Wolfgang H. Sommer

Genome-wide Association Study of Alcohol Dependence

CONTEXT Alcohol dependence is a serious and common public health problem. It is well established that genetic factors play a major role in the development of this disorder. Identification of genes that contribute to alcohol dependence will improve our understanding of the mechanisms that underlie this disorder. OBJECTIVE To identify susceptibility genes for alcohol dependence through a genome-wide association study (GWAS) and a follow-up study in a population of German male inpatients with an early age at onset. DESIGN The GWAS tested 524,396 single-nucleotide polymorphisms (SNPs). All SNPs with P < 10(-4) were subjected to the follow-up study. In addition, nominally significant SNPs from genes that had also shown expression changes in rat brains after long-term alcohol consumption were selected for the follow-up step. SETTING Five university hospitals in southern and central Germany. PARTICIPANTS The GWAS included 487 male inpatients with alcohol dependence as defined by the DSM-IV and an age at onset younger than 28 years and 1358 population-based control individuals. The follow-up study included 1024 male inpatients and 996 age-matched male controls. All the participants were of German descent. MAIN OUTCOME MEASURES Significant association findings in the GWAS and follow-up study with the same alleles. RESULTS The GWAS produced 121 SNPs with nominal P < 10(-4). These, together with 19 additional SNPs from homologues of rat genes showing differential expression, were genotyped in the follow-up sample. Fifteen SNPs showed significant association with the same allele as in the GWAS. In the combined analysis, 2 closely linked intergenic SNPs met genome-wide significance (rs7590720, P = 9.72 x 10(-9); rs1344694, P = 1.69 x 10(-8)). They are located on chromosome region 2q35, which has been implicated in linkage studies for alcohol phenotypes. Nine SNPs were located in genes, including the CDH13 and ADH1C genes, that have been reported to be associated with alcohol dependence. CONCLUSIONS This is the first GWAS and follow-up study to identify a genome-wide significant association in alcohol dependence. Further independent studies are required to confirm these findings.

Long-lasting increase in voluntary ethanol consumption and transcriptional regulation in the rat brain after intermittent exposure to alcohol

Prolonged exposure of the brain to ethanol is a prerequisite for developing ethanol dependence, but the underlying neural adaptations are unknown. Here we demonstrate that rats subjected to repeated cycles of intoxication and withdrawal develop a marked and long‐lasting increase in voluntary ethanol intake. Exposure‐induced but not spontaneous alcohol intake is antagonized by acamprosate, a compound clinically effective in human alcoholism. Expression analysis of cingulate cortex and amygdala reveals a set of long‐term up‐regulated transcripts in this model. These include members of pathways previously implicated in alcohol dependence (glutamatergic, endocan‐nabinoid, and monoaminergic neurotransmission), as well as pathways not previously thought to be involved in this disorder (e.g., members of the mitogen‐activated protein kinase pathway). Thus, alternating periods of ethanol intoxication and withdrawal are sufficient to induce an altered functional brain state, which is likely to be encoded by long‐term changes in gene expression. These observations may have important implications for how alcoholism is managed clinically. Novel clinically effective treatments may be possible to develop by targeting the products of genes found to be regulated in our model.—Rimondini, R., Arlinde, C., Sommer, W., Heilig, M. Long‐lasting increase in voluntary ethanol consumption and transcriptional regulation in the rat brain after intermittent exposure to alcohol. FASEB J. 16, 27–35 (2002)

Upregulation of Voluntary Alcohol Intake, Behavioral Sensitivity to Stress, and Amygdala Crhr1 Expression Following a History of Dependence

BACKGROUND A history of alcohol dependence recruits increased voluntary alcohol intake and sensitivity to stress. Corticotropin-releasing hormone (CRH) has been implicated in this transition, but underlying molecular mechanisms remain unclear. METHODS A postdependent state was induced using intermittent alcohol exposure. Experiments were carried out following > or =3 weeks of recovery to eliminate contributions of acute withdrawal. Voluntary alcohol consumption was assessed in a two-bottle, free choice procedure. Behavioral sensitivity to stress was examined using fear suppression of behavior in a punished drinking (Vogel) conflict test. Effects of forced swim stress on voluntary alcohol intake were examined as a function of exposure history. Expression of Crh, Crhr1, and Crhr2 transcripts was analyzed by in situ hybridization histochemistry. RESULTS Alcohol drinking was upregulated long-term following a history of dependence. Fear suppression of behavior was selectively potentiated in postdependent animals. This persisted 3 months after alcohol exposure and was reversed by the selective CRH-R1 antagonist 3-(4-Chloro-2-morpholin-4-yl-thiazol-5-yl)-8-(1-ethylpropyl)-2,6-dimethyl-imidazo[1,2-b]pyridazine (MTIP) (10 mg/kg). Forced swim stress increased alcohol intake in postdependent animals but not in control animals. Behavioral changes were paralleled by an upregulation of Crhr1 transcript expression within basolateral (BLA) and medial (MeA) amygdala and Crh messenger RNA (mRNA) in central amygdala (CeA). In contrast, Crhr2 expression was down in the BLA. CONCLUSIONS Neuroadaptations encompassing amygdala CRH signaling contribute to the behavioral phenotype of postdependent animals.

