Alanna C. Easton

RASGRF2 regulates alcohol-induced reinforcement by influencing mesolimbic dopamine neuron activity and dopamine release

The firing of mesolimbic dopamine neurons is important for drug-induced reinforcement, although underlying genetic factors remain poorly understood. In a recent genome-wide association metaanalysis of alcohol intake, we identified a suggestive association of SNP rs26907 in the ras-specific guanine-nucleotide releasing factor 2 (RASGRF2) gene, encoding a protein that mediates Ca2+-dependent activation of the ERK pathway. We performed functional characterization of this gene in relation to alcohol-related phenotypes and mesolimbic dopamine function in both mice and adolescent humans. Ethanol intake and preference were decreased in Rasgrf2−/− mice relative to WT controls. Accordingly, ethanol-induced dopamine release in the ventral striatum was blunted in Rasgrf2−/− mice. Recording of dopamine neurons in the ventral tegmental area revealed reduced excitability in the absence of Ras-GRF2, likely because of lack of inhibition of the IA potassium current by ERK. This deficit provided an explanation for the altered dopamine release, presumably linked to impaired activation of dopamine neurons firing. Functional neuroimaging analysis of a monetary incentive–delay task in 663 adolescent boys revealed significant association of ventral striatal activity during reward anticipation with a RASGRF2 haplotype containing rs26907, the SNP associated with alcohol intake in our previous metaanalysis. This finding suggests a link between the RASGRF2 haplotype and reward sensitivity, a known risk factor for alcohol and drug addiction. Indeed, follow-up of these same boys at age 16 y revealed an association between this haplotype and number of drinking episodes. Together, these combined animal and human data indicate a role for RASGRF2 in the regulation of mesolimbic dopamine neuron activity, reward response, and alcohol use and abuse.

aCaMKII Autophosphorylation Controls the Establishment of Alcohol Drinking Behavior.

The α-Ca2+/calmodulin-dependent protein kinase II (αCaMKII) is a crucial enzyme controlling plasticity in the brain. The autophosphorylation of αCaMKII works as a ‘molecular memory’ for a transient calcium activation, thereby accelerating learning. We investigated the role of αCaMKII autophosphorylation in the establishment of alcohol drinking as an addiction-related behavior in mice. We found that alcohol drinking was initially diminished in αCaMKII autophosphorylation-deficient αCaMKIIT286A mice, but could be established at wild-type level after repeated withdrawals. The locomotor activating effects of a low-dose alcohol (2 g/kg) were absent in αCaMKIIT286A mice, whereas the sedating effects of high-dose (3.5 g/kg) were preserved after acute and subchronic administration. The in vivo microdialysis revealed that αCaMKIIT286A mice showed no dopamine (DA) response in the nucleus accumbens to acute or subchronic alcohol administration, but enhanced serotonin (5-HT) responses in the prefrontal cortex. The attenuated DA response in αCaMKIIT286A mice was in line with altered c-Fos activation in the ventral tegmental area after acute and subchronic alcohol administration. In order to compare findings in mice with the human condition, we tested 23 single-nucleotide polymorphisms (SNPs) in the CAMK2A gene for their association with alcohol dependence in a population of 1333 male patients with severe alcohol dependence and 939 controls. We found seven significant associations between CAMK2A SNPs and alcohol dependence, one of which in an autophosphorylation-related area of the gene. Together, our data suggest αCaMKII autophosphorylation as a facilitating mechanism in the establishment of alcohol drinking behavior with changing the DA–5-HT balance as a putative mechanism.

αCaMKII autophosphorylation controls the establishment of alcohol-induced conditioned place preference in mice

The autophosphorylation of alpha Ca2+ /calmodulin dependent protein kinase II (αCaMKII) is important for memory formation and is becoming increasingly implicated in the development of drug addiction. Previous work suggests that αCaMKII acts via the monoaminergic systems to facilitate the establishment of alcohol drinking behaviour. The present study aims to investigate whether αCaMKII autophosphorylation deficient αCaMKII(T286A) mice show a difference in the rewarding properties of alcohol (2 g/kg, i.p.), as measured by conditioned place preference (CPP). We found that alcohol-induced CPP could be established at an accelerated rate in αCaMKII(T286A) compared to wild type (WT) mice. Hyperactivity/hyper-arousal induced by the test environment was normalised by alcohol in the αCaMKII(T286A), but not WT mice. This effect could be conditioned to the test environment and may suggest enhanced negative reinforcing action of alcohol in αCaMKII autophosphorylation deficient mice.

