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1.  Dysregulation of Nociceptin/Orphanin FQ activity in the amygdala is linked to excessive alcohol drinking in the rat 
Biological psychiatry  2008;64(3):211-218.
Background
Alcoholism is a complex behavioral disorder in which interactions between stressful life events and heritable susceptibility factors contribute to the initiation and progression of disease. Neural substrates of these interactions remain largely unknown. Here, we examined the role of the nociceptin/orphanin FQ (N/OFQ) system, using an animal model in which genetic selection for high alcohol preference has led to co-segregation of elevated behavioral sensitivity to stress (msP rats).
Methods
msP and Wistar rats trained to self-administer alcohol received central injections of N/OFQ. In situ hybridization, and receptor binding assays were also performed to evaluate N/OFQ receptor (NOP) function in naïve msP and Wistar rats.
Results
Intracerebroventricular (ICV) injection of N/OFQ significantly inhibited alcohol self-administration in msP but not in nonselected Wistar rats. NOP receptor mRNA expression and binding was upregulated across most brain regions in msP compared to Wistar rats. However, in msP rats [35S]GTPγS binding revealed a selective impairment of NOP receptor signaling in the central amygdala (CeA). Ethanol self-administration in msP rats was suppressed after N/OFQ microinjection into the CeA but not into the bed nucleus of the stria terminalis or the basolateral amygdala.
Conclusions
These findings indicate that dysregulation of N/OFQ-NOP receptor signaling in the CeA contributes to excessive alcohol intake in msP rats, and that this phenotype can be rescued by local administration of pharmacological doses of exogenous N/OFQ. Data are interpreted based of the anti-CRF actions of N/OFQ and the significance of the CRF system in promoting excessive alcohol drinking in msP rats.
doi:10.1016/j.biopsych.2008.02.004
PMCID: PMC4275225  PMID: 18367152
Addiction, Nociceptin/Orphanin FQ; NOP receptors; Central Amygdala; Alcohol Preferring Rats; Alcoholism
2.  β-arrestin 2 knockout mice exhibit sensitized dopamine release and increased reward in response to a low dose of alcohol 
Psychopharmacology  2013;230(3):10.1007/s00213-013-3166-x.
Rationale
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 β-arrestin 2 (Arrb2), a crucial regulator of μ-opioid receptor function, attenuates alcohol-induced hyperlocomotion and c-fos activation in the nucleus accumbens.
Objectives
Here, we further investigated the role of Arrb2 in modulating alcohol-induced dopamine (DA) release and conditioned place preference (CPP). We also assessed the functional importance of Arrb2 for μ-opioid receptor surface expression and signaling following an acute alcohol challenge.
Methods
Alcohol-evoked (0.375, 0.75 and 1.5 g/kg intraperitoneally, i.p.) DA release was measured by in vivo microdialysis in the shell of nucleus accumbens. Reward was assessed by the CPP paradigm. Receptor function was assessed by μ-receptor binding and [35S]GTP-γ-S autoradiography.
Results
In Arrb2 knockout mice accumbal DA levels reach maximum response at a lower dose compared to wild-type (wt) animals. In line with these results, Arrb2 knockout mice display increased CPP for alcohol as compared to wt mice. Finally, Arrb2 mutant mice display increased μ-opioid receptor signaling in the ventral and dorsal striatum and amygdala in response to a low dose of alcohol, indicating impaired desensitization mechanisms in these mice.
Conclusions
Our results show that Arrb2 modulates the response to low doses of alcohol on various levels including μ-opioid receptor signaling, DA release, and reward. They also reveal a clear dissociation between the effects of Arrb2 on psychomotor and reward behaviors.
doi:10.1007/s00213-013-3166-x
PMCID: PMC3817962  PMID: 23779257
Arrestin; opioid; dopamine; alcohol; reward; nucleus accumbens
3.  microRNA-206 in Rat Medial Prefrontal Cortex Regulates BDNF Expression and Alcohol Drinking 
The Journal of Neuroscience  2014;34(13):4581-4588.
Escalation of voluntary alcohol consumption is a hallmark of alcoholism, but its neural substrates remain unknown. In rats, escalation occurs following prolonged exposure to cycles of alcohol intoxication, and is associated with persistent, wide-ranging changes in gene expression within the medial prefrontal cortex (mPFC). Here, we examined whether induction of microRNA (miR) 206 in mPFC contributes to escalated alcohol consumption. Following up on a microarray screen, quantitative real-time reverse transcription PCR (qPCR) confirmed that a history of dependence results in persistent (>3weeks) up-regulation of miR-206 expression in the mPFC, but not in the ventral tegmental area, amygdala, or nucleus accumbens. Viral-mediated overexpression of miR-206 in the mPFC of nondependent rats reproduced the escalation of alcohol self-administration seen following a history of dependence and significantly inhibited BDNF expression. Bioinformatic analysis identified three conserved target sites for miR-206 in the 3′-UTR of the rat BDNF transcript. Accordingly, BDNF was downregulated in post-dependent rats on microarray analysis, and this was confirmed by qPCR. In vitro, BDNF expression was repressed by miR-206 but not miR-9 in a 3′-UTR reporter assay, confirming BDNF as a functional target of miR-206. Mutation analysis showed that repression was dependent on the presence of all three miR-206 target sites in the BDNF 3′-UTR. Inhibition of miR-206 expression in differentiated rat cortical primary neurons significantly increased secreted levels of BDNF. In conclusion, recruitment of miR-206 in the mPFC contributes to escalated alcohol consumption following a history of dependence, with BDNF as a possible mediator of its action.
doi:10.1523/JNEUROSCI.0445-14.2014
PMCID: PMC3965783  PMID: 24672003
addiction; alcohol dependence; BDNF; medial prefrontal cortex; microRNA; self-administration
4.  Reduced Cannabinoid CB1 Receptor Binding in Alcohol Dependence Measured with Positron Emission Tomographys 
Molecular psychiatry  2012;18(8):916-921.
