Rational development of novel therapeutic strategies for alcoholism requires understanding of its underlying neurobiology and pathophysiology. Obtaining this knowledge largely relies on animal studies. Thus, choosing the appropriate animal model is one of the most critical steps in pre-clinical medication development. Among the range of animal models that have been used to investigate excessive alcohol consumption in rodents, the postdependent model stands out. It was specifically developed to test the role of negative affect as a key driving force in a perpetuating addiction cycle for alcoholism. Here, we will describe our approach to make rats dependent via chronic intermittent exposure to alcohol, discuss the validity of this model, and compare it with other commonly used animal models of alcoholism. We will summarize evidence that postdependent rats fulfill several criteria of a 'Diagnostic and Statistical Manual of Mental Disorders IV/V-like' diagnostic system. Importantly, these animals show long-lasting excessive consumption of and increased motivation for alcohol, and evidence for loss of control over alcohol intake. Our conclusion that postdependent rats are an excellent model for medication development for alcoholism is underscored by a summary of more than two dozen pharmacological tests aimed at reversing these abnormal alcohol responses. We will end with open questions on the use of this model. In the tradition of the Sanchis-Segura and Spanagel review, we provide comic strips that illustrate the postdependent procedure and relevant phenotypes in this review.
The neuroanatomical and neurochemical basis of alcohol reward has been studied extensively, but global alterations of neural activity in reward circuits during chronic alcohol use remain poorly described. Here, we measured brain activity changes produced by long-term voluntary alcohol drinking in the alcohol-preferring AA (Alko alcohol) rats using manganese-enhanced magnetic resonance imaging (MEMRI). MEMRI is based on the ability of paramagnetic manganese ions to accumulate in excitable neurons and thereby enhance the T1-weighted signal in activated brain areas. Following 6 weeks of voluntary alcohol drinking, AA rats were allowed to drink alcohol for an additional week, during which they were administered manganese chloride (MnCl2 ) with subcutaneous osmotic minipumps before MEMRI. A second group with an identical alcohol drinking history received MnCl2 during the abstinence week following alcohol drinking. For comparing alcohol with a natural reinforcer, MEMRI was also performed in saccharin-drinking rats. A water-drinking group receiving MnCl2 served as a control. We found that alcohol drinking increased brain activity extensively in cortical and subcortical areas, including the mesocorticolimbic and nigrostriatal dopamine pathways and their afferents. Remarkably similar activation maps were seen after saccharin ingestion. Particularly in the prelimbic cortex, ventral hippocampus and subthalamic nucleus, activation persisted into early abstinence. These data show that voluntary alcohol recruits an extensive network that includes the ascending dopamine systems and their afferent connections, and that this network is largely shared with saccharin reward. The regions displaying persistent alterations after alcohol drinking could participate in brain networks underlying alcohol seeking and relapse.
Genetic factors have as large role as environmental factors in the etiology of alcohol dependence (AD). Although genome-wide association studies (GWAS) enable systematic searches for loci not hitherto implicated in the etiology of AD, many true findings may be missed owing to correction for multiple testing. The aim of the present study was to circumvent this limitation by searching for biological system-level differences, and then following up these findings in humans and animals. Gene-set-based analysis of GWAS data from 1333 cases and 2168 controls identified 19 significantly associated gene-sets, of which 5 could be replicated in an independent sample. Clustered in these gene-sets were novel and previously identified susceptibility genes. The most frequently present gene, ie in 6 out of 19 gene-sets, was X-ray repair complementing defective repair in Chinese hamster cells 5 (XRCC5). Previous human and animal studies have implicated XRCC5 in alcohol sensitivity. This phenotype is inversely correlated with the development of AD, presumably as more alcohol is required to achieve the desired effects. In the present study, the functional role of XRCC5 in AD was further validated in animals and humans. Drosophila mutants with reduced function of Ku80-the homolog of mammalian XRCC5-due to RNAi silencing showed reduced sensitivity to ethanol. In humans with free access to intravenous ethanol self-administration in the laboratory, the maximum achieved blood alcohol concentration was influenced in an allele-dose-dependent manner by genetic variation in XRCC5. In conclusion, our convergent approach identified new candidates and generated independent evidence for the involvement of XRCC5 in alcohol dependence.Neuropsychopharmacology advance online publication, 13 August 2014; doi:10.1038/npp.2014.178.
