnia (AIS with QIDS-SR-16 item 12 ρ =0.26, P = 0.03; MADRS item 10 ρ = 0.33, P = 0.003). Graphical sleep patterns showed that suicidal patients were enriched among the most fragmented sleep patterns, and this was confirmed by correlations of suicidal ideation with actigraphy data at 2 visits. Patients with lifetime suicide attempts (n = 8) had more varied objective sleep (a higher standard deviation of center of daily inactivity [0.64 vs. 0.26, P = 0.01], consolidation of daily inactivity [0.18 vs. 0.10, P = <0.001], sleep offset [3.02 hours vs. 1.90 hours, P = <0.001], and total sleep [105 vs. 69 minutes, P = 0.02], and a lower consolidation of daily inactivity [0.65 vs. 0.79, P = 0.03]). CONCLUSIONS: Subjective insomnia, a nonstigmatized symptom, can complement suicidality screens. Longer follow-ups and larger samples are warranted to understand whether real-time sleep monitoring predicts suicidal ideation in patient subgroups or individually.

Ultradian Secretion of Growth Hormone in Mice: Linking Physiology With Changes in Synapse Parameters Using Super-Resolution Microscopy.

Neuroendocrine circuits are orchestrated by the pituitary gland in response to hypothalamic hormone-releasing and inhibiting factors to generate an ultradian and/or circadian rhythm of hormone secretion. However, mechanisms that govern this rhythmicity are not fully understood. It has been shown that synaptic transmission in the rodent hypothalamus undergoes cyclical changes in parallel with rhythmic hormone secretion and a growing body of evidence suggests that rapid rewiring of hypothalamic neurons may be the source of these changes. For decades, structural synaptic studies have been utilizing electron microscopy, which provides the resolution suitable for visualizing synapses. However, the small field of view, limited specificity and manual analysis susceptible to bias fuel the search for a more quantitative approach. Here, we apply the fluorescence super-resolution microscopy approach direct Stochastic Optical Reconstruction Microscopy (dSTORM) to quantify and structurally characterize excitatory and inhibitory synapses that contact growth hormone-releasing-hormone (GHRH) neurons during peak and trough values of growth hormone (GH) concentration in mice. This approach relies on a three-color immunofluorescence staining of GHRH and pre- and post-synaptic markers, and a quantitative analysis with a Density-Based Spatial Clustering of Applications with Noise (DBSCAN) algorithm. With this method we confirm our previous findings, using electron microscopy, of increased excitatory synaptic input to GHRH neurons during peak levels of GH. Additionally, we find a shift in synapse numbers during low GH levels, where more inhibitory synaptic inputs are detected. Lastly, we utilize dSTORM to study novel aspects of synaptic structure. We show that more excitatory (but not inhibitory) pre-synaptic clusters associate with excitatory post-synaptic clusters during peaks of GH secretion and that the numbers of post-synaptic clusters increase during high hormone levels. The results presented here provide an opportunity to highlight dSTORM as a valuable quantitative approach to study synaptic structure in the neuroendocrine circuit. Importantly, our analysis of GH circuitry sheds light on the potential mechanism that drives ultradian changes in synaptic transmission and possibly aids in GH pulse generation in mice.

Inactograms and objective sleep measures as means to capture subjective sleep problems in patients with a bipolar disorder.

