Background Circadian gene disruptions are associated with the development of psychiatric disorders, including addiction. However, the mechanisms by which circadian genes regulate drug reward remain poorly understood. Methods We used mice with a mutation in Npas2, and AAV-shRNA mediated knock-down of Npas2 and Clock in the nucleus accumbens (NAc). We performed conditioned place preference (CPP) assays for cocaine. We utilized cell sorting techniques, qPCR and chromatin immunoprecipitation (ChIP) assays followed by deep sequencing (ChIP-seq). Results Npas2 mutants exhibit decreased sensitivity to cocaine reward which can be recapitulated with a knock-down of NPAS2 specifically in the NAc, demonstrating the functional importance of NPAS2 in this region. Interestingly, reducing CLOCK (a homologue of NPAS2) expression in the NAc had no effect, suggesting an important distinction in NPAS2 and CLOCK function. Furthermore, we find that NPAS2 expression is restricted to Drd1 expressing neurons, (i.e. “direct” pathway circuitry) while CLOCK is ubiquitous. Moreover, NPAS2 and CLOCK have distinct temporal patterns of DNA binding, and we identified novel and unique binding sites for each protein. We identified the Drd3 dopamine receptor as a direct transcriptional target of NPAS2 and find that NPAS2 knock-down in the NAc disrupts its diurnal rhythm in expression. Chronic cocaine treatment likewise disrupts the normal rhythm in Npas2 and Drd3 expression in the NAc, which may underlie behavioral plasticity in response to cocaine. Conclusions Together, these findings identify an important and novel role for the circadian protein, NPAS2, in the NAc in the regulation of dopamine receptor expression and drug reward.

Background Emerging evidence implicates circadian abnormalities as a component of the pathophysiology of major depressive disorder (MDD). The suprachiasmatic nucleus (SCN) of the hypothalamus coordinates rhythms throughout the brain and body. On a cellular level, rhythms are generated by transcriptional, translational, and post-translational feedback loops of core circadian genes and proteins. In patients with MDD, recent evidence suggests reduced amplitude of molecular rhythms in extra-SCN brain regions. We investigated whether unpredictable chronic mild stress (UCMS), an animal model that induces a depression-like physiological and behavioral phenotype, induces circadian disruptions similar to those seen with MDD. Methods Activity and temperature rhythms were recorded in C57BL/6J mice before, during, and after exposure to UCMS, and brain tissue explants were collected from Period2 luciferase (Per2::luc) mice following UCMS to assess cellular rhythmicity. Results UCMS significantly decreased circadian amplitude of activity and body temperature in mice, similar to findings in MDD patients and these changes directly correlate with depression-related behavior. While amplitude of molecular rhythms in the SCN was decreased following UCMS, surprisingly, rhythms in the nucleus accumbens were amplified with no changes seen in the prefrontal cortex or amygdala. These molecular rhythm changes in the SCN and the nucleus accumbens (NAc) also directly correlated with mood-related behavior. Conclusions These studies find that circadian rhythm abnormalities directly correlate with depression-related behavior following UCMS and suggest a desynchronization of rhythms in the brain with an independent enhancement of rhythms in the NAc.

Mice with a mutation in the Clock gene (ClockD19) exhibit increased preference for stimulant rewards and sucrose. They also have an increase in dopaminergic activity in the ventral tegmental area (VTA) and a general increase in glutamatergic tone that might underlie these behaviors. However, it is unclear if their phenotype would extend to a very different class of drug (ethanol), and if so, whether these systems might be involved in their response. Continuous access voluntary ethanol intake was evaluated in ClockD19 mutants and wild-type (WT) mice. We found that ClockD19 mice exhibited significantly increased ethanol intake in a two-bottle choice paradigm. Interestingly, this effect was more robust in female mice. Moreover, chronic ethanol experience resulted in a long-lasting decrease in VTA Clock expression. To determine the importance of VTA Clock expression in ethanol intake, we knocked down Clock expression in the VTA of WT mice via RNA interference. We found that reducing Clock expression in the VTA resulted in significantly increased ethanol intake similar to the ClockD19 mice. Interestingly, we also discovered that ClockD19 mice exhibit significantly augmented responses to the sedative effects of ethanol and ketamine, but not pentobarbital. However, their drinking behavior was not affected by acamprosate, an FDA-approved drug for the treatment of alcoholism, suggesting that their increased glutamatergic tone might underlie the increased sensitivity to the sedative/hypnotic properties of ethanol but not the rewarding properties of ethanol. Taken together, we have identified a significant role for Clock in the VTA as a negative regulator of ethanol intake and implicate the VTA dopamine system in this response.

