Reversal of Stress-Induced Social Interaction Deficits by Buprenorphine.

Background: Patients with post-traumatic stress disorder frequently report persistent problems with social interactions, emerging after a traumatic experience. Chronic social defeat stress is a widely used rodent model of stress that produces robust and sustained social avoidance behavior. The avoidance of other rodents can be reversed by 28 days of treatment with selective serotonin reuptake inhibitors, the only pharmaceutical class approved by the U.S. Food and Drug Administration for treating post-traumatic stress disorder. In this study, the sensitivity of social interaction deficits evoked by 10 days of chronic social defeat stress to prospective treatments for post-traumatic stress disorder was examined. Methods: The effects of acute and repeated treatment with a low dose of buprenorphine (0.25 mg/kg/d) on social interaction deficits in male C57BL/6 mice by chronic social defeat stress were studied. Another cohort of mice was used to determine the effects of the selective serotonin reuptake inhibitor fluoxetine (10 mg/kg/d), the NMDA antagonist ketamine (10 mg/kg/d), and the selective kappa opioid receptor antagonist CERC-501 (1 mg/kg/d). Changes in mRNA expression of Oprm1 and Oprk1 were assessed in a separate cohort. Results: Buprenorphine significantly reversed social interaction deficits produced by chronic social defeat stress following 7 days of administration, but not after acute injection. Treatment with fluoxetine for 7 days, but not 24 hours, also reinstated social interaction behavior in mice that were susceptible to chronic social defeat. In contrast, CERC-501 and ketamine failed to reverse social avoidance. Gene expression analysis found: (1) Oprm1 mRNA expression was reduced in the hippocampus and increased in the frontal cortex of susceptible mice and (2) Oprk1 mRNA expression was reduced in the amygdala and increased in the frontal cortex of susceptible mice compared to non-stressed controls and stress-resilient mice. Conclusions: Short-term treatment with buprenorphine and fluoxetine normalized social interaction after chronic social defeat stress. In concert with the changes in opioid receptor expression produced by chronic social defeat stress, we speculate that buprenorphine's efficacy in this model of post-traumatic stress disorder may be associated with the ability of this compound to engage multiple opioid receptors.

NPAS2 Regulation of Anxiety-Like Behavior and GABAA Receptors.

Abnormal circadian rhythms and circadian genes are strongly associated with several psychiatric disorders. Neuronal PAS Domain Protein 2 (NPAS2) is a core component of the molecular clock that acts as a transcription factor and is highly expressed in reward- and stress-related brain regions such as the striatum. However, the mechanism by which NPAS2 is involved in mood-related behaviors is still unclear. We measured anxiety-like behaviors in mice with a global null mutation in Npas2 (Npas2 null mutant mice) and found that Npas2 null mutant mice exhibit less anxiety-like behavior than their wild-type (WT) littermates (in elevated plus maze, light/dark box and open field assay). We assessed the effects of acute or chronic stress on striatal Npas2 expression, and found that both stressors increased levels of Npas2. Moreover, knockdown of Npas2 in the ventral striatum resulted in a similar reduction of anxiety-like behaviors as seen in the Npas2 null mutant mouse. Additionally, we identified Gabra genes as transcriptional targets of NPAS2, found that Npas2 null mutant mice exhibit reduced sensitivity to the GABAa positive allosteric modulator, diazepam and that knockdown of Npas2 reduced Gabra1 expression and response to diazepam in the ventral striatum. These results: (1) implicate Npas2 in the response to stress and the development of anxiety; and (2) provide functional evidence for the regulation of GABAergic neurotransmission by NPAS2 in the ventral striatum.

Functional Implications of the CLOCK 3111T/C Single-Nucleotide Polymorphism.

