Amphetamine regulates NR2B expression in Go2α knockout mice and thereby sustains behavioral sensitization.

The α-subunit of Go2 is a regulator of dopamine (DA) homeostasis. Deletion of the protein results in an imbalance of the direct and indirect DA pathway by reducing D1 and increasing D2 receptors. As a result, cocaine-induced behavioral sensitization is abolished. Here we show that repeated amphetamine injections in Go2α-/- mice induced a similar D1/D2 receptor ratio shift as cocaine but surprisingly the knockouts developed normal behavioral sensitization. DA receptor signaling following either cocaine or amphetamine treatment was also similar in Go2α-/- mice suggesting another mechanism involved in the differential behavioral response. Evidence is increasing that DA-glutamate interactions in the striatum determine psychostimulant action. In this line, repeated amphetamine injections led to a twofold increase in the amount of the NMDA receptor subunit NR2B in Go2α-/- mice resulting in an enhanced inhibition of the indirect DA pathway. This effect is not seen after cocaine treatment. Furthermore, amphetamine but not cocaine treatment maintained the ratio between the glutamate receptor mGluR1/5 interacting proteins Homer and Homer1a in the knockouts thereby sustaining the direct pathway. We conclude that amphetamine provokes behavioral sensitization in Go2α-/- mice by an enhanced inhibition of the indirect pathway without disturbing the direct pathway thereby overcoming the imbalance in the DArgic system.

Development of morphine-induced tolerance and withdrawal: involvement of the clock gene mPer2.

The present study has been designed to assess specifically the involvement of the clock gene mPer2 in morphine-induced tolerance and withdrawal. At first, we checked the absence of initial differences in the expression of several gene transcripts involved in the development of morphine dependence in Per2(Brdm1) mutant mice and in their respective wild-type (WT) control littermates. Morphine-induced tolerance as well as precipitated withdrawal was then assessed in these mice. The Per2(Brdm1) mutant mice clearly developed less tolerance and showed attenuated withdrawal signs compared to WT. These results show that mPER2 is involved in morphine-induced tolerance and withdrawal.

Ambiguous-cue interpretation is biased under stress- and depression-like states in rats.

Negative cognitive bias-the tendency to interpret ambiguous situations pessimistically-is a central feature of stress-related disorders such as depression. The underlying neurobiology of this bias, however, remains unclear, not least because of a lack of translational tools. We established a new ambiguous-cue interpretation paradigm and, with respect to the etiology of depression, evaluated if environmental and genetic factors contribute to a negative bias. Rats were trained to press a lever to receive a food reward contingent to one tone and to press another lever in response to a different tone to avoid punishment by electric foot-shock. In the ambiguous-cue test, the lever-press responses to tones with frequencies intermediate to the trained tones were taken as indicators for the rats' expectation of a positive or negative event. A negative response bias because of decreased positive and increased negative responding was found in congenitally helpless rats, a genetic animal model of depression. Moreover, treatment with a combined noradrenergic-glucocorticoid challenge, mimicking stress-related changes in endogenous neuromodulation, biased rats away from positive responding. This response shift was accompanied by neuronal activation in dentate gyrus and amygdala. Thus, environmental and genetic risk factors for depression induce a response bias, which resembles the pessimistic bias of patients suffering from depression. The behavioral paradigm described constitutes a useful tool to study the neuronal basis of decision making under ambiguous conditions and may promote innovative pharmaco- and psychotherapy for depression.

Modulation of chromatin modification facilitates extinction of cocaine-induced conditioned place preference.

BACKGROUND: Recent evidence suggests that epigenetic mechanisms have an important role in the development of addictive behavior. However, little is known about the role of epigenetic mechanisms in the extinction of drug-induced behavioral changes. In this study, we examined the ability of histone deacetylase (HDAC) inhibitors to facilitate extinction and attenuate reinstatement of cocaine-induced conditioned place preference (CPP). METHODS: C57BL/6 mice were subject to cocaine-induced CPP using 20 mg/kg dose. To facilitate extinction, mice were administered an HDAC inhibitor following nonreinforced exposure to the conditioned context. To measure persistence, mice were subject to a reinstatement test using 10 mg/kg dose of cocaine. RESULTS: We demonstrate that HDAC inhibition during extinction consolidation can facilitate extinction of cocaine-induced CPP. Animals treated with an HDAC inhibitor extinguished cocaine-induced CPP both more quickly and to a greater extent than did vehicle-treated animals. We also show that the extinction of cocaine seeking via HDAC inhibition modulates extinction learning such that reinstatement behavior is significantly attenuated. Acetylation of histone H3 in the nucleus accumbens following extinction was increased by HDAC inhibition. CONCLUSIONS: This study provides the first evidence that modulation of chromatin modification can facilitate extinction and prevent reinstatement of drug-induced behavioral changes. These findings provide a potential novel approach to the development of treatments that facilitate extinction of drug-seeking behavior.