Brain cannabinoid CB1 receptors contribute to alcohol-related behaviors in experimental animals, but their potential role in humans with alcohol dependence is poorly understood. We measured CB1 receptors in alcohol dependent patients in early and protracted abstinence, and in comparison with control subjects without alcohol use disorders, using positron emission tomography (PET) and [18F]FMPEP-d2, a radioligand for CB1 receptors. We scanned 18 male inpatients with alcohol dependence twice, within 3–7 days of admission from ongoing drinking, and after 2–4 weeks of supervised abstinence. Imaging data were compared with those from 19 age-matched healthy male control subjects. Data were also analyzed for potential influence of a common functional variation (rs2023239) in the CB1 receptor gene (CNR1) that may moderate CB1 receptor density. On the first scan, CB1 receptor binding was 20–30% lower in patients with alcohol dependence than in control subjects in all brain regions and was negatively correlated with years of alcohol abuse. After 2–4 weeks of abstinence, CB1 receptor binding remained similarly reduced in these patients. Irrespective of diagnostic status, C allele carriers at rs2023239 had higher CB1 receptor binding compared to non-carriers. Alcohol dependence is associated with a widespread reduction of cannabinoid CB1 receptor binding in the human brain and this reduction persists at least 2–4 weeks into abstinence. The correlation of reduced binding with years of alcohol abuse suggests an involvement of CB1 receptors in alcohol dependence in humans.
doi:10.1038/mp.2012.100
PMCID: PMC3594469  PMID: 22776901
alcohol dependence; alcoholism; cannabinoid CB1 receptor; positron emission tomography; brain imaging; withdrawal; endocannabinoid
5.  Restraint Stress Alters Nociceptin/Orphanin FQ and CRF Systems in the Rat Central Amygdala: Significance for Anxiety-Like Behaviors 
The Journal of Neuroscience  2014;34(2):363-372.
Corticotropin releasing factor (CRF) is the primary mediator of stress responses, and nociceptin/orphanin FQ (N/OFQ) plays an important role in the modulation of these stress responses. Thus, in this multidisciplinary study, we explored the relationship between the N/OFQ and the CRF systems in response to stress. Using in situ hybridization (ISH), we assessed the effect of body restraint stress on the gene expression of CRF and N/OFQ-related genes in various subdivisions of the amygdala, a critical brain structure involved in the modulation of stress response and anxiety-like behaviors. We found a selective upregulation of the NOP and downregulation of the CRF1 receptor transcripts in the CeA and in the BLA after body restraint. Thus, we performed intracellular electrophysiological recordings of GABAA-mediated IPSPs in the central nucleus of the amygdala (CeA) to explore functional interactions between CRF and N/OFQ systems in this brain region. Acute application of CRF significantly increased IPSPs in the CeA, and this enhancement was blocked by N/OFQ. Importantly, in stress-restraint rats, baseline CeA GABAergic responses were elevated and N/OFQ exerted a larger inhibition of IPSPs compared with unrestraint rats. The NOP antagonist [Nphe1]-nociceptin(1–13)NH2 increased the IPSP amplitudes in restraint rats but not in unrestraint rats, suggesting a functional recruitment of the N/OFQ system after acute stress. Finally, we evaluated the anxiety-like response in rats subjected to restraint stress and nonrestraint rats after N/OFQ microinjection into the CeA. Intra-CeA injections of N/OFQ significantly and selectively reduced anxiety-like behavior in restraint rats in the elevated plus maze. These combined results demonstrate that acute stress increases N/OFQ systems in the CeA and that N/OFQ has antistress properties.
doi:10.1523/JNEUROSCI.2400-13.2014
PMCID: PMC3870926  PMID: 24403138
6.  Coordinated dysregulation of mRNAs and microRNAs in the rat medial prefrontal cortex following a history of alcohol dependence 
The pharmacogenomics journal  2012;13(3):286-296.
Long-term changes in brain gene expression have been identified in alcohol dependence, but underlying mechanisms remain unknown. Here, we examined the potential role of microRNAs for persistent gene expression changes in the rat medial prefrontal cortex after a history of alcohol dependence. Two-bottle free-choice alcohol consumption increased following 7-week exposure to intermittent alcohol intoxication. A bioinformatic approach using microarray analysis, qPCR, bioinformatic analysis, and microRNA-mRNA integrative analysis identified expression patterns indicative of a disruption in synaptic processes and neuroplasticity. 41 rat-microRNAs and 165 mRNAs in the medial prefrontal cortex were significantly altered after chronic alcohol exposure. A subset of the microRNAs and mRNAs was confirmed by qPCR. Gene ontology categories of differential expression pointed to functional processes commonly associated with neurotransmission, neuroadaptation, and synaptic plasticity. microRNA-mRNA expression pairing identified 33 microRNAs putatively targeting 89 mRNAs suggesting transcriptional networks involved in axonal guidance and neurotransmitter signaling. Our results demonstrate a significant shift in microRNA expression patterns in the medial prefrontal cortex following a history of dependence. Due to their global regulation of multiple downstream target transcripts, microRNAs may play a pivotal role in the reorganization of synaptic connections and long term neuroadaptations in alcohol dependence. microRNA-mediated alterations of transcriptional networks may be involved in disrupted prefrontal control over alcohol-drinking observed in alcoholic patients.
doi:10.1038/tpj.2012.17
PMCID: PMC3546132  PMID: 22614244
alcohol; addiction; neuroadaptation; gene expression; microRNA; medial prefrontal cortex
7.  Childhood trauma exposure and alcohol dependence severity in adulthood: mediation by emotional abuse severity and neuroticism 
Background
Childhood trauma has been linked to a number of negative outcomes later in life, including alcohol dependence. Previous studies have suggested a mediating role for neuroticism in the relationship between childhood trauma and psychopathology. In this study we investigate the prevalence of multiple types of childhood trauma in treatment-seeking alcohol dependent patients, and the associations between childhood trauma and alcohol dependence severity using multiple mediation analysis.