Circadian rhythm disruptions are prominently associated with bipolar disorder (BD). Circadian rhythms are regulated by the molecular clock, a family of proteins that function together in a transcriptional-translational feedback loop. The CLOCK protein is a key transcription factor of this feedback loop, and previous studies have found that manipulations of the Clock gene are sufficient to produce manic-like behavior in mice (1). The CLOCK 3111T/C single-nucleotide polymorphism (SNP; rs1801260) is a genetic variation of the human CLOCK gene that is significantly associated with increased frequency of manic episodes in BD patients (2). The 3111T/C SNP is located in the 3'-untranslated region of the CLOCK gene. In this study, we sought to examine the functional implications of the human CLOCK 3111T/C SNP by transfecting a mammalian cell line (mouse embryonic fibroblasts isolated from Clock(-/-) knockout mice) with pcDNA plasmids containing the human CLOCK gene with either the T or C SNP at position 3111. We then measured circadian gene expression over a 24-h time period. We found that the CLOCK3111C SNP resulted in higher mRNA levels than the CLOCK 3111T SNP. Furthermore, we found that Per2, a transcriptional target of CLOCK, was also more highly expressed with CLOCK 3111C expression, indicating that the 3'-UTR SNP affects the expression, function, and stability of CLOCK mRNA.

Antidepressant-like Effects of Buprenorphine are Mediated by Kappa Opioid Receptors.

Previous studies have identified potential antidepressant effects of buprenorphine (BPN), a drug with high affinity for mu opioid receptor (MORs) and kappa opioid receptors (KORs) and some affinity at delta opioid receptor (DOR) and opioid receptor-like 1 (ORL-1) receptors. Therefore, these studies examined which opioid receptors were involved in BPN's effects on animal behavior tests sensitive to antidepressant drugs. The acute effects of BPN were tested in the forced swim test (FST) using mice with genetic deletion of individual opioid receptors or after pharmacological blockade of receptors. For evaluating the effects of BPN on chronic stress, separate groups of mice were exposed to unpredictable chronic mild stress (UCMS) for 3 weeks and treated with BPN for at least 7 days before behavioral assessment and subsequent measurement of Oprk1, Oprm1, and Pdyn mRNA expression in multiple brain regions. BPN did not reduce immobility in mice with KOR deletion or after pretreatment with norbinaltorphimine, even though desipramine remained effective. In contrast, BPN reduced immobility in MOR and DOR knockout mice and in mice pretreated with the ORL-1 antagonist JTC-801. UCMS reduced sucrose preference, decreased time in the light side of the light/dark box, increased immobility in the FST and induced region-specific alterations in Oprk1, Oprm1, and PDYN mRNA expression in the frontal cortex and striatum. All of these changes were normalized following BPN treatment. The KOR was identified as a key player mediating the effects of BPN in tests sensitive to antidepressant drugs in mice. These studies support further development of BPN as a novel antidepressant.

Direct regulation of diurnal Drd3 expression and cocaine reward by NPAS2.

BACKGROUND: Circadian gene disruptions are associated with the development of psychiatric disorders, including addiction. However, the mechanisms by which circadian genes regulate reward remain poorly understood. METHODS: We used mice with a mutation in Npas2 and adeno-associated virus-short hairpin RNA mediated knockdown of Npas2 and Clock in the nucleus accumbens (NAc). We performed conditioned place preference assays. We utilized cell sorting quantitative real-time polymerase chain reaction, and chromatin immunoprecipitation followed by deep sequencing. RESULTS: Npas2 mutants exhibit decreased sensitivity to cocaine reward, which is recapitulated with a knockdown of neuronal PAS domain protein 2 (NPAS2) specifically in the NAc, demonstrating the importance of NPAS2 in this region. Interestingly, reducing circadian locomotor output cycles kaput (CLOCK) (a homologue of NPAS2) in the NAc had no effect, suggesting an important distinction in NPAS2 and CLOCK function. Furthermore, we found that NPAS2 expression is restricted to Drd1 expressing neurons 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 found that NPAS2 knockdown 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 role for the circadian protein, NPAS2, in the NAc in the regulation of dopamine receptor expression and drug reward.

Effects of buprenorphine on behavioral tests for antidepressant and anxiolytic drugs in mice.