αCaMKII controls the establishment of cocaine's reinforcing effects in mice and humans

Although addiction develops in a considerable number of regular cocaine users, molecular risk factors for cocaine dependence are still unknown. It was proposed that establishing drug use and memory formation might share molecular and anatomical pathways. Alpha-Ca2+/calmodulin-dependent protein kinase-II (αCaMKII) is a key mediator of learning and memory also involved in drug-related plasticity. The autophosphorylation of αCaMKII was shown to accelerate learning. Thus, we investigated the role of αCaMKII autophosphorylation in the time course of establishing cocaine use-related behavior in mice. We found that αCaMKII autophosphorylation-deficient αCaMKIIT286A mice show delayed establishment of conditioned place preference, but no changes in acute behavioral activation, sensitization or conditioned hyperlocomotion to cocaine (20 mg kg−1, intraperitoneal). In vivo microdialysis revealed that αCaMKIIT286A mice have blunted dopamine (DA) and blocked serotonin (5-HT) responses in the nucleus accumbens (NAcc) and prefrontal cortex after acute cocaine administration (20 mg kg−1, intraperitoneal), whereas noradrenaline responses were preserved. Under cocaine, the attenuated DA and 5-HT activation in αCaMKIIT286A mice was followed by impaired c-Fos activation in the NAcc. To translate the rodent findings to human conditions, several CAMK2A gene polymorphisms were tested regarding their risk for a fast establishment of cocaine dependence in two independent samples of regular cocaine users from Brazil (n=688) and Switzerland (n=141). A meta-analysis across both samples confirmed that CAMK2A rs3776823 TT-allele carriers display a faster transition to severe cocaine use than C-allele carriers. Together, these data suggest that αCaMKII controls the speed for the establishment of cocaine’s reinforcing effects.

CAMK2A polymorphisms predict working memory performance in humans

Working memory is impaired in a number of psychiatric conditions.1, 2 An important role of alpha Ca2+/calmodulin dependent protein kinase II in learning and memory has been shown in rodent models. However, these findings have not been confirmed in humans yet. Here, we report genetic associations between the human CAMK2A gene and the performance in a spatial and a non-spatial working memory (non-SWM) task in two independent human samples.

αCaMKII autophosphorylation mediates neuronal activation in the hippocampal dentate gyrus after alcohol and cocaine in mice

Psychoactive drug-induced cellular activation is a key mechanism to promote neuronal plasticity and addiction. Alpha Ca(2+)/calmodulin-dependent protein kinase II (αCaMKII) and its autophosphorylation play a key role in the development of drug use associated behaviours. It has been suggested that αCaMKII autophosphorylation is necessary for drug-induced neuronal activation in the mesolimbic system. Here, we show an alcohol- and cocaine-induced increase in c-fos expression in the hippocampal dentate gyrus, which is absent in αCaMKII(T286A) autophosphorylation deficient mice. These findings may suggest a role in hippocampal αCaMKII autophosphorylation in the acute neuroplastic effects of alcohol and cocaine.

Lentiviral‐mediated gene delivery reveals distinct roles of nucleus accumbens dopamine D2 and D3 receptors in novelty‐ and light‐induced locomotor activity

The importance of the dopaminergic system for proper brain activity is demonstrated by findings that alterations in this system lead to severe disabilities, including motor impairment observed in various neurological and psychiatric disorders. Although the roles of specific dopamine receptors in behaviour have been extensively investigated using pharmacological agents and knockout mice, non‐specificity of ligands and compensatory molecular adaptations in mutated animals restrict the interpretation of the results. To overcome these limitations and further explore the role of the dopamine D2 and D3 receptors (D2R and D3R) in rats, we used lentivirus‐mediated gene knockdown and overexpression to specifically manipulate expression levels of these genes in the rat nucleus accumbens (NAcc), a brain area important for spontaneous and induced locomotor responses. Lentiviruses, inducing expression of rat D2R or D3R, or efficient knockdown of either receptor by small hairpin (sh)RNAs, were stereotaxically injected into the NAcc. While knockdown of either receptor significantly reduced spontaneous locomotor activity in a novel but not in a habituated environment, D2R and D3R appeared to contribute in opposite ways to light‐induced locomotor activity. D2R knockdown increased while D3R knockdown decreased locomotor activity in this test. Altogether, our findings suggest that D2R and D3R, expressed in the NAcc, have both shared and non‐overlapping roles in transduction of alerting signals elicited by potentially important sensory and environmental cues.

Rasgrf2 controls noradrenergic involvement in the acute and subchronic effects of alcohol in the brain.