Inhibition of cAMP responsive element binding protein in striatal neurons enhances approach and avoidance responses toward morphine--and morphine withdrawal-related cues.

To investigate the role of cAMP responsive element binding protein (CREB)-dependent gene expression in morphine induced behaviors, we examined bitransgenic mice expressing a dominant and strong inhibitor of the CREB family of transcription factors, A-CREB, in striatal neurons in a regulatable manner. The expression of A-CREB in the striatum enhanced both morphine-induced conditioned place preference and morphine withdrawal-induced conditioned place avoidance. Our experiments thereby support a role for CREB in striatal neurons regulating approach and avoidance responses toward drug-related cues.

Epigenetic mechanisms underlying extinction of memory and drug-seeking behavior.

An increasing body of evidence shows that structural modifications of chromatin, the DNA-protein complex that packages genomic DNA, do not only participate in maintaining cellular memory (e.g., cell fate), but they may also underlie the strengthening and maintenance of synaptic connections required for long-term changes in behavior. Accordingly, epigenetics has become a central topic in several neurobiology fields such as memory, drug addiction, and several psychiatric and mental disorders. This interest is justified as dynamic chromatin modifications may provide not only transient but also stable (or even potentially permanent) epigenetic marks to facilitate, maintain, or block transcriptional processes, which in turn may participate in the molecular neural adaptations underlying behavioral changes. Through epigenetic mechanisms the genome may be indexed in response to environmental signals, resulting in specific neural modifications that largely determine the future behavior of an organism. In this review we discuss recent advances in our understanding of how epigenetic mechanisms contribute to the formation of long-term memory and drug-seeking behavior and potentially how to apply that knowledge to the extinction of memory and drug-seeking behavior.

Selective boosting of transcriptional and behavioral responses to drugs of abuse by histone deacetylase inhibition.

Histone acetylation and other modifications of the chromatin are important regulators of gene expression and, consequently, may contribute to drug-induced behaviors and neuroplasticity. Earlier studies have shown that a reduction in histone deacetylase (HDAC) activity results in the enhancement of some psychostimulant-induced behaviors. In this study, we extend those seminal findings by showing that the administration of the HDAC inhibitor sodium butyrate enhances morphine-induced locomotor sensitization and conditioned place preference. In contrast, this compound has no effects on the development of morphine tolerance and dependence. Similar effects were observed for cocaine and ethanol-induced behaviors. These behavioral changes were accompanied by a selective boosting of a component of the transcriptional program activated by chronic morphine administration that included circadian clock genes and other genes relevant to addictive behavior. Our results support a specific function for histone acetylation and the epigenetic modulation of transcription at a reduced number of biologically relevant loci on non-homeostatic, long-lasting, drug-induced behavioral plasticity.

Automated scoring of fear-related behavior using EthoVision software.

Fear conditioning is a frequently used paradigm for assessing learning and memory in rodents. Traditionally researchers have relied upon scoring of fear-related behavior by human observation, which can be difficult and subjective and thus vary among investigators. The goal of this study was to evaluate the ability of EthoVision tracking software (Noldus Information Technology Inc.) to reliably and accurately score fear-related behavior in mice. Specifically, we were interested in its ability to accurately track mice and score immobility as a fear-related behavior during contextual and cued fear conditioning. Contextual and cued fear conditioning were performed in modified PhenoTyper chambers (Noldus Information Technology Inc.) fitted with grid floors to deliver a scrambled foot shock. Our results demonstrate that we have identified parameters in EthoVision that can accurately track mice and be used for automated scoring of immobility that is nearly identical to scoring by human observation. Together, EthoVision software and the modified PhenoTyper chambers provide an excellent system for the reliable and accurate measurement of fear-related behavior in a high-throughput manner.