BACKGROUND: Sleep problems are common in bipolar disorders (BDs). To objectively characterize these problems in BDs, further methodological development is needed to capture subjective insomnia. AIM: To test psychometric properties of the Athens Insomnia Scale (AIS), and associations with actigraphy-derived measures, applying modifications in actigraphy data processing to capture features of perturbed sleep in patients with a BD. METHODS: Seventy-four patients completed the AIS and the Quick Inventory of Depressive Symptomatology, self-report (QIDS-SR-16). Locomotor activity was continuously recorded by wrist actigraphy for ≥10 consecutive days. We computed the sleep onset/offset, the center of daily inactivity (CenDI), as a proxy for chronotype, and the degree of consolidation of daily inactivity (ConDI), as a proxy for sleep-wake rhythm strength. RESULTS: AIS showed good psychometric properties (Cronbach's alpha = 0.84; test-retest correlation = 0.84, P<.001). Subjective sleep problems correlated moderately with a later sleep phase (CenDI with AIS rho = 0.34, P = .003), lower consolidation (ConDI with AIS rho = -0.22, P = .05; with QIDS-SR-16 rho = -0.27, P = .019), later timing of sleep offset (with AIS rho = 0.49, P = ≤.001, with QIDS-SR-16 rho = 0.36, P = .002), and longer total sleep (with AIS rho = 0.29, P = .012, with QIDS-SR-16 rho = 0.41, P = ≤.001). While AIS was psychometrically more solid, correlations with objective sleep were more consistent across time for QIDS-SR-16. CONCLUSIONS: AIS and QIDS-SR-16 are suitable for clinical screening of sleep problems among patients with a BD. Subjective insomnia associated with objective measures. For clinical and research purposes, actigraphy and data visualization on inactograms are useful for accurate longitudinal characterization of sleep patterns.

Late and Instable Sleep Phasing is Associated With Irregular Eating Patterns in Eating Disorders.

BACKGROUND: Sleep problems are common in eating disorders (EDs). PURPOSE: We evaluated whether sleep-phasing regularity associates with the regularity of daily eating events. METHODS: ED patients (n = 29) completed hourly charts of mood and eating occasions for 2 weeks. Locomotor activity was recorded continuously by wrist actigraphy for a minimum of 10 days, and sleep was calculated based on periods of inactivity. We computed the center of daily inactivity (CenDI) as a measure of sleep phasing and consolidation of the daily inactivity (ConDI) as a measure of daily sleep rhythm strength. We assessed interday irregularities in the temporal structure of food intake using the standard deviation (SD) of frequency (IFRQ), timing (ITIM), and interval (IINT) of food intake. A self-evaluation of other characteristics included mood, anxiety, and early trauma. RESULTS: A later phasing of sleep associated with a lower frequency of eating (eating frequency with the CenDI rho = -0.49, p = .007). The phasing and rhythmic strength of sleep correlated with the degree of eating irregularity (CenDI with ITIM rho = 0.48, p = .008 and with IINT rho = 0.56, p = .002; SD of CenDI with ITIM rho = 0.47, p = .010, and SD of ConDI with IINT rho = 0.37, p = .048). Childhood Trauma Questionnaire showed associations with variation of sleep onset (rho = -0.51, p = .005) and with IFRQ (rho = 0.43, p = .023). CONCLUSIONS: Late and variable phasing of sleep associated robustly with irregular pattern of eating. Larger data sets are warranted to enable the analysis of diagnostic subgroups, current medication, and current symptomatology and to confirm the likely bidirectional association between eating pattern stability and the timing of sleep.

The eIF2α Kinase GCN2 Modulates Period and Rhythmicity of the Circadian Clock by Translational Control of Atf4.

The integrated stress response (ISR) is activated in response to diverse stress stimuli to maintain homeostasis in neurons. Central to this process is the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). Here, we report a critical role for ISR in regulating the mammalian circadian clock. The eIF2α kinase GCN2 rhythmically phosphorylates eIF2α in the suprachiasmatic circadian clock. Increased eIF2α phosphorylation shortens the circadian period in both fibroblasts and mice, whereas reduced eIF2α phosphorylation lengthens the circadian period and impairs circadian rhythmicity in animals. Mechanistically, phosphorylation of eIF2α promotes mRNA translation of Atf4. ATF4 binding motifs are identified in multiple clock genes, including Per2, Per3, Cry1, Cry2, and Clock. ATF4 binds to the TTGCAGCA motif in the Per2 promoter and activates its transcription. Together, these results demonstrate a significant role for ISR in circadian physiology and provide a potential link between dysregulated ISR and circadian dysfunction in brain diseases.

Morningness-Eveningness questionnaire in bipolar disorder.

