Molecular Rhythmicity in Glia: Importance for Brain Health and Relevance to Psychiatric Disease.

Circadian rhythms are approximate 24-hour rhythms present in nearly all aspects of human physiology, including proper brain function. These rhythms are produced at the cellular level through a transcriptional-translational feedback loop known as the molecular clock. Diurnal variation in gene expression has been demonstrated in brain tissue from multiple species, including humans, in both cortical and subcortical regions. Interestingly, these rhythms in gene expression have been shown to be disrupted across psychiatric disorders and may be implicated in their underlying pathophysiology. However, little is known regarding molecular rhythms in specific cell types in the brain and how they might be involved in psychiatric disease. Although glial cells (e.g., astrocytes, microglia, and oligodendrocytes) have been historically understudied compared to neurons, evidence of the molecular clock is found within each of these cell subtypes. Here, we review the current literature, which suggests that molecular rhythmicity is essential to functional physiologic outputs from each glial subtype. Furthermore, disrupted molecular rhythms within these cells and the resultant functional deficits may be relevant to specific phenotypes across psychiatric illnesses. Given that circadian rhythm disruptions have been so integrally tied to psychiatric disease, the molecular mechanisms governing these associations could represent exciting new avenues for future research and potential novel pharmacologic targets for treatment.

Cell-type and sex-specific rhythmic gene expression in the nucleus accumbens.

Circadian rhythms are critical for human health and are highly conserved across species. Disruptions in these rhythms contribute to many diseases, including psychiatric disorders. Previous results suggest that circadian genes modulate behavior through specific cell types in the nucleus accumbens (NAc), particularly dopamine D1-expressing medium spiny neurons (MSNs). However, diurnal rhythms in transcript expression have not been investigated in NAc MSNs. In this study we identified and characterized rhythmic transcripts in D1- and D2-expressing neurons and compared rhythmicity results to homogenate as well as astrocyte samples taken from the NAc of male and female mice. We find that all cell types have transcripts with diurnal rhythms and that top rhythmic transcripts are largely core clock genes, which peak at approximately the same time of day in each cell type and sex. While clock-controlled rhythmic transcripts are enriched for protein regulation pathways across cell type, cell signaling and signal transduction related processes are most commonly enriched in MSNs. In contrast to core clock genes, these clock-controlled rhythmic transcripts tend to reach their peak in expression about 2-h later in females than males, suggesting diurnal rhythms in reward may be delayed in females. We also find sex differences in pathway enrichment for rhythmic transcripts peaking at different times of day. Protein folding and immune responses are enriched in transcripts that peak in the dark phase, while metabolic processes are primarily enriched in transcripts that peak in the light phase. Importantly, we also find that several classic markers used to categorize MSNs are rhythmic in the NAc. This is critical since the use of rhythmic markers could lead to over- or under-enrichment of targeted cell types depending on the time at which they are sampled. This study greatly expands our knowledge of how individual cell types contribute to rhythms in the NAc.

Sexually dimorphic mechanisms of VGLUT-mediated protection from dopaminergic neurodegeneration.

Parkinson's disease (PD) targets some dopamine (DA) neurons more than others. Sex differences offer insights, with females more protected from DA neurodegeneration. The mammalian vesicular glutamate transporter VGLUT2 and Drosophila ortholog dVGLUT have been implicated as modulators of DA neuron resilience. However, the mechanisms by which VGLUT2/dVGLUT protects DA neurons remain unknown. We discovered DA neuron dVGLUT knockdown increased mitochondrial reactive oxygen species in a sexually dimorphic manner in response to depolarization or paraquat-induced stress, males being especially affected. DA neuron dVGLUT also reduced ATP biosynthetic burden during depolarization. RNA sequencing of VGLUT+ DA neurons in mice and flies identified candidate genes that we functionally screened to further dissect VGLUT-mediated DA neuron resilience across PD models. We discovered transcription factors modulating dVGLUT-dependent DA neuroprotection and identified dj-1ß as a regulator of sex-specific DA neuron dVGLUT expression. Overall, VGLUT protects DA neurons from PD-associated degeneration by maintaining mitochondrial health.

Acute exposure to artificial light at night alters hippocampal vascular structure in mice.