A genetic determinant of the striatal dopamine response to alcohol in men

Excessive alcohol use, a major cause of morbidity and mortality, is less well understood than other addictive disorders. Dopamine release in ventral striatum is a common element of drug reward, but alcohol has an unusually complex pharmacology, and humans vary greatly in their alcohol responses. This variation is related to genetic susceptibility for alcoholism, which contributes more than half of alcoholism risk. Here, we report that a functional OPRM1 A118G polymorphism is a major determinant of striatal dopamine responses to alcohol. Social drinkers recruited based on OPRM1 genotype were challenged in separate sessions with alcohol and placebo under pharmacokinetically controlled conditions, and examined for striatal dopamine release using positron emission tomography and [11C]-raclopride displacement. A striatal dopamine response to alcohol was restricted to carriers of the minor 118G allele. To directly establish the causal role of OPRM1 A118G variation, we generated two humanized mouse lines, carrying the respective human sequence variant. Brain microdialysis showed a fourfold greater peak dopamine response to an alcohol challenge in h/mOPRM1-118GG than in h/mOPRM1-118AA mice. OPRM1 A118G variation is a genetic determinant of dopamine responses to alcohol, a mechanism by which it likely modulates alcohol reward.

Genetic Impairment of Frontocortical Endocannabinoid Degradation and High Alcohol Preference

Endocannabinoid signaling has recently been implicated in ethanol-seeking behavior. We analyzed the expression of endocannabinoid-related genes in key brain regions of reward and dependence, and compared them between the alcohol-preferring AA (Alko Alcohol) and nonpreferring ANA (Alko Non-Alcohol) rat lines. A decreased expression of fatty acid amidohydrolase (FAAH), the main endocannabinoid-degrading enzyme, was found in prefrontal cortex (PFC) of AA rats, and was accompanied by decreased enzyme activity in this region. Binding of the endocannabinoid-cannabinoid 1 (CB1) receptor ligand 3[H]SR141716A, and [35S]GTPγS incorporation stimulated by the CB1 agonist WIN 55,212-2 were downregulated in the same area. Together, this suggests an overactive endocannabinoid transmission in the PFC of AA animals, and a compensatory downregulation of CB1 signaling. The functional role of impaired FAAH function for alcohol self-administration was validated in two independent ways. The CB1 antagonist SR141716A potently and dose-dependently suppressed self-administration in AA rats when given systemically, or locally into the PFC, but not in the striatum. Conversely, intra-PFC injections of the competitive FAAH inhibitor URB597 increased ethanol self-administration in nonselected Wistar rats. These results show for the first time that impaired FAAH function may confer a phenotype of high voluntary alcohol intake, and point to a FAAH both as a potential susceptibility factor and a therapeutic target.

The alcohol-preferring AA and alcohol-avoiding ANA rats: neurobiology of the regulation of alcohol drinking.

The AA (alko, alcohol) and ANA (alko, non‐alcohol) rat lines were among the earliest rodent lines produced by bidirectional selection for ethanol preference. The purpose of this review is to highlight the strategies for understanding the neurobiological factors underlying differential alcohol‐drinking behavior in these lines. Most early work evaluated functioning of the major neurotransmitter systems implicated in drug reward in the lines. No consistent line differences were found in the dopaminergic system either under baseline conditions or after ethanol challenges. However, increased opioidergic tone in the ventral striatum and a deficiency in endocannabinoid signaling in the prefrontal cortex of AA rats may comprise mechanisms leading to increased ethanol consumption. Because complex behaviors, such as ethanol drinking, are not likely to be controlled by single factors, system‐oriented molecular‐profiling strategies have been used recently. Microarray based expression analysis of AA and ANA brains and novel data‐mining strategies provide a system biological view that allows us to formulate a hypothesis on the mechanism underlying selection for ethanol preference. Two main factors appear active in the selection: a recruitment of signal transduction networks, including mitogen‐activated protein kinases and calcium pathways and involving transcription factors such as Creb, Myc and Max, to mediate ethanol reinforcement and plasticity. The second factor acts on the mitochondrion and most likely provides metabolic flexibility for alternative substrate utilization in the presence of low amounts of ethanol.