Method
The prevalence of five types of childhood trauma – emotional abuse, sexual abuse, physical abuse, emotional neglect, and physical neglect – was assessed in treatment seeking alcohol dependent patients (n = 280) and healthy controls (n = 137) using the Childhood Trauma Questionnaire. Multiple mediation analyses were used to model associations between childhood trauma measures and alcohol-related outcomes, primarily the severity of alcohol dependence in the alcohol dependent sample.
Results
Childhood trauma was significantly more prevalent and more severe in the alcohol dependent subjects. In addition, childhood trauma was found to influence alcohol dependence severity, an effect that was mediated by neuroticism. When individual trauma types were examined, emotional abuse was found to be the primary predictor of alcohol dependence severity, both directly and through the mediating effects of the impulsivity subfacet of neuroticism. Physical abuse also had a moderate direct effect on alcohol dependence severity. Mediation analysis did not reveal any association between childhood trauma and AUDIT score in the non-dependent control sample.
Conclusions
Childhood trauma is highly prevalent in treatment-seeking alcoholics and may play a significant role in the development and severity of alcohol dependence through an internalizing pathway involving negative affect. Our findings suggest that alcoholics with a history of childhood emotional abuse may be particularly vulnerable to severe dependence.
doi:10.1111/acer.12053
PMCID: PMC3620963  PMID: 23278300
8.  Conditioned Preference to a Methamphetamine-Associated Contextual Cue in Humans 
Neuropsychopharmacology  2013;38(6):921-929.
Classical conditioning is widely used to study motivational properties of addictive drugs in animals, but has rarely been used in humans. We established a procedure suitable for studying the neurobiology and individual determinants of classical conditioning in humans. Healthy volunteers were randomly assigned to four groups that received methamphetamine or placebo in the presence of distinctive environmental cues under paired or unpaired conditions. During each session, subjects performed tasks known to activate the ventral striatum. Tasks were performed in the presence of a distinctive context, consisting of a screen background image of a beach or mountains, accompanied by corresponding sounds. Separate groups of subjects carried out the tasks under high ($35–50) or low ($5–20) reward conditions. Within each of the two reward conditions, one group (paired) received methamphetamine (20 mg, oral) or placebo consistently associated with one of the contexts, while the other (unpaired) received drug or placebo unrelated to context. A fifth group (paired) performed the tasks with contextual cues but in the absence of monetary incentives. Before and after conditioning, participants carried out a series of forced choice tasks for the contextual cues, and change of preference over time was analyzed. All paired groups showed a significant increase in preference for the drug-associated context, with a linear trend for increase across the levels of reward. Preference was unrelated to subjective drug effects, and did not change in the unpaired group. These data support the translational utility of our conditioning procedure for studies of reward mechanisms in humans.
doi:10.1038/npp.2013.3
PMCID: PMC3629404  PMID: 23416857
addiction & substance abuse; learning & memory; dopamine; behavioral science; conditioned place preference; methamphetamine; conditioning; place preference; stimulant; amphetamine; reward
9.  The NK1 Receptor Antagonist L822429 Reduces Heroin Reinforcement 
Neuropsychopharmacology  2013;38(6):976-984.
Genetic deletion of the neurokinin 1 receptor (NK1R) has been shown to decrease the reinforcing properties of opioids, but it is unknown whether pharmacological NK1R blockade has the same effect. Here, we examined the effect of L822429, a rat-specific NK1R antagonist, on the reinforcing properties of heroin in rats on short (1 h: ShA) or long (12 h: LgA) access to intravenous heroin self-administration. ShA produces heroin self-administration rates that are stable over time, whereas LgA leads to an escalation of heroin intake thought to model important dependence-related aspects of addiction. L822429 reduced heroin self-administration and the motivation to consume heroin, measured using a progressive-ratio schedule, in both ShA and LgA rats. L822429 also decreased anxiety-like behavior in both groups, measured on the elevated plus maze, but did not affect mechanical hypersensitivity observed in LgA rats. Expression of TacR1 (the gene encoding NK1R) was decreased in reward- and stress-related brain areas both in ShA and LgA rats compared with heroin-naïve rats, but did not differ between the two heroin-experienced groups. In contrast, passive exposure to heroin produced increases in TacR1 expression in the prefrontal cortex and nucleus accumbens. Taken together, these results show that pharmacological NK1R blockade attenuates heroin reinforcement. The observation that animals with ShA and LgA to heroin were similarly affected by L822429 indicates that the SP/NK1R system is not specifically involved in neuroadaptations that underlie escalation resulting from LgA self-administration. Instead, the NK1R antagonist appears to attenuate acute, positively reinforcing properties of heroin and may be useful as an adjunct to relapse prevention in detoxified opioid-dependent subjects.
doi:10.1038/npp.2012.261
PMCID: PMC3629386  PMID: 23303056
addiction & substance abuse; opioids; animal models; neuropeptides; heroin; self-administration; substance P; neurokinin receptor; opioids; heroin; self-administration; substance P; neurokinin receptor
10.  Tacr1 Gene Variation and Neurokinin 1 Receptor Expression Is Associated with Antagonist Efficacy in Genetically Selected Alcohol-Preferring Rats 
Biological psychiatry  2013;73(8):774-781.