The structure and function of the cardiovascular system are modulated across the day by circadian rhythms, making this system susceptible to circadian rhythm disruption. Recent evidence demonstrated that short-term exposure to a pervasive circadian rhythm disruptor, artificial light at night (ALAN), increased inflammation and altered angiogenic transcripts in the hippocampi of mice. Here, we examined the effects of four nights of ALAN exposure on mouse hippocampal vascular networks. To do this, we analyzed 2D and 3D images of hippocampal vasculature and hippocampal transcriptomic profiles of mice exposed to ALAN. ALAN reduced vascular density in the CA1 and CA2/3 of female mice and the dentate gyrus of male mice. Network structure and connectivity were also impaired in the CA2/3 of female mice. These results demonstrate the rapid and potent effects of ALAN on cerebrovascular networks, highlighting the importance of ALAN mitigation in the context of health and cerebrovascular disease.

Differential Effects of Cocaine and Morphine on the Diurnal Regulation of the Mouse Nucleus Accumbens Proteome.

Substance use disorders are associated with disruptions in sleep and circadian rhythms that persist during abstinence and may contribute to relapse risk. Repeated use of substances such as psychostimulants and opioids may lead to significant alterations in molecular rhythms in the nucleus accumbens (NAc), a brain region central to reward and motivation. Previous studies have identified rhythm alterations in the transcriptome of the NAc and other brain regions following the administration of psychostimulants or opioids. However, little is known about the impact of substance use on the diurnal rhythms of the proteome in the NAc. We used liquid chromatography coupled to tandem mass spectrometry-based quantitative proteomics, along with a data-independent acquisition analysis pipeline, to investigate the effects of cocaine or morphine administration on diurnal rhythms of proteome in the mouse NAc. Overall, our data reveal cocaine and morphine differentially alter diurnal rhythms of the proteome in the NAc, with largely independent differentially expressed proteins dependent on time-of-day. Pathways enriched from cocaine altered protein rhythms were primarily associated with glucocorticoid signaling and metabolism, whereas morphine was associated with neuroinflammation. Collectively, these findings are the first to characterize the diurnal regulation of the NAc proteome and demonstrate a novel relationship between the phase-dependent regulation of protein expression and the differential effects of cocaine and morphine on the NAc proteome. The proteomics data in this study are available via ProteomeXchange with identifier PXD042043.

Differential Effects of Cocaine and Morphine on the Diurnal Regulation of the Mouse Nucleus Accumbens Proteome.

Substance use disorders (SUDs) are associated with disruptions in sleep and circadian rhythms that persist during abstinence and may contribute to relapse risk. Repeated use of substances such as psychostimulants and opioids may lead to significant alterations in molecular rhythms in the nucleus accumbens (NAc), a brain region central to reward and motivation. Previous studies have identified rhythm alterations in the transcriptome of the NAc and other brain regions following the administration of psychostimulants or opioids. However, little is known about the impact of substance use on the diurnal rhythms of the proteome in the NAc. We used liquid chromatography coupled to tandem mass spectrometry-based (LC-MS/MS) quantitative proteomics, along with a data-independent acquisition (DIA) analysis pipeline, to investigate the effects of cocaine or morphine administration on diurnal rhythms of proteome in the mouse NAc. Overall, our data reveals cocaine and morphine differentially alters diurnal rhythms of the proteome in the NAc, with largely independent differentially expressed proteins dependent on time-of-day. Pathways enriched from cocaine altered protein rhythms were primarily associated with glucocorticoid signaling and metabolism, whereas morphine was associated with neuroinflammation. Collectively, these findings are the first to characterize the diurnal regulation of the NAc proteome and demonstrate a novel relationship between phase-dependent regulation of protein expression and the differential effects of cocaine and morphine on the NAc proteome.

Machine learning and deep learning frameworks for the automated analysis of pain and opioid withdrawal behaviors.

The automation of behavioral tracking and analysis in preclinical research can serve to advance the rate of research outcomes, increase experimental scalability, and challenge the scientific reproducibility crisis. Recent advances in the efficiency, accuracy, and accessibility of deep learning (DL) and machine learning (ML) frameworks are enabling this automation. As the ongoing opioid epidemic continues to worsen alongside increasing rates of chronic pain, there are ever-growing needs to understand opioid use disorders (OUDs) and identify non-opioid therapeutic options for pain. In this review, we examine how these related needs can be advanced by the development and validation of DL and ML resources for automated pain and withdrawal behavioral tracking. We aim to emphasize the utility of these tools for automated behavioral analysis, and we argue that currently developed models should be deployed to address novel questions in the fields of pain and OUD research.