Loss of the Ca2+/calmodulin-dependent protein kinase type IV in dopaminoceptive neurons enhances behavioral effects of cocaine.

The persistent nature of addiction has been associated with activity-induced plasticity of neurons within the striatum and nucleus accumbens (NAc). To identify the molecular processes leading to these adaptations, we performed Cre/loxP-mediated genetic ablations of two key regulators of gene expression in response to activity, the Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) and its postulated main target, the cAMP-responsive element binding protein (CREB). We found that acute cocaine-induced gene expression in the striatum was largely unaffected by the loss of CaMKIV. On the behavioral level, mice lacking CaMKIV in dopaminoceptive neurons displayed increased sensitivity to cocaine as evidenced by augmented expression of locomotor sensitization and enhanced conditioned place preference and reinstatement after extinction. However, the loss of CREB in the forebrain had no effect on either of these behaviors, even though it robustly blunted acute cocaine-induced transcription. To test the relevance of these observations for addiction in humans, we performed an association study of CAMK4 and CREB promoter polymorphisms with cocaine addiction in a large sample of addicts. We found that a single nucleotide polymorphism in the CAMK4 promoter was significantly associated with cocaine addiction, whereas variations in the CREB promoter regions did not correlate with drug abuse. These findings reveal a critical role for CaMKIV in the development and persistence of cocaine-induced behaviors, through mechanisms dissociated from acute effects on gene expression and CREB-dependent transcription.

Glutamate receptors on dopamine neurons control the persistence of cocaine seeking.

Cocaine strengthens excitatory synapses onto midbrain dopamine neurons through the synaptic delivery of GluR1-containing AMPA receptors. This cocaine-evoked plasticity depends on NMDA receptor activation, but its behavioral significance in the context of addiction remains elusive. Here, we generated mice lacking the GluR1, GluR2, or NR1 receptor subunits selectively in dopamine neurons. We report that in midbrain slices of cocaine-treated mice, synaptic transmission was no longer strengthened when GluR1 or NR1 was abolished, while in the respective mice the drug still induced normal conditioned place preference and locomotor sensitization. In contrast, extinction of drug-seeking behavior was absent in mice lacking GluR1, while in the NR1 mutant mice reinstatement was abolished. In conclusion, cocaine-evoked synaptic plasticity does not mediate concurrent short-term behavioral effects of the drug but may initiate adaptive changes eventually leading to the persistence of drug-seeking behavior.

Deletion of Go2alpha abolishes cocaine-induced behavioral sensitization by disturbing the striatal dopamine system.

The alpha-subunits of the trimeric Go class of GTPases, comprising the splice variants Go1alpha and Go2alpha, are abundantly expressed in brain and reside on both plasma membrane and synaptic vesicles. Go2alpha is involved in the vesicular storage of monoamines but its physiological relevance is still obscure. We now show that genetic depletion of Go2alpha reduces motor activity induced by dopamine-enhancing drugs like cocaine, as repeated injections of cocaine fail to provoke behavioral sensitization in Go2alpha(-/-) mice. In Go2alpha(-/-) mice, D1 receptor signaling in the striatum is attenuated due to a reduced expression of Golf alpha and Gs alpha. Following cocaine treatment, Go2alpha(-/-) mice have lower D1 and higher D2 receptor amounts compared to wild-type mice. The lack of behavioral sensitization correlates with reduced dopamine levels in the striatum and decreased expression of tyrosine hydroxylase. One reason for the neurochemical changes may be a reduced uptake of monoamines by synaptic vesicles from Go2alpha(-/-) mice as a consequence of a lowered set point for filling. We conclude that Go2alpha optimizes vesicular filling which is instrumental for normal dopamine functioning and for the development of drug-induced behavioral sensitization.

A phenotype-driven ENU mutagenesis screen for the identification of dominant mutations involved in alcohol consumption.