Background
Genetic deletion or antagonism of the neurokinin 1 receptor (NK1R) decreases alcohol intake, alcohol reward, and stress-induced alcohol relapse in rodents, while TACR1 variation is associated with alcoholism in humans.
Methods
We used L822429, a specific antagonist with high affinity for the rat NK1R, and examined whether sensitivity to NK1R blockade is altered in alcohol-preferring (P) rats. Operant alcohol self-administration and progressive ratio responding were analyzed in P-rats and their founder Wistar line. We also analyzed Tacr1 expression and binding and sequenced the Tacr1 promoter from both lines.
Results
Systemic L822429 decreased alcohol self-administration in P-rats but did not affect the lower rates of alcohol self-administration in Wistar rats. Tacr1 expression was elevated in the prefrontal cortex and the amygdala of P-rats. In central amygdala, elevated Tacr1 expression was accompanied by elevated NK1R binding. Central amygdala (but not prefrontal cortex) infusion of L822429 replicated the systemic antagonist effects on alcohol self-administration in P-rats. All P-rats, but only 18% of their founder Wistar population, were CC homozygous for a −1372G/C single nucleotide polymorphism. In silico analysis indicated that the Tacr1 −1372 genotype could modulate binding of the transcription factors GATA-2 and E2F-1. Electromobility shift and luciferase reporter assays suggested that the −1372C allele confers increased transcription factor binding and transcription.
Conclusions
Genetic variation at the Tacr1 locus may contribute to elevated rates of alcohol self-administration, while at the same time increasing sensitivity to NK1R antagonist treatment.
doi:10.1016/j.biopsych.2012.12.027
PMCID: PMC3773538  PMID: 23419547
Alcohol; amygdala; neurokinin; P-rat; self-administration; Substance P
11.  Enhanced GABAergic transmission in the central nucleus of the amygdala of genetically selected Marchigian Sardinian rats: alcohol and CRF effects 
Neuropharmacology  2012;67C:337-348.
The GABAergic system in the central amygdala (CeA) plays a major role in ethanol dependence and the anxiogenic-like response to ethanol withdrawal. Alcohol dependence is associated with increased corticotropin releasing factor (CRF) influence on CeA GABA release and CRF type 1 receptor (CRF1) antagonists prevent the excessive alcohol consumption associated with dependence. Genetically-selected Marchigian Sardinian (msP) rats have an overactive extrahypothalamic CRF1 system, are highly sensitive to stress, and display an innate preference for alcohol. The present study examined differences in CeA GABAergic transmission and the effects of ethanol, CRF and a CRF1 antagonist in msP, Sprague-Dawley, and Wistar rats using an electrophysiological approach. We found no significant differences in membrane properties or mean amplitude of evoked GABAA-inhibitory postsynaptic potentials (IPSPs). However, paired-pulse facilitation (PPF) ratios of evoked IPSPs were significantly lower and spontaneous miniature inhibitory postsynaptic current (mIPSC) frequencies were higher in msP rats, suggesting increased CeA GABA release in msP as compared to Sprague-Dawley and Wistar rats. The sensitivity of spontaneous GABAergic transmission to ethanol (44 mM), CRF (200 nM) and CRF1 antagonist (R121919, 1 μM) was comparable in msP, Sprague Dawley, and Wistar rats. However, a history of ethanol drinking significantly increased the baseline mIPSC frequency and decreased the effects of a CRF1 antagonist in msP rats, suggesting increased GABA release and decreased CRF1 sensitivity. These results provide electrophysiological evidence that msP rats display distinct CeA GABAergic activity as compared to Sprague Dawley and Wistar rats. The elevated GABAergic transmission observed in naïve mSP rats is consistent with the neuroadaptations reported in Sprague Dawley rats after the development of ethanol dependence.
doi:10.1016/j.neuropharm.2012.11.026
PMCID: PMC3562384  PMID: 23220399
amygdala; GABA; alcohol; CRF; CRF1 antagonist; electrophysiology
12.  Behavioral, biological, and chemical perspectives on targeting CRF1 receptor antagonists to treat alcoholism 
Drug and alcohol dependence  2013;128(3):175-186.
Background
Alcohol use disorders are chronic disabling conditions for which existing pharmacotherapies have only modest efficacy. In the present review, derived from the 2012 Behavior, Biology and Chemistry “Translational Research in Addiction” symposium, we summarize the anti-relapse potential of corticotropin-releasing factor type 1 (CRF1) receptor antagonists to reduce negative emotional symptoms of acute and protracted alcohol withdrawal and stress-induced relapse to alcohol seeking.
Methods
We review the biology of CRF1 systems, the activity of CRF1 receptor antagonists in animal models of anxiolytic and antidepressant activity, and experimental findings in alcohol addiction models. We also update the clinical trial status of CRF1 receptor antagonists, including pexacerfont (BMS-562086), emicerfont (GW876008), verucerfont (GSK561679), CP316311, SSR125543A, R121919/NBI30775, R317573/19567470/CRA5626, and ONO-2333Ms. Finally, we discuss the potential heterogeneity and pharmacogenomics of CRF1 receptor pharmacotherapy for alcohol dependence.
Results
The evidence suggests that brain penetrant-CRF1 receptor antagonists have therapeutic potential for alcohol dependence. Lead compounds with clinically desirable pharmacokinetic properties now exist, and longer receptor residence rates (i.e., slow dissociation) may predict greater CRF1 receptor antagonist efficacy. Functional variants in genes that encode CRF system molecules, including polymorphisms in Crhr1 (rs110402, rs1876831, rs242938) and Crhbp genes (rs10055255, rs3811939) may promote alcohol seeking and consumption by altering basal or stress-induced CRF system activation.