Time of day as a critical variable in biology.

BACKGROUND: Circadian rhythms are important for all aspects of biology; virtually every aspect of biological function varies according to time of day. Although this is well known, variation across the day is also often ignored in the design and reporting of research. For this review, we analyzed the top 50 cited papers across 10 major domains of the biological sciences in the calendar year 2015. We repeated this analysis for the year 2019, hypothesizing that the awarding of a Nobel Prize in 2017 for achievements in the field of circadian biology would highlight the importance of circadian rhythms for scientists across many disciplines, and improve time-of-day reporting. RESULTS: Our analyses of these 1000 empirical papers, however, revealed that most failed to include sufficient temporal details when describing experimental methods and that few systematic differences in time-of-day reporting existed between 2015 and 2019. Overall, only 6.1% of reports included time-of-day information about experimental measures and manipulations sufficient to permit replication. CONCLUSIONS: Circadian rhythms are a defining feature of biological systems, and knowing when in the circadian day these systems are evaluated is fundamentally important information. Failing to account for time of day hampers reproducibility across laboratories, complicates interpretation of results, and reduces the value of data based predominantly on nocturnal animals when extrapolating to diurnal humans.

The Ventral Tegmental Area and Nucleus Accumbens as Circadian Oscillators: Implications for Drug Abuse and Substance Use Disorders.

Circadian rhythms convergently evolved to allow for optimal synchronization of individuals' physiological and behavioral processes with the Earth's 24-h periodic cycling of environmental light and temperature. Whereas the suprachiasmatic nucleus (SCN) is considered the primary pacemaker of the mammalian circadian system, many extra-SCN oscillatory brain regions have been identified to not only exhibit sustainable rhythms in circadian molecular clock function, but also rhythms in overall region activity/function and mediated behaviors. In this review, we present the most recent evidence for the ventral tegmental area (VTA) and nucleus accumbens (NAc) to serve as extra-SCN oscillators and highlight studies that illustrate the functional significance of the VTA's and NAc's inherent circadian properties as they relate to reward-processing, drug abuse, and vulnerability to develop substance use disorders (SUDs).

Astrocyte Molecular Clock Function in the Nucleus Accumbens Is Important for Reward-Related Behavior.

BACKGROUND: Substance use disorders are associated with disruptions in circadian rhythms. Both human and animal work have shown the integral role for circadian clocks in the modulation of reward behaviors. Astrocytes have emerged as key regulators of circadian rhythmicity. However, no studies to date have identified the role of circadian astrocyte function in the nucleus accumbens (NAc), a hub for reward regulation, or determined the importance of these rhythms for reward-related behavior. METHODS: Using astrocyte-specific RNA sequencing across time of day, we first characterized diurnal variation of the NAc astrocyte transcriptome. We then investigated the functional significance of this circadian regulation through viral-mediated disruption of molecular clock function in NAc astrocytes, followed by assessment of reward-related behaviors, metabolic-related molecular assays, and whole-cell electrophysiology in the NAc. RESULTS: Strikingly, approximately 43% of the astrocyte transcriptome has a diurnal rhythm, and key metabolic pathways were enriched among the top rhythmic genes. Moreover, mice with a viral-mediated loss of molecular clock function in NAc astrocytes show a significant increase in locomotor response to novelty, exploratory drive, operant food self-administration, and motivation. At the molecular level, these animals also show disrupted metabolic gene expression, along with significant downregulation of both lactate and glutathione levels in the NAc. Loss of NAc astrocyte clock function also significantly altered glutamatergic signaling onto neighboring medium spiny neurons, alongside upregulated glutamate-related gene expression. CONCLUSIONS: Taken together, these findings demonstrate a novel role for astrocyte circadian molecular clock function in the regulation of the NAc and reward-related behaviors.

Circadian transcription factor NPAS2 and the NAD+ -dependent deacetylase SIRT1 interact in the mouse nucleus accumbens and regulate reward.