Gluco- and mineralocorticoid receptor-mediated regulation of neurotrophic factor gene expression in the dorsal hippocampus and the neocortex of the rat

Gluco‐ and mineralocorticoid receptors (GR and MR) act via common promoter elements but may exert different effects on gene regulation in various regions of the forebrain. In order to separately analyse the role of GR and MR in the regulation of neurotrophic factor genes and their receptors, we used adrenalectomy and subsequent hormone injections in the rat as a model system. Twenty‐four hours after adrenalectomy rats were injected with a single dose of corticosterone (2 and 10 mg/kg), aldosterone (0.5 mg/kg) or the synthetic glucocorticoid agonist RU 28362 (4 mg/kg). Gene expression of basic fibroblast growth factor (bFGF) and its high‐affinity receptors [fibroblast growth factor receptor subtypes 1–3 (FGF‐R1, FGF‐R2, FGF‐R3)], as well as brain‐derived growth factor (BDNF) and neurotrophin‐3 (NT‐3) was analysed at 4 h after the hormone injection in CA1–CA4 (cornus of Ammon areas of the hippocampus) and dentate gyrus of the dorsal hippocampus and in neocortex by means of in situ hybridization. We found that bFGF is regulated in CA2, CA3 and dentate gyrus by GR and MR together, and in CA1, CA4 and neocortex by GR alone. FGF‐R2 expression in the hippocampus seems to be regulated only by MR, while BDNF expression appears to depend on both receptors. FGF‐R1, FGF‐R3 and NT‐3 were only moderately affected by the hormone activation of GR and MR acting in concert or alone in the various regions. Thus, the present findings suggest that the adrenal cortical system through GR and MR participate in the control of neurotrophic factor signalling in a highly subregion‐ and cellular‐dependent manner.

Anxiogenic-Like Action of Galanin after Intra-Amygdala Administration in the Rat

The neuropeptide galanin is expressed in brain structures implicated in regulation of emotionality. The amygdala is known to play a central role in mechanisms of fear and anxiety. We therefore examined the effects of galanin (0.2 and 0.6 nmol/side) on experimental anxiety upon microinjection into the amygdala. Two established animal models of anxiety were used: a punished drinking test, and the elevated plus-maze. Punished responding was dose dependently suppressed by intra-amygdala galanin, whereas unpunished responding, drinking motivation, locomotor activity, and shock thresholds were unaffected. No effects on experimental anxiety were seen in the plus- maze following galanin injection. When injected into parietal cortex, no anxiety promoting properties of galanin were detected. These data suggest that activation of galanin receptors in amygdala modulates neurotransmission involved in fear and experimental anxiety.

Translational magnetic resonance spectroscopy reveals excessive central glutamate levels during alcohol withdrawal in humans and rats.

BACKGROUND In alcoholism, excessive glutamatergic neurotransmission has long been implicated in the acute withdrawal syndrome and as a key signal for dependence-related neuroplasticity. Our understanding of this pathophysiological mechanism originates largely from animal studies, but human data are needed for translation into successful medication development. METHODS We measured brain glutamate levels during detoxification in alcohol-dependent patients (n = 47) and in healthy control subjects (n = 57) as well as in a rat model of alcoholism by state-of-the-art ¹H-magnetic magnetic resonance spectroscopy at 3 and 9.4 T, respectively. RESULTS We found significantly increased glutamate levels during acute alcohol withdrawal in corresponding prefrontocortical regions of treatment-seeking alcoholic patients and alcohol-dependent rats versus respective control subjects. The augmented spectroscopic glutamate signal is likely related to increased glutamatergic neurotransmission because, enabled by the high field strength of the animal scanner, we detected a profoundly elevated glutamate/glutamine ratio in alcohol-dependent rats during acute withdrawal. All dependence-induced metabolic alterations normalize within a few weeks of abstinence in both humans and rats. CONCLUSIONS Our data provide first-time direct support from humans for the glutamate hypothesis of alcoholism, demonstrate the comparability of human and animal magnetic resonance spectroscopy responses, and identify the glutamate/glutamine ratio as potential biomarker for monitoring disease progression.

Translating the neuroscience of alcoholism into clinical treatments : from blocking the buzz to curing the blues

Understanding the pathophysiology of addictive disorders is critical for development of new treatments. A major focus of addiction research has for a long time been on systems that mediate acute positively reinforcing effects of addictive drugs, most prominently the mesolimbic dopaminergic (DA) system and its connections. This research line has been successful in shedding light on the physiology of both natural and drug reward, but has not led to therapeutic breakthroughs. The role of classical reward systems is perhaps least clear in alcohol addiction. Here, recent work is summarized that points to some clinically important conclusions. First, important pharmacogenetic differences exist with regard to positively reinforcing effects of alcohol and the ability of this drug to activate classical reward pathways. This offers an opportunity for personalized treatment approaches in alcoholism. Second, brain stress and fear systems become pathologically activated in later stages of alcoholism and their activation is a major influence in escalation of alcohol intake, sensitization of stress responses, and susceptibility to relapse. These findings offer a new category of treatment mechanisms. Corticotropin-releasing hormone (CRH) signaling through CRH1 receptors is a major candidate target in this category, but recent data indicate that antagonists for substance P (SP) neurokinin 1 (NK1) receptors may have a similar potential.