RATIONALE: Buprenorphine (BPN) has been shown to rapidly improve mood in treatment-resistant depressed patients in small clinical studies. However, BPN's effects in preclinical tests for mood and antidepressant efficacy are largely unexplored. OBJECTIVE: The current study examined the effects of BPN in the forced swim test (FST) and novelty-induced hypophagia (NIH) test as measures of antidepressant and anxiolytic-like effects in C57BL/6 J mice. Microdialysis was used to measure whether BPN engaged kappa-opioid receptor (KORs) in the nucleus accumbens shell (NAcSh) at a behaviorally active dose (0.25 mg/kg). METHODS: BPN was tested in the FST at both 30 min and 24 h post-administration. Also measured in the FST at 24 h post-administration were the KOR antagonist norbinaltorphimine (nor-BNI), the MOR agonist morphine and the reference antidepressant desipramine. The anxiolytic effects of BPN were examined in the NIH test 24 h after treatment. The effects of acute injection of BPN and the KOR agonist U50,488 were measured on extracellular dopamine (DA) levels in the NAcSh. RESULTS: BPN produced significant reductions in FST immobility without changing locomotor activity and reduced approach latencies in the novel environment of the NIH test when tested 24 h after treatment. Repeated daily BPN injections for 6 days did not produce tolerance to these behavioral effects. nor-BNI produced a similar antidepressant-like response in the FST 24 h post-injection but morphine and desipramine were ineffective. BPN (0.25 mg/kg) did not alter DA levels when given alone but prevented the KOR agonist U50,488 from reducing DA levels. CONCLUSIONS: Acute and subchronic treatment with BPN produced antidepressant and anxiolytic-like responses in mice at doses that engage KORs. These studies support the clinical evidence that BPN may be a novel rapid-acting antidepressant medication and provides rodent models for investigating associated neurochemical mechanisms.

The role of clock in ethanol-related behaviors.

Mice with a mutation in the Clock gene (ClockΔ19) 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 ClockΔ19 mutants and wild-type (WT) mice. We found that ClockΔ19 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 ClockΔ19 mice. Interestingly, we also discovered that ClockΔ19 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.

Differential regulation of the period genes in striatal regions following cocaine exposure.

Several studies have suggested that disruptions in circadian rhythms contribute to the pathophysiology of multiple psychiatric diseases, including drug addiction. In fact, a number of the genes involved in the regulation of circadian rhythms are also involved in modulating the reward value for drugs of abuse, like cocaine. Thus, we wanted to determine the effects of chronic cocaine on the expression of several circadian genes in the Nucleus Accumbens (NAc) and Caudate Putamen (CP), regions of the brain known to be involved in the behavioral responses to drugs of abuse. Moreover, we wanted to explore the mechanism by which these genes are regulated following cocaine exposure. Here we find that after repeated cocaine exposure, expression of the Period (Per) genes and Neuronal PAS Domain Protein 2 (Npas2) are elevated, in a somewhat regionally selective fashion. Moreover, NPAS2 (but not CLOCK (Circadian Locomotor Output Cycles Kaput)) protein binding at Per gene promoters was enhanced following cocaine treatment. Mice lacking a functional Npas2 gene failed to exhibit any induction of Per gene expression after cocaine, suggesting that NPAS2 is necessary for this cocaine-induced regulation. Examination of Per gene and Npas2 expression over twenty-four hours identified changes in diurnal rhythmicity of these genes following chronic cocaine, which were regionally specific. Taken together, these studies point to selective disruptions in Per gene rhythmicity in striatial regions following chronic cocaine treatment, which are mediated primarily by NPAS2.

Circadian genes Period 1 and Period 2 in the nucleus accumbens regulate anxiety-related behavior.