Substance use disorders are associated with disruptions to both circadian rhythms and cellular metabolic state. At the molecular level, the circadian molecular clock and cellular metabolic state may be interconnected through interactions with the nicotinamide adenine dinucleotide (NAD+ )-dependent deacetylase, sirtuin 1 (SIRT1). In the nucleus accumbens (NAc), a region important for reward, both SIRT1 and the circadian transcription factor neuronal PAS domain protein 2 (NPAS2) are highly enriched, and both are regulated by the metabolic cofactor NAD+ . Substances of abuse, like cocaine, greatly disrupt cellular metabolism and promote oxidative stress; however, their effects on NAD+ in the brain remain unclear. Interestingly, cocaine also induces NAc expression of both NPAS2 and SIRT1, and both have independently been shown to regulate cocaine reward in mice. However, whether NPAS2 and SIRT1 interact in the NAc and/or whether together they regulate reward is unknown. Here, we demonstrate diurnal expression of Npas2, Sirt1 and NAD+ in the NAc, which is altered by cocaine-induced upregulation. Additionally, co-immunoprecipitation reveals NPAS2 and SIRT1 interact in the NAc, and cross-analysis of NPAS2 and SIRT1 chromatin immunoprecipitation sequencing reveals several reward-relevant and metabolic-related pathways enriched among shared gene targets. Notably, NAc-specific Npas2 knock-down or a functional Npas2 mutation in mice attenuates SIRT1-mediated increases in cocaine preference. Together, our data reveal an interaction between NPAS2 and SIRT1 in the NAc, which may serve to integrate cocaine's effects on circadian and metabolic factors, leading to regulation of drug reward.

Light at night disrupts biological clocks, calendars, and immune function.

Light at night is a pervasive problem in our society; over 80% of the world's population experiences significant light pollution. Exacerbating this issue is the reality that artificially lit outdoor areas are growing by 2.2% per year and continuously lit areas brighten by 2.2% each year due to the rapid growths in population and urbanization. Furthermore, the increase in the prevalence of night shift work and smart device usage contributes to the inescapable nature of artificial light at night (ALAN). Although previously assumed to be innocuous, ALAN has deleterious effects on the circadian system and circadian-regulated physiology, particularly immune function. Due to the relevance of ALAN to the general population, it is important to understand its roles in disrupting immune function. This review presents a synopsis of the effects of ALAN on circadian clocks and immune function. We delineate the role of ALAN in altering clock gene expression and suppressing melatonin. We review the effects of light at night on inflammation and the innate and adaptive immune systems in various species to demonstrate the wide range of ALAN consequences. Finally, we propose future directions to provide further clarity and expansion of the field.

The Suprachiasmatic Nucleus Regulates Anxiety-Like Behavior in Mice.

Individuals suffering from mood and anxiety disorders often show significant disturbances in sleep and circadian rhythms. Animal studies indicate that circadian rhythm disruption can cause increased depressive- and anxiety-like behavior, but the underlying mechanisms are unclear. One potential mechanism to explain how circadian rhythms are contributing to mood and anxiety disorders is through dysregulation of the suprachiasmatic nucleus (SCN) of the hypothalamus, known as the "central pacemaker." To investigate the role of the SCN in regulating depressive- and anxiety-like behavior in mice, we chronically manipulated the neural activity of the SCN using two optogenetic stimulation paradigms. As expected, chronic stimulation of the SCN late in the active phase (circadian time 21, CT21) resulted in a shortened period and dampened amplitude of homecage activity rhythms. We also repeatedly stimulated the SCN at unpredictable times during the active phase of mice when SCN firing rates are normally low. This resulted in dampened, fragmented, and unstable homecage activity rhythms. In both chronic SCN optogenetic stimulation paradigms, dampened homecage activity rhythms (decreased amplitude) were directly correlated with increased measures of anxiety-like behavior. In contrast, we only observed a correlation between behavioral despair and homecage activity amplitude in mice stimulated at CT21. Surprisingly, the change in period of homecage activity rhythms was not directly associated with anxiety- or depressive-like behavior. Finally, to determine if anxiety-like behavior is affected during a single SCN stimulation session, we acutely stimulated the SCN in the active phase (zeitgeber time 14-16, ZT14-16) during behavioral testing. Unexpectedly this also resulted in increased anxiety-like behavior. Taken together, these results indicate that SCN-mediated dampening of rhythms is directly correlated with increased anxiety-like behavior. This work is an important step in understanding how specific SCN neural activity disruptions affect depressive- and anxiety-related behavior.