Conclusions
Ongoing clinical trials with pexacerfont and verucerfont in moderately to highly severe dependent anxious alcoholics may yield insight as to the role of CRF1 receptor antagonists in a personalized medicine approach to treat drug or alcohol dependence.
doi:10.1016/j.drugalcdep.2012.12.017
PMCID: PMC3596012  PMID: 23294766
corticotropin-releasing factor or hormone receptor antagonist; CRF or CRH; anxiety disorder; major depression; alcohol or ethanol; drug addiction or alcoholism or alcohol dependence or alcohol use disorder or binge drinking; acute or protracted withdrawal or abstinence; treatment or clinical trial; stress-induced relapse or reinstatement or craving
13.  A Novel Brain Penetrant NPS Receptor Antagonist, NCGC00185684, Blocks Alcohol-Induced ERK-Phosphorylation in the Central Amygdala and Decreases Operant Alcohol Self-Administration in Rats 
The Journal of Neuroscience  2013;33(24):10132-10142.
The Neuropeptide S receptor, a Gs/Gq-coupled GPCR expressed in brain regions involved in mediating drug reward, has recently emerged as a candidate therapeutic target in addictive disorders. Here, we describe the in vitro and in vivo pharmacology of a novel, selective and brain penetrant NPSR antagonist with nanomolar affinity for the NPSR, NCGC00185684. In vitro, NCGC00185684 shows biased antagonist properties, and preferentially blocks ERK-phosphorylation over intracellular cAMP or calcium responses to NPS. In vivo, systemic NCGC00185684 blocks alcohol-induced ERK-phosphorylation in the rat central amygdala, a region involved in regulation of alcohol intake. NCGC00185684 also decreases operant alcohol self-administration, and lowers motivation for alcohol reward as measured using progressive ratio responding. These effects are behaviorally specific, in that they are observed at doses that do not influence locomotor activity or reinstatement responding following extinction. Together, these data provide an initial validation of the NPSR as a therapeutic target in alcoholism.
doi:10.1523/JNEUROSCI.4742-12.2013
PMCID: PMC3682378  PMID: 23761908
14.  Stress related neuropeptides and addictive behaviors: Beyond the usual suspects 
Neuron  2012;76(1):192-208.
Summary
Addictive disorders are chronic, relapsing conditions that cause extensive disease burden. Genetic factors partly account for susceptibility to addiction, but environmental factors such as stressful experiences and prolonged exposure of the brain to addictive drugs promote its development. Progression to addiction involves neuroadaptations within neurocircuitry that mediates stress responses, and is influenced by several peptidergic neuromodulators. While corticotropin releasing factor is the prototypic member of this class, recent work has identified several additional stress-related neuropeptides that play an important role in regulation of drug intake and relapse, including the urocortins, nociceptin, substance P and neuropeptide S. Here, we review this emerging literature, discussing to what extent the properties of these neuromodulators are shared or distinct and considering their potential as drug targets.
doi:10.1016/j.neuron.2012.09.026
PMCID: PMC3495179  PMID: 23040815
15.  Correction: Low Vitamin D Status and Suicide: A Case-Control Study of Active Duty Military Service Members 
PLoS ONE  2013;8(9):10.1371/annotation/9af84cbe-5576-4c4b-871c-f7ab0c64b9fd.
doi:10.1371/annotation/9af84cbe-5576-4c4b-871c-f7ab0c64b9fd
PMCID: PMC3782564
16.  Rescue of infralimbic mGluR2 deficit restores control over drug-seeking behavior in alcohol dependence 
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 mGluR2. Also, alcohol dependent rats do not respond to mGluR2/3 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 mGluR2 expression in the infralimbic cortex via viral-mediated gene transfer. Human anterior cingulate cortex from alcoholic patients shows a significant reduction in mGluR2 transcripts compared to control subjects suggesting that mGluR2 loss in the rodent and human cortico-accumbal neurocircuitry may be a major consequence of alcohol dependence and a key pathophysiological mechanism mediating increased propensity to relapse. Normalization of mGluR2 function within this brain circuit may be of therapeutic value.
doi:10.1523/JNEUROSCI.4062-12.2013
PMCID: PMC3711176  PMID: 23407939
17.  Reduced alcohol intake and reward associated with impaired endocannabinoid signaling in mice with a deletion of the glutamate transporter GLAST 
Neuropharmacology  2012;63(2):181-189.
A hyperglutamatergic state has been hypothesized to drive escalation of alcohol intake. This hypothesis predicts that an impairment of glutamate clearance through inactivation of the astrocytic glutamate transporter, GLAST (EAAT1), will result in escalation of alcohol consumption. Here, we used mice with a deletion of GLAST to test this prediction. WT and GLAST KO mice were tested for alcohol consumption using two-bottle free-choice drinking. Alcohol reward was evaluated using conditioned place preference (CPP). Sensitivity to depressant alcohol effects was tested using the accelerating rotarod, alcohol-induced hypothermia, and loss of righting reflex. Extracellular glutamate was measured using microdialysis, and striatal slice electrophysiology was carried out to examine plasticity of the cortico-striatal pathway as a model system in which adaptations to the constitutive GLAST deletion can be studied. Contrary to our hypothesis, GLAST KO mice showed markedly decreased alcohol consumption, and lacked CPP for alcohol, despite a higher locomotor response to this drug. Alcohol-induced ataxia, hypothermia, and sedation were unaffected. In striatal slices from GLAST KO mice, long-term depression (LTD) induced by high frequency stimulation, or by post-synaptic depolarization combined with the L-type calcium channel activator FPL 64176 was absent. In contrast, normal synaptic depression was observed after application of the cannabinoid 1 (CB1) receptor agonist WIN55,212-2. Constitutive deletion of GLAST unexpectedly results in markedly reduced alcohol consumption and preference, associated with markedly reduced alcohol reward. Endocannabinoid signaling appears to be down-regulated upstream of the CB1 receptor as a result of the GLAST deletion, and is a candidate mechanism behind the reduction of alcohol reward observed.