The aim of this study was the application of a phenotype-driven N-ethyl-N-nitrosourea (ENU) mutagenesis screen in mice for the identification of dominant mutations involved in the regulation and modulation of alcohol-drinking behavior. The chemical mutagen ENU was utilized in the generation of 131 male ENU-mutant C57BL/6J mice (G0). These ENU-treated mice were paired with wild-type C57BL/6J mice to generate G1 and subsequent generations. In total, 3327 mice were generated. Starting with G1, mice were screened for voluntary oral self-administration of 10% (v/v) alcohol vs. water in a two-bottle paradigm. From these mice, after a total period of 5 weeks of drinking, 43 mutants fulfilled the criteria of an "alcohol phenotype," that is, high or low ethanol intake. They were then selected for breeding and tested in a "confirmation cross" (G2-G4) for inheritance. Although we did not establish stable high or low drinking lines, several results were obtained in the context of alcohol consumption. First, female mice drank more alcohol than their male counterparts. Second, the former demonstrated greater infertility. Third, all animals displayed relatively stable alcohol intake, although significantly different in two different laboratories. Finally, seasonal and monthly variability was observed, with the highest alcohol consumption occurring in spring and the lowest in autumn. In conclusion, it seems difficult to identify dominant mutations involved in the modulation or regulation of voluntary alcohol consumption via a phenotype-driven ENU mutagenesis screen. In accordance with the findings from knockout studies, we suggest that mainly recessive mutations contribute to an alcohol-drinking or alcohol-avoiding phenotype.

Etomidate and propofol-hyposensitive GABAA receptor beta3(N265M) mice show little changes in acute alcohol sensitivity but enhanced tolerance and withdrawal.

Gamma-aminobutyric acid-A (GABAA) receptors are ligand-gated ion channels comprised of subunits from several classes (alpha, beta, gamma, delta). Recent studies have clearly demonstrated that the functional properties of GABAA receptors are altered following chronic ethanol administration that could provide the molecular basis for the previously proposed role of these receptors in ethanol tolerance and dependence. Because the subunit composition of GABAA receptors determines receptor pharmacology, the present study was devoted to assess if the behavioral sensitivity after acute and chronic ethanol exposure depends on beta3-containing GABAA receptors. In the present study, we used knock-in mice harboring a point mutation (N265M) in the second transmembrane region of the beta3 subunit of the GABAA receptor in order to study acute and chronic behavioral effects of ethanol. More specifically, we tested tolerance to loss of righting reflex (LORR) and the development of withdrawal signs after chronic ethanol exposure using ethanol vapor chambers. Our results show that the beta3(N265M) mutation does not play a major modulatory role of acute ethanol-induced LORR. However, following repeated LORR testing, enhanced tolerance to the intoxicating effects of ethanol was observed--a finding which was unrelated to the pharmacokinetics of ethanol as both genotypes had the same blood alcohol concentrations following repeated LORR testing. In addition, following chronic alcohol vapor exposure, mouse mutants displayed increased handling-induced convulsions during withdrawal. The results of the present study suggest that the alcohol effects abolished by the beta3(N265M) mutation do not play a dominant role in acute alcohol intoxication but influence ethanol tolerance and withdrawal.

Ethanol self-administration and reinstatement of ethanol-seeking behavior in Per1(Brdm1) mutant mice.

RATIONALE: Alcohol consumption shows circadian rhythmicity, i.e., alcohol preference and intake change with circadian time. Circadian rhythmicity is controlled by a biological clock, which has been shown to govern behavioral, physiological, and hormonal processes in synchronization with internal as well as external cues. Molecular components of the clock include circadian clock genes such as period (Per) 1, 2, and 3. Previously, our lab demonstrated the involvement of mouse Per1 (mPer1) and Per2 (mPer2) in modulating cocaine sensitization and reward. What is more, we investigated voluntary alcohol consumption in Per2 ( Brdm1 ) mice with the results suggesting a relationship between this circadian clock gene and ethanol consumption. Objective To further complement the mPer2 study, our lab proceeded to assess mPer1's possible role on alcohol intake using operant and free choice two bottle paradigms. METHODS: Using operant conditions, Per1 ( Brdm1 ) and wild type mice were trained to self-administer ethanol (10%) under a fixed ratio 1 (FR1) paradigm. This was ensued by a progressive ratio (PR) schedule. Furthermore, extinction sessions were introduced, followed by reinstatement measures of ethanol-seeking behavior. In another set of animals, the mice were exposed to voluntary long-term alcohol consumption, ensued by a 2-month deprivation phase, after which the alcohol deprivation effect (ADE) was measured. RESULTS: Mutant mice did not display a significantly divergent number of reinforced lever presses (FR1 and PR) than wild type animals. Furthermore, no significant differences between groups were obtained regarding reinstatement of ethanol-seeking behavior. Similar results were obtained in the two bottle free choice paradigm. Specifically, no genotype differences concerning consumption and preference were observed over a broad range of different ethanol concentrations. Moreover, after the deprivation phase, both groups exhibited significant ADEs, yet no genotype differences. CONCLUSIONS: Contrary to the mPer2 data, the present findings do not suggest a relationship between the circadian clock gene mPer1 and ethanol reinforcement, seeking, and relapse behavior.