It has been suggested for some time that circadian rhythm abnormalities underlie the development of multiple psychiatric disorders. However, it is unclear how disruptions in individual circadian genes might regulate mood and anxiety. Here we found that mice lacking functional mPeriod 1 (mPer1) or mPeriod 2 (mPer2) individually did not have consistent behavioral abnormalities in measures of anxiety-related behavior. However, mice deficient in both mPer1 and mPer2 had an increase in levels of anxiety-like behavior in multiple measures. Moreover, we found that mPer1 and mPer2 expression was reduced in the nucleus accumbens (NAc) after exposure to chronic social defeat stress, a paradigm that led to increased anxiety-related behavior. Following social defeat, chronic treatment with fluoxetine normalized Per gene expression towards wild-type levels. Knockdown of both mPer1 and mPer2 expression via RNA interference specifically in the NAc led to a similar increase in anxiety-like behavior as seen in the mutant animals. Taken together, these results implicate the Per genes in the NAc in response to stress and the development of anxiety.

A mutation in CLOCK leads to altered dopamine receptor function.

Mice with a mutation in the Clock gene (ClockΔ19) have a number of behavioral phenotypes that suggest alterations in dopaminergic transmission. These include hyperactivity, increased exploratory behavior, and increased reward value for drugs of abuse. However, the complex changes in dopaminergic transmission that underlie the behavioral abnormalities in these mice remain unclear. Here we find that a loss of CLOCK function increases dopamine release and turnover in striatum as indicated by increased levels of metabolites HVA and DOPAC, and enhances sensitivity to dopamine receptor antagonists. Interestingly, this enlarged dopaminergic tone results in downstream changes in dopamine receptor (DR) levels with a surprising augmentation of both D1- and D2-type DR protein, but a significant shift in the ratio of D1 : D2 receptors in favor of D2 receptor signaling. These effects have functional consequences for both behavior and intracellular signaling, with alterations in locomotor responses to both D1-type and D2-type specific agonists and a blunted response to cAMP activation in the ClockΔ19 mutants. Taken together, these studies further elucidate the abnormalities in dopaminergic transmission that underlie mood, activity, and addictive behaviors.

Specific role of VTA dopamine neuronal firing rates and morphology in the reversal of anxiety-related, but not depression-related behavior in the ClockΔ19 mouse model of mania.

Lithium has been used extensively for mood stabilization, and it is particularly efficacious in the treatment of bipolar mania. Like other drugs used in the treatment of psychiatric diseases, it has little effect on the mood of healthy individuals. Our previous studies found that mice with a mutation in the Clock gene (ClockΔ19) have a complete behavioral profile that is very similar to human mania, which can be reversed with chronic lithium treatment. However, the cellular and physiological effects that underlie its targeted therapeutic efficacy remain unknown. Here we find that ClockΔ19 mice have an increase in dopaminergic activity in the ventral tegmental area (VTA), and that lithium treatment selectively reduces the firing rate in the mutant mice with no effect on activity in wild-type mice. Furthermore, lithium treatment reduces nucleus accumbens (NAc) dopamine levels selectively in the mutant mice. The increased dopaminergic activity in the Clock mutants is associated with cell volume changes in dopamine neurons, which are also rescued by lithium treatment. To determine the role of dopaminergic activity and morphological changes in dopamine neurons in manic-like behavior, we manipulated the excitability of these neurons by overexpressing an inwardly rectifying potassium channel subunit (Kir2.1) selectively in the VTA of ClockΔ19 mice and wild-type mice using viral-mediated gene transfer. Introduction of this channel mimics the effects of lithium treatment on the firing rate of dopamine neurons in ClockΔ19 mice and leads to a similar change in dopamine cell volume. Furthermore, reduction of dopaminergic firing rates in ClockΔ19 animals results in a normalization of locomotor- and anxiety-related behavior that is very similar to lithium treatment; however, it is not sufficient to reverse depression-related behavior. These results suggest that abnormalities in dopamine cell firing and associated morphology underlie alterations in anxiety-related behavior in bipolar mania, and that the therapeutic effects of lithium come from a reversal of these abnormal phenotypes.

Intra-accumbens shell injections of SR48692 enhanced cocaine self-administration intake in rats exposed to an environmentally-elicited reinstatement paradigm.