doi:10.1016/j.neuropharm.2012.01.027
PMCID: PMC3372600  PMID: 22342743
glutamate transporter; alcohol; reward; endocannabinoid
18.  Impact of Multiple Types of Childhood Trauma Exposure on Risk of Psychiatric Comorbidity among Alcoholic Inpatients 
Background
This study examined the prevalence of single- and multiple-type childhood trauma exposure (CTE) among alcoholic patients undergoing in-patient detoxification and treatment. The relationships between various types of CTE and lifetime psychiatric comorbidites and suicide attempts were also explored.
Methods
A total of 196 alcoholic inpatients were assessed by Structured Clinical Interview for DSM-IV Axis I Disorders (SCID) and Childhood Trauma Questionnaire (CTQ) for CTE history.
Results
The overall prevalence of CTE of the entire sample was high (55.1%). Specifically, the prevalence of emotional abuse was 21.4%, physical abuse 31.1%, sexual abuse 24.0%, emotional neglect 20.4%, and physical neglect 19.9%. Regarding multiple types of CTE, 31.7% and 18.9% reported at least two and at least three CTE types respectively. Strikingly, among those with at least one positive CTQ category, more than half reported two or more CTE types. A history of emotional abuse increased the risk of mood disorder, in particular major depressive disorder, as well as PTSD. Physical abuse contributed to the prediction of suicide attempts, while sexual abuse was associated with a diagnosis of anxiety disorder, PTSD, and multiple comobidities (e.g., anxiety and mood disorder).The number of reported CTE types or the total scores of the CTQ predicted an increased risk of having single or multiple psychiatric comorbidities as well as suicide attempts.
Conclusions
We observed high rates of a broad range of CTE types and a trend for CTE-specific enhancement of risk for various psychiatric outcomes among alcoholic inpatients. Of notion, a dose-response relationship between number of CTE types and risk of psychiatric comorbidities as well as suicide attempts was found. We suggest a wide range of CTE should be included when exploring the effects of CTE or developing prevention and treatment strategies among alcoholic subjects.
doi:10.1111/j.1530-0277.2011.01695.x
PMCID: PMC3370064  PMID: 22420670
Childhood Trauma; Alcohol Dependence; Psychiatric Comorbidity; Suicide
19.  The kappa opioid receptor antagonist JDTic attenuates alcohol seeking and withdrawal anxiety 
Addiction Biology  2012;17(3):634-647.
The role of kappa-opioid receptors (KOR) in regulation of alcohol-related behaviors is not completely understood. For example, alcohol consumption has been reported to increase following treatment with KOR antagonists in rats, but was decreased in mice with genetic deletion of KOR. Recent studies have further suggested that KOR antagonists may selectively decrease alcohol self-administration in rats following a history of dependence. We assessed the effects of the KOR antagonist JDTic on alcohol self-administration, reinstatement of alcohol seeking induced by alcohol-associated cues or stress, and acute alcohol withdrawal-induced anxiety (“hangover anxiety”). JDTic dose-dependently reversed hangover anxiety when given 48 h prior to testing, a time interval corresponding to the previously demonstrated anxiolytic efficacy of this drug. In contrast, JDTic decreased alcohol self-administration and cue-induced reinstatement of alcohol seeking when administered 2 h prior to testing, but not at longer pretreatment times. For comparison, we determined that the prototypical KOR antagonist nor-BNI can suppress self-administration of alcohol at 2h pretreatment time, mimicking our observations with JDTic. The effects of JDTic were behaviorally specific, as it had no effect on stress-induced reinstatement of alcohol seeking, self administration of sucrose, or locomotor activity. Further, we demonstrate that at a 2h pretreatment time JDTic antagonized the antinociceptive effects of the KOR agonist U50,488H but had no effect on morphine-induced behaviors. Our results provide additional evidence for the involvement of KOR in regulation of alcohol-related behaviors and provide support for KOR antagonists, including JDTic, to be evaluated as medications for alcoholism.
doi:10.1111/j.1369-1600.2012.00455.x
PMCID: PMC3334348  PMID: 22515275
Alcoholism; Dynorphin; Ethanol; Reinstatement; Self-administration; Stress
20.  Medications Development to Treat Alcohol Dependence: A Vision for the Next Decade 
Addiction biology  2012;17(3):513-527.
More than 76 million people worldwide are estimated to have diagnosable Alcohol Use Disorders (AUDs) (alcohol abuse or dependence), making these disorders a major global health problem. Pharmacotherapy offers promising means for treating AUDs, and significant progress has been made in the past 20 years. The U.S. Food and Drug Administration approved three of the four medications for alcoholism in the last two decades. Unfortunately, these medications do not work for everyone, prompting the need for a personalized approach to optimize clinical benefit or more efficacious medications that can treat a wider range of patients, or both. To promote global health, the potential reorganization of the National Institutes of Health (NIH) must continue to support the National Institute on Alcohol Abuse and Alcoholism’s (NIAAA’s) vision of ensuring the development and delivery of new and more efficacious medications to treat AUDs in the coming decade. To achieve this objective, the NIAAA Medications Development Team has identified three fundamental long-range goals: 1) to make the drug development process more efficient; 2) to identify more efficacious medications, personalize treatment approaches, or both, and 3) to facilitate the implementation and adaptation of medications in real-world treatment settings. These goals will be carried out through seven key objectives. This paper describes those objectives in terms of rationale and strategy. Successful implementation of these objectives will result in the development of more efficacious and safe medications, provide a greater selection of therapy options, and ultimately lessen the impact of this devastating disorder.