Circadian-Dependent and Sex-Dependent Increases in Intravenous Cocaine Self-Administration in Npas2 Mutant Mice.

Substance use disorder (SUD) is associated with disruptions in circadian rhythms. The circadian transcription factor neuronal PAS domain protein 2 (NPAS2) is enriched in reward-related brain regions and regulates reward, but its role in SU is unclear. To examine the role of NPAS2 in drug taking, we measured intravenous cocaine self-administration (acquisition, dose-response, progressive ratio, extinction, cue-induced reinstatement) in wild-type (WT) and Npas2 mutant mice at different times of day. In the light (inactive) phase, cocaine self-administration, reinforcement, motivation and extinction responding were increased in all Npas2 mutants. Sex differences emerged during the dark (active) phase with Npas2 mutation increasing self-administration, extinction responding, and reinstatement only in females as well as reinforcement and motivation in males and females. To determine whether circulating hormones are driving these sex differences, we ovariectomized WT and Npas2 mutant females and confirmed that unlike sham controls, ovariectomized mutant mice showed no increase in self-administration. To identify whether striatal brain regions are activated in Npas2 mutant females, we measured cocaine-induced ΔFosB expression. Relative to WT, ΔFosB expression was increased in D1+ neurons in the nucleus accumbens (NAc) core and dorsolateral (DLS) striatum in Npas2 mutant females after dark phase self-administration. We also identified potential target genes that may underlie the behavioral responses to cocaine in Npas2 mutant females. These results suggest NPAS2 regulates reward and activity in specific striatal regions in a sex and time of day (TOD)-specific manner. Striatal activation could be augmented by circulating sex hormones, leading to an increased effect of Npas2 mutation in females.SIGNIFICANCE STATEMENT Circadian disruptions are a common symptom of substance use disorders (SUDs) and chronic exposure to drugs of abuse alters circadian rhythms, which may contribute to subsequent SU. Diurnal rhythms are commonly found in behavioral responses to drugs of abuse with drug sensitivity and motivation peaking during the dark (active) phase in nocturnal rodents. Emerging evidence links disrupted circadian genes to SU vulnerability and drug-induced alterations to these genes may augment drug-seeking. The circadian transcription factor neuronal PAS domain protein 2 (NPAS2) is enriched in reward-related brain regions and regulates reward, but its role in SU is unclear. To examine the role of NPAS2 in drug taking, we measured intravenous cocaine self-administration in wild-type (WT) and Npas2 mutant mice at different times of day.

Valproate reverses mania-like behaviors in mice via preferential targeting of HDAC2.

Valproate (VPA) has been used in the treatment of bipolar disorder since the 1990s. However, the therapeutic targets of VPA have remained elusive. Here we employ a preclinical model to identify the therapeutic targets of VPA. We find compounds that inhibit histone deacetylase proteins (HDACs) are effective in normalizing manic-like behavior, and that class I HDACs (e.g., HDAC1 and HDAC2) are most important in this response. Using an RNAi approach, we find that HDAC2, but not HDAC1, inhibition in the ventral tegmental area (VTA) is sufficient to normalize behavior. Furthermore, HDAC2 overexpression in the VTA prevents the actions of VPA. We used RNA sequencing in both mice and human induced pluripotent stem cells (iPSCs) derived from bipolar patients to further identify important molecular targets. Together, these studies identify HDAC2 and downstream targets for the development of novel therapeutics for bipolar mania.

Mood-related central and peripheral clocks.