RationaleAlcohol addiction is a major psychiatric disease, and yet, the underlying molecular adaptations in the brain remain unclear. Recent evidence suggests a functional role for the ras-specific guanine-nucleotide releasing factor 2 (Rasgrf2) in alcoholism. Rasgrf2−/− mice consume less alcohol and show entirely absent dopamine responses to an alcohol challenge compared to wild types (WT).ObjectiveIn order to further investigate how Rasgrf2 modifies the acute and subchronic effects of alcohol in the brain, we investigated its effects on the noradrenergic and serotonergic systems.MethodsWe measured noradrenaline and serotonin activity in the brain by in vivo microdialysis and RNA expression by chip analysis and RT-PCR after acute and sub-chronic alcohol exposure in Rasgrf2−/− and WT mice.ResultsIn vivo microdialysis showed a significantly reduced noradrenergic response and an absent serotonergic response in the nucleus accumbens (NAcc) and caudate putamen (CPu) after an alcohol challenge in Rasgrf2−/− mice. A co-expression analysis showed that there is a high correlation between Rasgrf2 and α2 adrenoceptor RNA expression in the ventral striatum in naïve animals. Accordingly, we further assessed the role of Rasgrf2 in the response of the noradrenergic system to subchronic alcohol exposure. A decrease in β1 adrenoceptor gene expression was seen in Rasgrf2+/+, but not Rasgrf2−/− mice following alcohol exposure. Conversely, alcohol resulted in a decrease in both β2 and α2 adrenoceptor gene expression in knockout but not WT Rasgrf2 mice.ConclusionsThese findings suggest that adaptations in the noradrenergic system contribute to the Rasgrf2 enhanced risk of alcoholism.

Rasgrf2 controls dopaminergic adaptations to alcohol in mice.

Alcohol abuse leads to serious health problems with no effective treatment available. Recent evidence suggests a role for ras-specific guanine-nucleotide releasing factor 2 (RASGRF2) in alcoholism. Rasgrf2 is a calcium sensor and MAPK/ERK activating protein, which has been linked to neurotransmitter release and monoaminergic receptor adaptations. Rasgrf2 knock out (KO) mice do not develop a dopamine response in the nucleus accumbens after an alcohol challenge and show a reduced consumption of alcohol. The present study aims to further characterise the role of Rasgrf2 in dopaminergic activation beyond the nucleus accumbens following alcohol treatment. Using in vivo microdialysis we found that alcohol induces alterations in dopamine levels in the dorsal striatum between wildtype (WT) and Rasgrf2 KO mice. There was no difference in the expression of dopamine transporter (DAT), dopamine receptor regulating factor (DRRF), or dopamine D2 receptor (DRD2) mRNA in the brain between Rasgrf2 KO and WT mice. After sub-chronic alcohol treatment, DAT and DRRF, but not DRD2 mRNA expression differed between WT and Rasgrf2 KO mice. Brain adaptations were positively correlated with splenic expression levels. These data suggest that Rasgrf2 controls dopaminergic signalling and adaptations to alcohol also in other brain regions, beyond the nucleus accumbens.

Corrigendum: ACaMKII autophosphorylation controls the establishment of alcohol drinking behavior (Neuropsychopharmacology (2013) 38 (2735))

Correction to: Neuropsychopharmacology (2013) 38, 1636–1647; doi:10.1038/npp.2013.60 In this article, the following collaborators of The GESGA Consortium are being added to the byline: Jens Treutlein, Sven Cichon, Monika Ridinger, Manuel Mattheisen, Stefan Herms, Norbert Wodarz, Peter Zill, Wolfgang Maier, Rainald Mössner, Wolfgang Gaebel, Norbert Dahmen, Norbert Scherbaum, Christine Schmäl, Michael Steffens, Susanne Lucae, Marcus Ising, Bertram Müller-Myhsok, Markus M Nöthen, Karl Mann, and Marcella Rietschel Department of Genetic Epidemiology in Psychiatry, Central Institute of Mental Health, Medical Faculty Mannheim/Heidelberg University, Heidelberg, Germany; Department of Genomics, Life and Brain Center, University of Bonn, Bonn, Germany; Institute of Human Genetics, University of Bonn, Bonn, Germany; Institute of Neuroscience and Medicine (INM-1), Research Center Juelich, Juelich, Germany; Division of Medical Genetics, Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Germany; Department of Psychiatry, University Medical Center Regensburg, University of Regensburg, Regensburg, Germany; Institute for Medical Biometry, Informatics, and Epidemiology, University of Bonn, Bonn, Germany; Department of Biostatistics, Harvard School of Public Health, Boston, MA, USA; Department of Psychiatry, University of Munich, Munich, Germany; Department of Psychiatry, University of Bonn, Bonn, Germany; Department of Psychiatry and Psychotherapy, University of Düsseldorf, Düsseldorf, Germany; Department of Psychiatry, University of Mainz, Mainz, Germany; Addiction Research Group at the Department of Psychiatry and Psychotherapy, LVR Hospital Essen, University of Duisburg-Essen, Essen, Germany; Department of Psychiatric Pharmacogenetics, Max-Planck-Institute of Psychiatry, Munich, Germany; Department of Molecular Psychology, Max-Planck-Institute of Psychiatry, Munich, Germany; Department of Statistical Genetics, Max-PlanckInstitute of Psychiatry, Munich, Germany; German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany; Department of Addictive Behavior and Addiction Medicine, Central Institute of Mental Health, Medical Faculty Mannheim/Heidelberg University, Heidelberg, Germany.