doi:10.1111/j.1369-1600.2012.00454.x
PMCID: PMC3484365  PMID: 22458728
alcohol; medications; drug development; Alcohol Use Disorders; alcohol dependence
21.  Role of a Genetic Polymorphism in the Corticotropin-Releasing Factor Receptor 1 Gene in Alcohol Drinking and Seeking Behaviors of Marchigian Sardinian Alcohol-Preferring Rats 
Marchigian Sardinian alcohol-preferring (msP) rats exhibit innate preference for alcohol, are highly sensitive to stress and stress-induced alcohol seeking. Genetic analysis showed that over-expression of the corticotropin-releasing factor (CRF) system of msP rats is correlated with the presence of two single nucleotide polymorphisms (SNPs) occurring in the promoter region (position −1836 and −2097) of the CRF1 receptor (CRF1-R) gene. Here we examined whether these point mutations were associated to the innate alcohol preference, stress-induced drinking, and seeking. We have recently re-derived the msP rats to obtain two distinct lines carrying the wild type (GG) and the point mutations (AA), respectively. The phenotypic characteristics of these two lines were compared with those of unselected Wistar rats. Both AA and GG rats showed similar patterns of voluntary alcohol intake and preference. Similarly, the pharmacological stressor yohimbine (0.0, 0.625, 1.25, and 2.5 mg/kg) elicited increased operant alcohol self-administration under fixed and progressive ratio reinforcement schedules in all three lines. Following extinction, yohimbine (0.0, 0.625, 1.25, and 2.5 mg/kg) significantly reinstated alcohol seeking in the three groups. However, at the highest dose this effect was no longer evident in AA rats. Treatment with the CRF1-R antagonist antalarmin (0, 5, 10, and 20 mg/kg) significantly reduced alcohol-reinforced lever pressing in the AA line (10 and 20 mg/kg) while a weaker or no effect was observed in the Wistar and the GG group, respectively. Finally, antalarmin significantly reduced yohimbine-induced increase in alcohol drinking in all three groups. In conclusion, these specific SNPs in the CRF1-R gene do not seem to play a primary role in the expression of the msP excessive drinking phenotype or stress-induced drinking but may be associated with a decreased threshold for stress-induced alcohol seeking and an increased sensitivity to the effects of pharmacological blockade of CRF1-R on alcohol drinking.
doi:10.3389/fpsyt.2013.00023
PMCID: PMC3624086  PMID: 23630503
CRF; SNP; self-administration; msP; yohimbine; relapse
22.  Relationship between liver function and brain shrinkage in patients with alcohol dependence 
Background
Oxidative stress has been proposed as one of the mechanisms of alcohol-induced brain shrinkage and alcohol-induced hepatotoxicity. The study aim was to assess the correlations between liver function and brain volume measurements in patients with alcohol dependence.
Methods
We recruited 124 patients with alcohol dependence and 111 healthy control subjects from National Institute of Health, National Institute on Alcohol Abuse and Alcoholism inpatient alcohol treatment program. Gamma glutamyltransferase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT) as well as hematocrit (Hct) and albumin were assayed shortly after admission. MRI examination was conducted in both groups (after 3-week abstinence in the patient group). We used stepwise linear regression analyses to determine the variables most strongly correlated with brain shrinkage.
Results
Patients with alcohol dependence had lower brain volume (BV), and greater brain shrinkage as measured by gray matter ratio (GMR), white matter ratio (WMR), and brain ratio (BR) and higher CSF ratio (CSFR) compared to their healthy counterparts. Age and sex were significantly correlated with some brain volume measurements in both patient and control groups. Body mass index (BMI) was significantly correlated with CSFR, BR, GMR, and WMR; Hct with CSFR and BR; serum GGT level with BV, CSFR, BR, GMR, and WMF in the patient group. No biological variables were correlated with brain volume indices in the control group. In gender-stratified analysis, age was significantly correlated with brain shrinkage in male patients, but not in female patients. Serum GGT level in male and female patients, Hct in male patients, and AST levels in female patients were significantly correlated with brain shrinkage.
Conclusions
Our results showed that the higher levels of liver function indices, especially GGT, correlated with brain volume shrinkage as measured using CSFR, BR, GMR, and WMR in patients with alcohol dependence, but not in controls. Serum GGT level outweighed aging effect on brain shrinkage in female patients.
doi:10.1111/j.1530-0277.2011.01662.x
PMCID: PMC3262075  PMID: 21995416
alcohol dependence; MRI; liver function; brain shrinkage
23.  Brain-Specific Inactivation of the Crhr1 Gene Inhibits Post-Dependent and Stress-Induced Alcohol Intake, but Does Not Affect Relapse-Like Drinking 
Neuropsychopharmacology  2011;37(4):1047-1056.