IL-6 knockout mice exhibit resistance to stress-induced development of depression-like behaviors.

Cytokine-dependent mechanisms in the CNS have been implicated in the pathogenesis of depression. Interleukin-6 is upregulated in depressed patients and dowregulated by antidepressants. It is, however, unknown whether IL-6 is involved in the pathogenesis of depression. We subjected IL-6-deficient mice (IL-6(-/-)) to depression-related tests (learned helplessness, forced swimming, tail suspension, sucrose preference). We also investigated IL-6 in the hippocampus of stressed wild-type mice. IL-6(-/-) mice showed reduced despair in the forced swim, and tail suspension test, and enhanced hedonic behavior. Moreover, IL-6(-/-) mice exhibited resistance to helplessness. This resistance may be caused by the lack of IL-6, because stress increased IL-6 expression in wild-type hippocampi. This suggests that IL-6 is a component in molecular mechanisms in the pathogenesis of depression. IL-6(-/-) mice represent tools to study IL-6-dependent signaling pathways in the pathophysiology of depression in vivo. Moreover, these mice may support the screening of compounds for depression by altering cytokine-mediated signaling.

Behavioural assessment of drug reinforcement and addictive features in rodents: an overview.

Some psychoactive drugs are abused because of their ability to act as reinforcers. As a consequence behavioural patterns (such as drug-seeking/drug-taking behaviours) are promoted that ensure further drug consumption. After prolonged drug self-administration, some individuals lose control over their behaviour so that these drug-seeking/taking behaviours become compulsive, pervading almost all life activities and precipitating the loss of social compatibility. Thus, the syndrome of addictive behaviour is qualitatively different from controlled drug consumption. Drug-induced reinforcement can be assessed directly in laboratory animals by either operant or non-operant self-administration methods, by classical conditioning-based paradigms such as conditioned place preference or sign tracking, by facilitation of intracranial electric self-stimulation, or, alternatively by drug-induced memory enhancement. In contrast, addiction cannot be modelled in animals, at least as a whole, within the constraints of the laboratory. However, various procedures have been proposed as possible rodent analogues of addiction's major elements including compulsive drug seeking, relapse, loss of control/impulsivity, and continued drug consumption despite negative consequences. This review provides an extensive overview and a critical evaluation of the methods currently used for studying drug-induced reinforcement as well as specific features of addictive behaviour. In addition, comic strips that illustrate behavioural methods used in the drug abuse field are provided given for free download under http://www.zi-mannheim/psychopharmacology.de.

Involvement of the AMPA receptor GluR-C subunit in alcohol-seeking behavior and relapse.

Craving and relapse are core symptoms of drug addiction and alcoholism. It is suggested that, after chronic drug consumption, long-lasting neuroplastic changes within the glutamatergic system are important determinants of addictive behavior. Here, we show that the AMPA type glutamate receptor plays a crucial role in alcohol craving and relapse. We observed, in two animal models of alcohol craving and relapse, that the AMPA antagonist GYKI 52466 [1-(4-aminophenyl)-4-methyl-7, 8-methylenedioxy-5H-2, 3-benzodiazepine] dose-dependently reduced cue-induced reinstatement of alcohol-seeking behavior and the alcohol deprivation effect. The involvement of the AMPA receptor in these phenomena was further studied using mice deficient for the GluR-C AMPA subunit [GluR-C knock-out (KO)]. GluR-C KOs displayed a blunted, cue-induced reinstatement response and alcohol deprivation effect, when compared with wild-type controls; however, no differences between genotypes could be observed regarding ethanol self-administration under operant or home cage drinking conditions. These results imply a role for GluR-C in alcohol relapse, although this phenotype could also be attributable to a reduction in the total number of AMPA receptors in specific brain areas. In conclusion, AMPA receptors seem to be involved in the neuroplastic changes underlying alcohol seeking behavior and relapse. Thus, AMPA receptors represent a novel therapeutic target in preventing relapse.