The Morningness-Eveningness Questionnaire (MEQ) is among the most commonly used scales to measure chronotype. We aimed to evaluate psychometric properties and clinical correlates of MEQ in bipolar disorder. Patients with a clinical diagnosis of bipolar disorder (n = 53) answered questionnaires for chronotype (MEQ), mood (Quick Inventory of Depressive Symptoms-16, Altman Self-Rating Mania Scale), insomnia (Athens Insomnia Scale, AIS), and sleepiness (Epworth Sleepiness Scale). Mood was evaluated using Montgomery-Åsberg Depression Rating Scale and Young Mania Rating Scale. The MEQ showed high internal consistency with Cronbach's alpha of .85. Lower MEQ scores (eveningness) correlated with insomnia (AIS) (r = -.34, p = .013). The estimate for eveningness (13/53, 24.5%) in our study was higher than in comparable studies in the general population. Patients on lithium exhibited a higher mean MEQ score (56.0 on lithium vs 46.9 with no lithium, p = .007), whereas this score was lower for patients on an antidepressant (46.0 on antidepressants vs 52.6 with no antidepressants, p = .023). We conclude that the MEQ score is psychometrically reliable. However, future studies should further evaluate the association of medication with chronotype. Validation of categorical cut-offs for MEQ in a larger sample of bipolar patients is needed to increase clinical utility.

Control of Rest:Activity by a Dopaminergic Ultradian Oscillator and the Circadian Clock.

There is long-standing evidence for rhythms in locomotor activity, as well as various other aspects of physiology, with periods substantially shorter than 24 h in organisms ranging from fruit flies to humans. These ultradian oscillations, whose periods frequently fall between 2 and 6 h, are normally well integrated with circadian rhythms; however, they often lack the period stability and expression robustness of the latter. An adaptive advantage of ultradian rhythms has been clearly demonstrated for the common vole, suggesting that they may have evolved to confer social synchrony. The cellular substrate and mechanism of ultradian rhythm generation have remained elusive so far, however recent findings-the subject of this review-now indicate that ultradian locomotor rhythms rely on an oscillator based on dopamine, dubbed the dopaminergic ultradian oscillator (DUO). These findings also reveal that the DUO period can be lengthened from <4 to >48 h by methamphetamine treatment, suggesting that the previously described methamphetamine-sensitive (circadian) oscillator represents a long-period manifestation of the DUO.

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.

DCC Confers Susceptibility to Depression-like Behaviors in Humans and Mice and Is Regulated by miR-218.

BACKGROUD: Variations in the expression of the Netrin-1 guidance cue receptor DCC (deleted in colorectal cancer) appear to confer resilience or susceptibility to psychopathologies involving prefrontal cortex (PFC) dysfunction. METHODS: With the use of postmortem brain tissue, mouse models of defeat stress, and in vitro analysis, we assessed microRNA (miRNA) regulation of DCC and whether changes in DCC levels in the PFC lead to vulnerability to depression-like behaviors. RESULTS: We identified miR-218 as a posttranscriptional repressor of DCC and detected coexpression of DCC and miR-218 in pyramidal neurons of human and mouse PFC. We found that exaggerated expression of DCC and reduced levels of miR-218 in the PFC are consistent traits of mice susceptible to chronic stress and of major depressive disorder in humans. Remarkably, upregulation of Dcc in mouse PFC pyramidal neurons causes vulnerability to stress-induced social avoidance and anhedonia. CONCLUSIONS: These data are the first demonstration of microRNA regulation of DCC and suggest that, by regulating DCC, miR-218 may be a switch of susceptibility versus resilience to stress-related disorders.

Adult NG2-Glia Are Required for Median Eminence-Mediated Leptin Sensing and Body Weight Control.

While leptin is a well-known regulator of body fat mass, it remains unclear how circulating leptin is sensed centrally to maintain energy homeostasis. Here we show that genetic and pharmacological ablation of adult NG2-glia (also known as oligodendrocyte precursors), but not microglia, leads to primary leptin resistance and obesity in mice. We reveal that NG2-glia contact the dendritic processes of arcuate nucleus leptin receptor (LepR) neurons in the median eminence (ME) and that these processes degenerate upon NG2-glia elimination, which explains the consequential attenuation of these neurons' molecular and electrical responses to leptin. Our data therefore indicate that LepR dendrites in the ME represent the principal conduits of leptin's anorexigenic action and that NG2-glia are essential for their maintenance. Given that ME-directed X-irradiation confirmed the pharmacological and genetically mediated ablation effects on body weight, our findings provide a rationale for the known obesity risk associated with cranial radiation therapy.