According to the World Health Organization, about 2 billion people drink alcohol. Excessive alcohol consumption can result in alcohol addiction, which is one of the most prevalent neuropsychiatric diseases afflicting our society today. Prevention and intervention of alcohol binging in adolescents and treatment of alcoholism are major unmet challenges affecting our health-care system and society alike. Our newly formed German SysMedAlcoholism consortium is using a new systems medicine approach and intends (1) to define individual neurobehavioral risk profiles in adolescents that are predictive of alcohol use disorders later in life and (2) to identify new pharmacological targets and molecules for the treatment of alcoholism. To achieve these goals, we will use omics-information from epigenomics, genetics transcriptomics, neurodynamics, global neurochemical connectomes and neuroimaging (IMAGEN; Schumann et?al. ) to feed mathematical prediction modules provided by two Bernstein Centers for Computational Neurosciences (Berlin and Heidelberg/Mannheim), the results of which will subsequently be functionally validated in independent clinical samples and appropriate animal models. This approach will lead to new early intervention strategies and identify innovative molecules for relapse prevention that will be tested in experimental human studies. This research program will ultimately help in consolidating addiction research clusters in Germany that can effectively conduct large clinical trials, implement early intervention strategies and impact political and healthcare decision makers.
A key deficit in alcohol dependence is disrupted prefrontal function leading to excessive alcohol seeking, but the molecular events underlying the emergence of addictive responses remain unknown. Here we show by convergent transcriptome analysis that the pyramidal neurons of the infralimbic cortex are particularly vulnerable for the long-term effects of chronic intermittent ethanol intoxication. These neurons exhibit a pronounced deficit in metabotropic glutamate receptor subtype 2 (mGluR(2)). Also, alcohol-dependent rats do not respond to mGluR(2/3) agonist treatment with reducing extracellular glutamate levels in the nucleus accumbens. Together these data imply a loss of autoreceptor feedback control. Alcohol-dependent rats show escalation of ethanol seeking, which was abolished by restoring mGluR(2) expression in the infralimbic cortex via viral-mediated gene transfer. Human anterior cingulate cortex from alcoholic patients shows a significant reduction in mGluR(2) transcripts compared to control subjects, suggesting that mGluR(2) loss in the rodent and human corticoaccumbal neurocircuitry may be a major consequence of alcohol dependence and a key pathophysiological mechanism mediating increased propensity to relapse. Normalization of mGluR(2) function within this brain circuit may be of therapeutic value.
The rewarding effects of alcohol have been attributed to interactions between opioid and dopaminergic system within the mesolimbic reward pathway. We have previously shown that ablation of ?-arrestin 2 (Arrb2), a crucial regulator of ?-opioid receptor function, attenuates alcohol-induced hyperlocomotion and c-fos activation in the nucleus accumbens.
The Rhodopsin family of G protein coupled receptors (GPCRs) includes the phylogenetic ?-group consisting of about 100 human members. The ?-group is the only group of GPCRs that has many receptors for biogenic amines which are major drug targets. Several members of this group are orphan receptors and their functions are elusive. In this study we present a detailed phylogenetic and anatomical characterization of the Gpr153 receptor and also attempt to study its functional role. We identified the homologue of Gpr153 in the elephant shark genome and phylogenetic and synteny analyses revealed that Gpr162 and Gpr153 share a common ancestor that split most likely through a duplication event before the divergence of the tetrapods and the teleost lineage. A quantitative real-time PCR study reveals widespread expression of Gpr153 in the central nervous system and all the peripheral tissues investigated. Detailed in?situ hybridization on mouse brain showed specifically high expression in the thalamus, cerebellum and the arcuate nucleus. The antisense oligodeoxynucleotide knockdown of Gpr153 caused a slight reduction in food intake and the elevated plus maze test showed significant reduction in the percentage of time spent in the centre square, which points towards a probable role in decision making. This report provides the first detailed characterization of the evolution, expression and primary functional properties of the Gpr153 gene.