Corticotropin-releasing hormone (CRH) and its receptor, CRH receptor-1 (CRHR1), have a key role in alcoholism. Especially, post-dependent and stress-induced alcohol intake involve CRH/CRHR1 signaling within extra-hypothalamic structures, but a contribution of the hypothalamic–pituitary–adrenal (HPA) axis activity might be involved as well. Here we examined the role of CRHR1 in various drinking conditions in relation to HPA and extra-HPA sites, and studied relapse-like drinking behavior in the alcohol deprivation model (ADE). To dissect CRH/CRHR1 extra-HPA and HPA signaling on a molecular level, a conditional brain-specific Crhr1-knockout (Crhr1NestinCre) and a global knockout mouse line were studied for basal alcohol drinking, stress-induced alcohol consumption, deprivation-induced intake, and escalated alcohol consumption in the post-dependent state. In a second set of experiments, we tested CRHR1 antagonists in the ADE model. Stress-induced augmentation of alcohol intake was lower in Crhr1NestinCre mice as compared with control animals. Crhr1NestinCre mice were also resistant to escalation of alcohol intake in the post-dependent state. Contrarily, global Crhr1 knockouts showed enhanced stress-induced alcohol consumption and a more pronounced escalation of intake in the post-dependent state than their control littermates. Basal intake and deprivation-induced intake were unaltered in both knockout models when compared with their respective controls. In line with these findings, CRHR1 antagonists did not affect relapse-like drinking after a deprivation period in rats. We conclude that CRH/CRHR1 extra-HPA and HPA signaling may have opposing effects on stress-related alcohol consumption. CRHR1 does not have a role in basal alcohol intake or relapse-like drinking situations with a low stress load.
doi:10.1038/npp.2011.297
PMCID: PMC3280644  PMID: 22113086
alcoholism; stress; relapse; post-dependent drinking; alcohol deprivation effect (ADE); conditional Crhr1NestinCre-knockout mice; alcohol and alcoholism; biological psychiatry; neurochemistry; animal models; stress; relapse; post-dependent drinking; alcohol deprivation effect; CRH
24.  Acamprosate suppresses magnetic resonance spectroscopy measures of central glutamate in detoxified alcoholics: A randomized controlled experimental medicine study 
Archives of general psychiatry  2010;67(10):1069-1077.
Context
Acamprosate is approved for treatment of alcoholism, but its mechanism of action remains unclear. Animal studies suggest that a persistent hyperglutamatergic state contributes to the pathophysiology of alcoholism, and that acamprosate may exert its actions by intervening in this process. Human translation of these findings is lacking.
Objective
To examine whether acamprosate modulates indices of central glutamate levels in recently abstinent alcohol dependent patients, as measured by proton nuclear magnetic resonance spectroscopy (1H-MRS).
Design
A 4 week, double-blind, placebo-controlled randomized controlled experimental medicine study, with 1H-MRS measures obtained on day 4 and 25.
Setting
NIAAA inpatient research unit at the NIH Clinical Center.
Patients
Thirty three patients who met the DSM-IV criteria for alcohol dependence and were admitted for medically supervised withdrawal from ongoing alcohol use.
Intervention
Four weeks of acamprosate (initial oral loading followed by 1998mg daily) or matched placebo, initiated at the time of admission.
Outcome measures
The main outcome was the glutamate/creatine ratio (Glu) as determined by single voxel 1H-MRS within the anterior cingulate. Exploratory neuroendocrine, biochemical and behavioral outcomes were also collected, as well as safety/tolerability – related measures.
Result
There was a highly significant suppression of Glu over time by acamprosate (time × treatment interaction: F[1, 29]=13.5, p<0.001). Cerebrospinal fluid (CSF) levels of glutamate obtained in a subset of patients 4 weeks into abstinence were uncorrelated with the MRS measures and were unaffected by treatment, but were strongly correlated (R2=0.48, p<0.001) with alcohol dependence severity. Other exploratory outcomes, including repeated Dex/CRH tests, as well as psychiatric ratings were unaffected. Among tolerability measures, gastrointestinal symptoms were significantly greater in acamprosate treated subjects, in agreement with the established profile of acamprosate.
Conclusion
MRS measures of central glutamate are reduced over time when acamprosate is initiated at the onset of alcohol abstinence.
Trial registration
www.clinicaltrials.gov Identifier: NCT00106106
doi:10.1001/archgenpsychiatry.2010.125
PMCID: PMC3583213  PMID: 20921123
acamprosate; alcohol; magnetic resonance spectroscopy; glutamate
25.  Low Vitamin D Status and Suicide: A Case-Control Study of Active Duty Military Service Members 
PLoS ONE  2013;8(1):e51543.
Objective
Considering that epidemiological studies show that suicide rates in many countries are highest in the spring when vitamin D status is lowest, and that low vitamin D status can affect brain function, we sought to evaluate if a low level of 25-hydroxyvitamin D [25(OH)D] could be a predisposing factor for suicide.
Method
We conducted a prospective, nested, case-control study using serum samples stored in the Department of Defense Serum Repository. Participants were previously deployed active duty US military personnel (2002–2008) who had a recent archived serum sample available for analysis. Vitamin D status was estimated by measuring 25(OH) D levels in serum samples drawn within 24 months of the suicide. Each verified suicide case (n = 495) was matched to a control (n = 495) by rank, age and sex. We calculated odds ratio of suicide associated with categorical levels (octiles) of 25(OH) D, adjusted by season of serum collection.
Findings
More than 30% of all subjects had 25(OH)D values below 20 ng/mL. Although mean serum 25(OH)D concentrations did not differ between suicide cases and controls, risk estimates indicated that subjects in the lowest octile of season-adjusted 25(OH)D (<15.5 ng/mL) had the highest risk of suicide, with subjects in the subsequent higher octiles showing approximately the same level of decreased risk (combined odds ratio compared to lowest octile  = 0.49; 95% C.I.: 0.315–0.768).
Conclusions
Low vitamin D status is common in active duty service members. The lowest 25(OH)D levels are associated with an increased risk for suicide. Future studies could determine if additional sunlight exposure and vitamin D supplementation might reduce suicide by increasing 25(OH) D levels.
doi:10.1371/journal.pone.0051543
PMCID: PMC3537724  PMID: 23308099

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