Mood disorders, including major depression, bipolar disorder, and seasonal affective disorder, are debilitating disorders that affect a significant portion of the global population. Individuals suffering from mood disorders often show significant disturbances in circadian rhythms and sleep. Moreover, environmental disruptions to circadian rhythms can precipitate or exacerbate mood symptoms in vulnerable individuals. Circadian clocks exist throughout the central nervous system and periphery, where they regulate a wide variety of physiological processes implicated in mood regulation. These processes include monoaminergic and glutamatergic transmission, hypothalamic-pituitary-adrenal axis function, metabolism, and immune function. While there seems to be a clear link between circadian rhythm disruption and mood regulation, the mechanisms that underlie this association remain unclear. This review will touch on the interactions between the circadian system and each of these processes and discuss their potential role in the development of mood disorders. While clinical studies are presented, much of the review will focus on studies in animal models, which are attempting to elucidate the molecular and cellular mechanisms in which circadian genes regulate mood.

Cell-Type-Specific Regulation of Nucleus Accumbens Synaptic Plasticity and Cocaine Reward Sensitivity by the Circadian Protein, NPAS2.

The circadian transcription factor neuronal PAS domain 2 (NPAS2) is linked to psychiatric disorders associated with altered reward sensitivity. The expression of Npas2 is preferentially enriched in the mammalian forebrain, including the nucleus accumbens (NAc), a major neural substrate of motivated and reward behavior. Previously, we demonstrated that downregulation of NPAS2 in the NAc reduces the conditioned behavioral response to cocaine in mice. We also showed that Npas2 is preferentially enriched in dopamine receptor 1 containing medium spiny neurons (D1R-MSNs) of the striatum. To extend these studies, we investigated the impact of NPAS2 disruption on accumbal excitatory synaptic transmission and strength, along with the behavioral sensitivity to cocaine reward in a cell-type-specific manner. Viral-mediated knockdown of Npas2 in the NAc of male and female C57BL/6J mice increased the excitatory drive onto MSNs. Using Drd1a-tdTomato mice in combination with viral knockdown, we determined these synaptic adaptations were specific to D1R-MSNs relative to non-D1R-MSNs. Interestingly, NAc-specific knockdown of Npas2 blocked cocaine-induced enhancement of synaptic strength and glutamatergic transmission specifically onto D1R-MSNs. Last, we designed, validated, and used a novel Cre-inducible short-hairpin RNA virus for MSN-subtype-specific knockdown of Npas2 Cell-type-specific Npas2 knockdown in D1R-MSNs, but not D2R-MSNs, in the NAc reduced cocaine conditioned place preference. Together, our results demonstrate that NPAS2 regulates excitatory synapses of D1R-MSNs in the NAc and cocaine reward-related behavior.SIGNIFICANCE STATEMENT Drug addiction is a widespread public health concern often comorbid with other psychiatric disorders. Disruptions of the circadian clock can predispose or exacerbate substance abuse in vulnerable individuals. We demonstrate a role for the core circadian protein, NPAS2, in mediating glutamatergic neurotransmission at medium spiny neurons (MSNs) in the nucleus accumbens (NAc), a region critical for reward processing. We find that NPAS2 negatively regulates functional excitatory synaptic plasticity in the NAc and is necessary for cocaine-induced plastic changes in MSNs expressing the dopamine 1 receptor (D1R). We further demonstrate disruption of NPAS2 in D1R-MSNs produces augmented cocaine preference. These findings highlight the significance of cell-type-specificity in mechanisms underlying reward regulation by NPAS2 and extend our knowledge of its function.

NAD+ cellular redox and SIRT1 regulate the diurnal rhythms of tyrosine hydroxylase and conditioned cocaine reward.

The diurnal regulation of dopamine is important for normal physiology and diseases such as addiction. Here we find a novel role for the CLOCK protein to antagonize CREB-mediated transcriptional activity at the tyrosine hydroxylase (TH) promoter, which is mediated by the interaction with the metabolic sensing protein, Sirtuin 1 (SIRT1). Additionally, we demonstrate that the transcriptional activity of TH is modulated by the cellular redox state, and daily rhythms of redox balance in the ventral tegmental area (VTA), along with TH transcription, are highly disrupted following chronic cocaine administration. Furthermore, CLOCK and SIRT1 are important for regulating cocaine reward and dopaminergic (DAergic) activity, with interesting differences depending on whether DAergic activity is in a heightened state and if there is a functional CLOCK protein. Taken together, we find that rhythms in cellular metabolism and circadian proteins work together to regulate dopamine synthesis and the reward value for drugs of abuse.

Antimanic Efficacy of a Novel Kv3 Potassium Channel Modulator.

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.