The most common functional single nucleotide polymorphism of the human OPRM1 gene, A118G, has been shown to be associated with interindividual differences in opioid analgesic requirements, particularly with morphine, in patients with acute postoperative pain. The purpose of this study was to examine whether this polymorphism would modulate the morphine and fentanyl pharmacological profile of sensory neurons isolated from a humanized mouse model homozygous for either the 118A or 118G allele.
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.
Alcohol consumption is a moderately heritable trait, but the genetic basis in humans is largely unknown, despite its clinical and societal importance. We report a genome-wide association study meta-analysis of ?2.5 million directly genotyped or imputed SNPs with alcohol consumption (gram per day per kilogram body weight) among 12 population-based samples of European ancestry, comprising 26,316 individuals, with replication genotyping in an additional 21,185 individuals. SNP rs6943555 in autism susceptibility candidate 2 gene (AUTS2) was associated with alcohol consumption at genome-wide significance (P = 4 × 10(-8) to P = 4 × 10(-9)). We found a genotype-specific expression of AUTS2 in 96 human prefrontal cortex samples (P = 0.026) and significant (P < 0.017) differences in expression of AUTS2 in whole-brain extracts of mice selected for differences in voluntary alcohol consumption. Down-regulation of an AUTS2 homolog caused reduced alcohol sensitivity in Drosophila (P < 0.001). Our finding of a regulator of alcohol consumption adds knowledge to our understanding of genetic mechanisms influencing alcohol drinking behavior.
A major hypothesis of depression postulates that a dysregulation of the neurotrophin systems is directly involved in the pathophysiology of depression, and that restoration of such deficits may underlie the therapeutic efficacy of antidepressant treatment. One key finding supporting this hypothesis is upregulation of brain derived neurotrophic factor (BDNF) in the hippocampus after antidepressant treatment. Here, we further test the hypothesis of BDNF involvement in antidepressant action in a genetic rat model of depression after chronic oral treatment with escitalopram, nortriptyline or placebo. Active treatments had significant behavioural antidepressant-like actions in female rats of the Flinders Sensitive Line (FSL) and non-selected Sprague Dawley (SD) rats, while Flinders Resistant Line (FRL) rats were unaffected. Escitalopram, but not nortriptyline, markedly reduced BDNF mRNA levels in the dentate gyrus of FSL rats. The BDNF downregulation was common to the four major promoters of the gene. Treatments did not affect BDNF expression in FRL or SD strains. We conclude that the antidepressant effects of escitalopram and nortriptyline, two common drugs with different pharmacological profiles, appear to be unrelated to the regulation of hippocampal BDNF expression in female rats. These results indicate that the tropic hypothesis of depression has limitations and emphasize the need for validated disease models of depression to assess potential treatment targets.
Alcohol drinking is highly prevalent in many cultures and contributes to the global burden of disease. In fact, it was shown that alcohol constitutes 3.2% of all worldwide deaths in the year 2006 and is linked to more than 60 diseases, including cancers, cardiovascular diseases, liver cirrhosis, neuropsychiatric disorders, injuries and foetal alcohol syndrome. Alcoholism, which has been proven to have a high genetic load, is one potentially fatal consequence of chronic heavy alcohol consumption, and may be regarded as one of the most prevalent neuropsychiatric diseases afflicting our society today. The aim of the integrated genome research network Genetics of Alcohol Addiction--which is a German inter-/trans-disciplinary life science consortium consisting of molecular biologists, behavioural pharmacologists, system biologists with mathematicians, human geneticists and clinicians--is to better understand the genetics of alcohol addiction by identifying and validating candidate genes and molecular networks involved in the aetiology of this pathology. For comparison, addictive behaviour to other drugs of abuse (e.g. cocaine) is studied as well. Here, we present an overview of our research consortium, the current state of the art on genetic research in the alcohol field, and list finally several of our recently published research highlights. As a result of our scientific efforts, better insights into the molecular and physiological processes underlying addictive behaviour will be obtained, new targets and target networks in the addicted brain will be defined, and subsequently, novel and individualized treatment strategies for our patients will be delivered.