Circadian rhythms prepare organisms for predictable events during the Earth's 24-h day. These rhythms are entrained by a variety of stimuli. Light is the most ubiquitous and best known zeitgeber, but a number of others have been identified, including food, social cues, locomotor activity, and, most recently drugs of abuse. Given the diversity of zeitgebers, it is probably not surprising that genes capable of clock functions are located throughout almost all organs and tissues. Recent evidence suggests that drugs of abuse can directly entrain some circadian rhythms. We have report here that entrainment by drugs of abuse is independent of the suprachiasmatic nucleus and the light/dark cycle, is not dependent on direct locomotor stimulation, and is shared by a variety of classes of drugs of abuse. We suggest that drug-entrained rhythms reflect variations in underlying neurophysiological states. This could be the basis for known daily variations in drug metabolism, tolerance, and sensitivity to drug reward. These rhythms could also take the form of daily periods of increased motivation to seek and take drugs, and thus contribute to abuse, addiction and relapse.

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Both chronic stress and antidepressant medications have been associated with changes in body weight. In the current study, we investigate mechanisms by which stress and antidepressant medications interact to affect meal patterns. A group of mice was subjected to the chronic social defeat stress model of major depression followed by fluoxetine treatment and was subsequently analyzed for food intake using metabolic cages. Here, we report that chronic social defeat stress increases food intake by specifically increasing meal size, an effect that is reversed by fluoxetine treatment. In an attempt to gain mechanistic insight into changes in meal patterning induced by stress and fluoxetine, fasting serum samples were collected every four hours over a 24-hour period and acyl-ghrelin, leptin, and corticosterone levels were measured. Chronic stress induces a peak in acyl-ghrelin levels just prior to lights off, which is shifted in mice treated with fluoxetine. Taken together, these results indicate that stress increases food intake by decreasing satiation, and that fluoxetine can reverse stress-induced changes in meal patterns.

Kv3.1 and Kv3.2 voltage-gated potassium channels are expressed on parvalbumin-positive GABAergic interneurons in corticolimbic brain regions and contribute to high-frequency neural firing. The channels are also expressed on GABAergic neurons of the basal ganglia, substantia nigra, and ventral tegmental area (VTA) where they regulate firing patterns critical for movement control, reward, and motivation. Modulation of Kv3.1 and Kv3.2 channels may therefore have potential in the treatment of disorders in which these systems have been implicated, such as bipolar disorder. Following the recent development of a potassium channel modulator, AUT1-an imidazolidinedione compound that specifically increases currents mediated by Kv3.1 and Kv3.2 channels in recombinant systems-we report that the compound is able to reverse 'manic-like' behavior in two mouse models: amphetamine-induced hyperactivity and ClockΔ19 mutants. AUT1 completely prevented amphetamine-induced hyperactivity in a dose-dependent manner, similar to the atypical antipsychotic, clozapine. Similar efficacy was observed in Kv3.2 knockout mice. In contrast, AUT1 was unable to prevent amphetamine-induced hyperactivity in mice lacking Kv3.1 channels. Notably, Kv3.1-null mice displayed baseline hyperlocomotion, reduced anxiety-like behavior, and antidepressant-like behavior. In ClockΔ19 mice, AUT1 reversed hyperactivity. Furthermore, AUT1 application modulated firing frequency and action potential properties of ClockΔ19 VTA dopamine neurons potentially through network effects. Kv3.1 protein levels in the VTA of ClockΔ19 and WT mice were unaltered by acute AUT1 treatment. Taken together, these results suggest that the modulation of Kv3.1 channels may provide a novel approach to the treatment of bipolar mania.

A new transplantable ovarian tumor model is presented using a novel folate receptor (FR) positive, murine ovarian cancer cell line that emulates the human disease and induces widespread intraperitoneal (i.p.) tumors in immunocompetent mice within 4–8 weeks of implantation. Tumor development was monitored using a new positron emission tomography (PET) FR-targeting reporter with PET/computerized tomography (PET/CT) and fluorescence molecular tomography (FMT) using a commercial FR-targeting reporter. Conventional structural magnetic resonance imaging (MRI) was also performed. Adult female C57BL/6 mice were injected i.p. with 6 × 106 MKP-L FR+ cells. Imaging was performed weekly beginning 2 weeks after tumor induction. The albumin-binding, FR-targeting ligand cm09 was radiolabeled with the positron emitter 68Ga and used to image the tumors with a small animal PET/CT. The FR-reporter FolateRSense 680 (PerkinElmer) was used for FMT and flow cytometry. Preclinical MRI (7 T) without FR-targeting was compared with the PET and FMT molecular imaging. Tumors were visible by all three imaging modalities. PET/CT had the highest imaging sensitivity at 3–3.5 h postadministration (mean %IA/g mean > 6) and visualized tumors earlier than the other two modalities with lower kidney uptake (mean %IA/g mean < 17) than previously reported FR-targeting agents in late stage disease. FMT showed relatively low FR-targeted agent in the bladder and kidneys, but yielded the lowest anatomical image resolution. MRI produced the highest resolution images, but it was difficult to distinguish tumors from abdominal organs during early progression since a FR-targeting MRI reporter was not used. Nevertheless, there was good correlation of imaging biomarkers between the three modalities. Tumors in the mouse ovarian cancer model could be detected using FR-targeted imaging as early as 2 weeks post i.p. injection of tumor cells. An imaging protocol should combine one or more of the modalities, e.g., PET/CT or PET/MRI for optimal tumor detection and delineation from surrounding tissues.