Clocks within the Master Gland: Hypophyseal Rhythms and Their Physiological Significance.

Various aspects of mammalian endocrine physiology show a time-of-day variation with a period of 24 h, which represents an adaptation to the daily environmental fluctuations resulting from the rotation of the earth. These 24-h rhythms in hormone abundance and consequently hormone function may rely on rhythmic signals produced by the master circadian clock, which resides in the suprachiasmatic nucleus and is thought to chiefly dictate the pattern of rest and activity in mammals in conjunction with the light/dark (LD) cycle. However, it is likely that clocks intrinsic to elements of the endocrine axes also contribute to the 24-h rhythms in hormone function. Here we review the evidence for rhythm generation in the endocrine master gland, the pituitary, and its physiological significance in the context of endocrine axes regulation and function.

Light-regulated translational control of circadian behavior by eIF4E phosphorylation.

The circadian (∼24 h) clock is continuously entrained (reset) by ambient light so that endogenous rhythms are synchronized with daily changes in the environment. Light-induced gene expression is thought to be the molecular mechanism underlying clock entrainment. mRNA translation is a key step of gene expression, but the manner in which clock entrainment is controlled at the level of mRNA translation is not well understood. We found that a light- and circadian clock-regulated MAPK/MNK pathway led to phosphorylation of the cap-binding protein eIF4E in the mouse suprachiasmatic nucleus of the hypothalamus, the locus of the master circadian clock in mammals. Phosphorylation of eIF4E specifically promoted translation of Period 1 (Per1) and Period 2 (Per2) mRNAs and increased the abundance of basal and inducible PER proteins, which facilitated circadian clock resetting and precise timekeeping. Together, these results highlight a critical role for light-regulated translational control in the physiology of the circadian clock.

Circadian mechanisms of food anticipatory rhythms in rats fed once or twice daily: clock gene and endocrine correlates.

Circadian clocks in many brain regions and peripheral tissues are entrained by the daily rhythm of food intake. Clocks in one or more of these locations generate a daily rhythm of locomotor activity that anticipates a regular mealtime. Rats and mice can also anticipate two daily meals. Whether this involves 1 or 2 circadian clocks is unknown. To gain insight into how the circadian system adjusts to 2 daily mealtimes, male rats in a 12∶12 light-dark cycle were fed a 2 h meal either 4 h after lights-on or 4 h after lights-off, or a 1 h meal at both times. After 30 days, brain, blood, adrenal and stomach tissue were collected at 6 time points. Multiple clock genes from adrenals and stomachs were assayed by RT-PCR. Blood was assayed for corticosterone and ghrelin. Bmal1 expression was quantified in 14 brain regions by in situ hybridization. Clock gene rhythms in adrenal and stomach from day-fed rats oscillated in antiphase with the rhythms in night-fed rats, and at an intermediate phase in rats fed twice daily. Corticosterone and ghrelin in 1-meal rats peaked at or prior to the expected mealtime. In 2-meal rats, corticosterone peaked only prior the nighttime meal, while ghrelin peaked prior to the daytime meal and then remained elevated. The olfactory bulb, nucleus accumbens, dorsal striatum, cerebellum and arcuate nucleus exhibited significant daily rhythms of Bmal1 in the night-fed groups that were approximately in antiphase in the day-fed groups, and at intermediate levels (arrhythmic) in rats anticipating 2 daily meals. The dissociations between anticipatory activity and the peripheral clocks and hormones in rats anticipating 2 daily meals argue against a role for these signals in the timing of behavioral rhythms. The absence of rhythmicity at the tissue level in brain regions from rats anticipating 2 daily meals support behavioral evidence that circadian clock cells in these tissues may reorganize into two populations coupled to different meals.