Much research on experimental animals that is aimed to decipher genetic factors involved in alcoholism has been devoted to either models of innate alcohol-related phenotypes or responses after acute alcohol challenge. Such focus has, however, limitations when it comes to the pathogenetic mechanism underlying alcohol addiction, because the progression into the disorder takes years and genetic as well as environmental factors may exert different influences along this trajectory. Animal models of the neuroadaptations involved in the development of dependence exist, but have been difficult to implement for genetic and genomics analysis. Consequently, currently available data have been difficult to reconcile with the human condition and could be misleading in predicting targets for medication development. This review will illustrate strengths and pitfalls of genomic approaches in rodent models of alcoholism and emphasize the need for convergent lines of evidence to improve the predictive value of such studies. Examples of a convergent research approach include validation studies for Agt, Arrb2, Crhr1, Grin3a, and Npy.
Studies in humans and animals suggest a role for NPY in the mediation of behavioral stress responses. Here, we examined whether the NPY promoter variant rs16147:T>C is functional for expression of NPY in a brain region relevant for behavioral control, anxiety and depression, the anterior cingulate cortex. In silico analysis of DNA structural profile changes produced by rs16147 variation suggests allelic differences in protein binding at the rs16147 site. This was confirmed by electrophoretic mobility shift assay, demonstrating that the rs16147 C-allele has strongly reduced affinity for a yet unknown factor compared to the T-allele. Analyzing 107 human post-mortem brain samples we show that allelic variation at rs16147 contributes to regulation of NPY mRNA and peptide levels in this region. Specifically, the C-allele leads to increased gene expression. In agreement with the molecular findings, rs16147:T>C is associated with anxiety and depressive symptoms in 314 young adults via a gene x environment interaction with early childhood adversity, replicating the recent finding of rs16147-C as a risk factor for stress related psychopathology. Our results show the importance of rs16147:T>C for regulation of NPY gene expression and brain function.
The reinforcing properties of ethanol are in part attributed to interactions between opioid and dopaminergic signaling pathways, but intracellular mediators of such interactions are poorly understood. Here we report that an acute ethanol challenge induces a robust phosphorylation of two key signal transduction kinases, AKT and DARPP-32, in the striatum of mice. Ethanol-induced AKT phosphorylation was blocked by the opioid receptor antagonist naltrexone but unaffected by blockade of dopamine D2 receptors via sulpiride. In contrast, DARPP-32 phosphorylation was abolished by both antagonists. These data suggest that ethanol acts via two distinct but potentially synergistic striatal signaling cascades. One of these is D2-dependent, while the other is not. These findings illustrate that pharmacology of ethanol reward is likely more complex than that for other addictive drugs.
Neuropeptide Y (NPY) counters stress and is involved in neuroadaptations that drive escalated alcohol drinking in rodents. In humans, low NPY expression predicts amygdala response and emotional reactivity. Genetic variation that affects the NPY system could moderate stress resilience and susceptibility to alcohol dependence.
Residual dysfunction of multiple neurotransmitter systems due to chronic alcohol use is likely responsible for the occurrence of compulsive alcohol seeking during abstinence and relapse behavior. There is increasing evidence that glycine, which activates both glycine and N-methyl-D-aspartate receptors, contributes to excessive alcohol consumption. We therefore hypothesized that the blockade of glycine transporter 1 might interfere with compulsive alcohol consumption and relapse behavior.