Neurotensin (NT) is a neuropeptide involved in cocaine reward, and in learning and memory processes related to drug use within the mesolimbic dopamine (DA) system. Studies have demonstrated that NT receptor antagonists have potential as pharmacotherapeutical tools for cocaine abuse. Therefore, it is important to understand the molecular profile of NT within mesolimbic neurons and the behavioral effects of NT receptor inhibitors on environmentally-elicited cocaine seeking behavior. To address this issue, male Sprague Dawley rats were trained to self-administer cocaine and to discriminate between environmental cues signaling cocaine vs. saline availability. Then, following extinction, these cues were used to induce reinstatement of cocaine seeking behavior. A differential expression profile was observed throughout the experiment. Particularly, a significant increase of NT levels was observed within the nucleus accumbens (NAc) shell subregion during the acquisition phase of training. To further examine the implications of this increase, separate groups of animals received intra NAc shell injections of one of three doses (25, 50, 100 nM) of the NT1 receptor antagonist SR48692 after reaching stable self-administration. Animals were injected prior to placement in the operant conditioning chambers for four consecutive sessions. An increase in lever pressing was observed following antagonist treatment, whereas no major changes in locomotor activity were observed. We propose that the observed increase in lever pressing may be a compensatory response to a decrease in reinforcement, possibly due to decreased DA release, as previous studies show that chronic SR48692 decreases basal DA release in the NAc shell.

A role for the circadian genes in drug addiction.

Diurnal and circadian rhythms are prominent in nearly all bodily functions. Chronic disruptions in normal sleep wake and social schedules can lead to serious health problems such as those seen in shift worker's syndrome. Moreover, genetic disruptions in normal circadian gene functions have recently been linked to a variety of psychiatric conditions including depression, bipolar disorder, seasonal affective disorder and alcoholism. Recent studies are beginning to determine how these circadian genes and rhythms are involved in the development of drug addiction. Several of these studies suggest an important role for these genes in limbic regions of the brain, outside of the central circadian pacemaker in the suprachiasmatic nucleus (SCN). This review summarizes some of the basic research into the importance of circadian genes in drug addiction.

Role of GluR1 expression in nucleus accumbens neurons in cocaine sensitization and cocaine-seeking behavior.

Chronic cocaine use reduces glutamate levels in the nucleus accumbens (NAc), and is associated with experience-dependent changes in (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) glutamate receptor membrane expression in NAc neurons. These changes accompany behavioral sensitization to cocaine and increased susceptibility to cocaine relapse. The functional relationship between neuroplasticity in AMPA receptors and the behavioral manifestation of cocaine addiction remains unclear. Thus, we examined the behavioral effects of up- and downregulating basal AMPA receptor function in the NAc core and shell using viral-mediated gene transfer of wild-type glutamate receptor 1 (wt-GluR1) or a dominant-negative pore-dead GluR1 (pd-GluR1), respectively. Transient increases in wt-GluR1 during or after cocaine treatments diminished the development of cocaine sensitization, while pd-GluR1 expression exacerbated cocaine sensitization. Parallel changes were found in D2, but not D1, receptor-mediated behavioral responses. As a correlate of the sensitization experiments, we overexpressed wt- or pd-GluR1 in the NAc core during cocaine self-administration, and tested the effects on subsequent drug-seeking behavior 3 weeks after overexpression declined. wt-GluR1 overexpression during self-administration had no effect on cocaine intake, but subsequently reduced cocaine seeking in extinction and cocaine-induced reinstatement, whereas pd-GluR1 facilitated cocaine-induced reinstatement. When overexpressed during reinstatement tests, wt-GluR1 directly attenuated cocaine- and D2 agonist-induced reinstatement, while pd-GluR1 enhanced reinstatement. In both experimental procedures, neither wt- nor pd-GluR1 expression affected cue-induced reinstatement. Together, these results suggest that degrading basal AMPA receptor function in NAc neurons is sufficient to facilitate relapse via sensitization in D2 receptor responses, whereas elevating basal AMPA receptor function attenuates these behaviors.