Role of the endogenous opioid system on the neuropsychopharmacological effects of ethanol: new insights about an old question.

This article presents the proceedings of the symposium "Endogenous Opioids and Voluntary Ethanol Consumption: What Have We Learnt From Knock-out Mice?" presented at the meeting of the International Society for Biomedical Research on Alcoholism held in Heidelberg/Mannheim, Germany, in September/October 2004. The organizers and chairpersons were Michael S. Cowen and Carles Sanchis-Segura. The presentations were as follows: (1) Regulation of the Opioid System by Alcohol: Comparison of Alcohol-Preferring and -Nonpreferring Strains by Michael S. Cowen; (2) Endogenous Opioids and Alcohol: Lessons From Microdialysis and Knock-out Mice by M. Foster Olive; (3) From Neurochemistry to Neuroanatomy: The Hypothalamic Arcuate Nucleus as a Main Site for Ethanol-Opioids Interaction by Carles Sanchis-Segura; (4) Sensitivity to Ethanol Is Modulated by beta-Endorphin in Transgenic Mice by Judy E. Grisel, Amanda J. Roberts, and George F. Koob; and () The mu-Opioid Receptor Modulates Acute Ethanol Sensitivity and Ethanol Withdrawal Severity by Sandra Ghozland.

Social and structural housing conditions influence the development of a depressive-like phenotype in the learned helplessness paradigm in male mice.

Structural and social factors are known to play a crucial role in the pathogenesis of depression. Since animal models of depression are a major tool to gain insights into the mechanisms involved in the pathophysiology of this disease it is important not only to exploit but also to be aware of factors that may affect these models. As housing represents a fundamental external factor, which is controversially debated to affect the animals' emotionality, this study aimed to investigate the impact of different social and structural housing conditions on the development of a depressive-like syndrome in the learned helplessness paradigm. Group housing in an impoverished environment led to an increased vulnerability in the learned helplessness paradigm. Groups that were housed enriched, however, were less helpless. Furthermore impoverished conditions did not increase the vulnerability in single housed animals. Regarding emotionality in the animals, basal anxiety was reduced and the exploration was enhanced by group housing and enriched environment. These results suggest that housing conditions significantly influence the outcome of learned helplessness studies.

Effect of selective antagonism of mu(1)-, mu(1/2)-, mu(3)-, and delta-opioid receptors on the locomotor-stimulating actions of ethanol.

Previous studies have demonstrated that administration of nonspecific opioid antagonists such as naltrexone or naloxone reduces ethanol-induced locomotor activity in mice. However, because of their broad pharmacological profile, it remains unclear through which opioid receptor this antagonism is achieved. Therefore, the present study was aimed at further investigating the role of the different opioid receptors in ethanol-induced (2.5 g/kg) locomotion in mice. First, we compared the effect of naltrexone (0-2 mg/kg) on ethanol-induced locomotion with that of the selective delta-opioid receptor antagonist, naltrindole (0-10 mg/kg). Results of this first set of data revealed that naltrexone completely blocked this effect of ethanol at doses suggested to occupy only mu-opioid receptors, and naltrindole did not modify ethanol-induced locomotion. In a second set of experiments, we further investigated the involvement of mu-opioid receptors in ethanol-stimulated motor activity by assessing the implication of mu(1)-, mu(1/2)-, and mu(3)-opioid receptor subtypes. Results revealed that mu(1/2)-, and to a lesser extent mu(3)-, but not mu(1)-opioid receptor subtypes are involved in the psychomotor actions of ethanol. Data are discussed together with previous results which have emphasized the critical dependence of ethanol-induced motor behaviors on opioid receptors, as well as, of the integrity of beta-endorphin synthesizing neurons from the hypothalamic Arcuate Nucleus.