Alcohol dependence leads to persistent neuroadaptations, potentially related to structural plasticity. Previous work has shown that hippocampal neurogenesis is modulated by alcohol, but effects of chronic alcohol on neurogenesis in the forebrain subventricular zone (SVZ) have not been reported. Effects in this region may be relevant for the impairments in olfactory discrimination present in alcoholism. Here, we examined the effects of prolonged alcohol dependence on neurogenesis. Rats were sacrificed directly after 7 wk of intermittent alcohol vapour exposure, or 3, 7 or 21 d into abstinence. Proliferation was assessed using BrdU and Ki67 immunoreactivity, newly differentiated neurons (neurogenesis) as doublecortin-immunoreactivity (DCX-IR), and neural stem cells using the SOX2 marker. In the dentate gyrus, chronic dependence resulted in a pattern similar to that previously reported for acute alcohol exposure: proliferation and neurogenesis were suppressed by the end of exposure, rebounded on day 3 of abstinence, and returned to control levels by days 7 and 21. In the SVZ, proliferation was also suppressed at the end of alcohol exposure, followed by a proliferation burst 3 d into abstinence. However, in this area, there was a trend for reduced proliferation on days 7 and 21 of abstinence, and this was accompanied by significant suppression of DCX-IR, indicating a long-term suppression of forebrain neurogenesis. Finally, a decrease in the SOX2 stem cell marker was detected at days 7 and 21, suggesting long-term reduction of the SVZ stem cell pool. While suppression of hippocampal neurogenesis by alcohol dependence is transient, the suppression in the forebrain SVZ appears long-lasting.
Reinstatement of responding to a previously alcohol-associated lever following extinction is an established model of relapse-like behavior and can be triggered by stress exposure. Here, we examined whether neuropeptide Y (NPY), an endogenous anti-stress mediator, blocks reinstatement of alcohol-seeking induced by the pharmacological stressor yohimbine.
Corticotropin-releasing factor (CRF), encoded by the CRH gene, is a key integrator of stress responses, and, as such, CRH gene variation may contribute to individual differences in susceptibility to stress-related pathology. In rhesus macaques, a single nucleotide polymorphism (SNP) is found within the CRH promoter (-248C--> T). Here, we assessed whether this variant influenced stress responding and, because increased CRF system activity drives alcohol drinking in rodents, we examined whether it predicted voluntary alcohol consumption as a function of prior stress exposure. Using a hypothalamic nuclear extract, we showed that the -248 T allele resulted in increased DNA protein interactions relative to the C allele. In vitro, the T allele resulted in CRH promoter activity that was higher following both stimulation with forskolin and treatment with dexamethasone. Endocrine and behavioral responses to social separation stress (release of ACTH and cortisol, and suppression of environmental exploration, respectively) were higher among carriers of the T allele, particularly among those exposed to early adversity in the form of peer rearing. We also found that T allele carriers with a history of early life adversity consumed more alcohol in a limited-access paradigm. Our data suggest that CRH promoter variation that confers increased stress reactivity increases the risk for alcohol use disorders in stress-exposed individuals.
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.
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.
Intracellular signalling pathways emerge as key mediators of the molecular and behavioural effects of addictive drugs including ethanol. Previously, we demonstrated that the innate high ethanol preference in AA rats is driven by dysfunctional endocannabinoid signalling in the medial prefrontal cortex (mPFC). Here, we report that acute ethanol challenge, at a dose commonly regarded as reinforcing, strongly phosphorylates glycogen synthase kinase-3beta (GSK-3beta) in this region with corresponding increased phosphorylation of AKT, a major regulator of GSK-3beta. In the non-preferring counterpart ANA line we found a weaker, AKT-independent phosphorylation of GSK-3beta by ethanol. Furthermore, AA rats showed rapid and transient dephosphorylation of ERK1/2 upon acute ethanol challenge in the medial prefrontal cortex (mPFC) and to a lesser degree in the nucleus accumbens; ANA rats were completely non-responsive for this mechanism. Together, these results identify candidate pathways for mediating high ethanol preference and emphasize the importance of the mPFC in controlling this behaviour.
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 Ca(2+)-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 I(A) 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.
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