Neuropeptide Y (NPY) and Protein Kinase A (PKA) have been implicated in neurobiological responses to ethanol. We have previously reported that mutant mice lacking normal production of the RIIβ subunit of PKA (RIIβ−/− mice) show enhanced sensitivity to the locomotor stimulant effects of ethanol and increased behavioral sensitization relative to littermate wild-type RIIβ+/+ mice. We now report that RIIβ−/− mice also show increased NPY immunoreactivity in the nucleus accumbens (NAc) core and the ventral striatum relative to RIIβ+/+ mice. These observations suggest that elevated NPY signaling in the NAc and/or striatum may contribute to the increased sensitivity to ethanol-induced behavioral sensitization that is characteristic of RIIβ−/− mice. Consistently, NPY−/− mice failed to display ethanol-induced behavioral sensitization that was evident in littermate NPY+/+ mice. To more directly examine the role of NPY in the locomotor stimulant effects of ethanol, we infused a recombinant adeno-associated virus (rAAV) into the region of the NAc core of DBA/2J mice. The rAAV-FIB-NPY13-36 vector expresses and constitutively secretes the NPY fragment NPY13-36 (a selective Y2 receptor agonist) from infected cells in vivo. Mice treated with the rAAV-FIB-NPY13-36 vector exhibited reduced expression of ethanol-induced behavioral sensitization compared to mice treated with a control vector. Taken together, the current data provide the first evidence that NPY signaling in the NAc core and the Y2 receptor modulate ethanol-induced behavioral sensitization.

Environmental enrichment (EE) during a period of forced abstinence attenuates incentive motivational effects of cocaine-paired stimuli. Here we examined whether EE during forced abstinence from cocaine self-administration would prevent time-dependent increases in cue-elicited cocaine-seeking behavior (i.e., the incubation effect). Rats were trained to self-administer cocaine, which was paired with light/tone cues, for 15 days while living in isolated conditions (IC). Controls received yoked saline infusions. Subsequently, rats were assigned to live in either continued IC or EE for either 1 or 21 days of forced abstinence prior to a test for cocaine-seeking behavior. During testing, responding resulted only in presentation of the light/tone cues. Contrary to our prediction, cocaine-seeking behavior increased over time regardless of living condition during abstinence; however, EE attenuated cocaine-seeking behavior relative to IC regardless of length of abstinence. Brains were harvested and trunk blood was collected immediately after the 60-min test and later assayed. Results indicated that short-term EE elevated hippocampal brain-derived neurotrophic factor and reduced plasma corticosterone compared to IC. Furthermore, 21 days of EE during forced abstinence prevented increases in the cue-elicited amygdala phosphorylated extracellular signal-regulated kinase expression that was observed in IC rats. These findings suggest that EE attenuates incentive motivational effects of cocaine cues through a mechanism other than preventing the incubation effect, perhaps involving reduction of stress and neural activity in response to cocaine-paired cues during acute withdrawal.

Alcohol activates orosensory circuits that project to motivationally relevant limbic forebrain areas that control appetite, feeding and drinking. To date, limited data exists regarding the contribution of chemosensory-derived ethanol reinforcement to ethanol preference and consumption. Measures of taste reactivity to intra-orally infused ethanol have not found differences in initial orofacial responses to alcohol between alcohol-preferring (P) and – nonpreferring (NP) genetically selected rat lines. Yet, in voluntary intake tests P rats prefer highly-concentrated ethanol upon initial exposure, suggesting an early sensory-mediated attraction. Here, we directly compared self-initiated chemosensory responding for alcohol and prototypic sweet, bitter, and oral trigeminal stimuli among selectively bred P, NP, and non-selected Wistar (WI) outbred lines to determine whether differential sensory responsiveness to ethanol and its putative sensory components are phenotypically associated with genetically-influenced alcohol preference. Rats were tested for immediate short-term lick responses to alcohol (3–40%), sucrose (0.01–1 M), quinine (0.01–3 mM) and capsaicin (0.003–1 mM) in a brief-access assay designed to index orosensory-guided behavior. P rats exhibited elevated short-term lick responses to both alcohol and sucrose relative to NP and WI lines across a broad range of concentrations of each stimulus and in the absence of blood alcohol levels that would produce significant postabsorptive effects. There was no consistent relationship between genetically-mediated alcohol preference and orosensory avoidance of quinine or capsaicin. These data indicate that enhanced initial chemosensory attraction to ethanol and sweet stimuli are phenotypes associated with genetic alcohol preference and are considered within the framework of downstream activation of oral appetitive reward circuits.

Cocaine-induced neuroadaptation of stress-related circuitry and increased access to cocaine each putatively contribute to the transition from cocaine use to cocaine dependence. The present study tested the hypothesis that rats receiving extended versus brief daily access to cocaine would exhibit regional differences in levels of the stress-regulatory neuropeptide corticotropin-releasing factor (CRF). A secondary goal was to explore how CRF levels change in relation to the time since cocaine self-administration. Male Wistar rats acquired operant self-administration of cocaine and were assigned to receive daily long access (6 hours/day, LgA, n = 20) or short access (1 hour/day, ShA, n = 18) to intravenous cocaine self-administration (fixed ratio 1, ~0.50 mg/kg/infusion). After at least 3 weeks, tissue CRF immunoreactivity was measured at one of three timepoints: pre-session, post-session or 3 hours post-session. LgA, but not ShA, rats showed increased total session and first-hour cocaine intake. CRF immunoreactivity increased within the dorsal raphe (DR) and basolateral, but not central, nucleus of the amygdala (BLA, CeA) of ShA rats from pre-session to 3 hours post-session. In LgA rats, CRF immunoreactivity increased from pre-session to 3 hours post-session within the CeA and DR but tended to decrease in the BLA. LgA rats showed higher CRF levels than ShA rats in the DR and, pre-session, in the BLA. Thus, voluntary cocaine intake engages stress-regulatory CRF systems of the DR and amygdala. Increased availability of cocaine promotes greater tissue CRF levels in these extrahypothalamic brain regions, changes associated here with a model of cocaine dependence.

Stress both contributes to the development of ethanol dependence and is a consequence of dependence. However, the complexity of physiological interactions between activation of the hypothalamic-pituitary-adrenal (HPA) axis and ethanol itself is not well delineated. Emerging evidence derived from examination of corticotropin releasing factor systems and glucocorticoid receptor systems in ethanol dependence suggests a role for pharmacological manipulation of the HPA axis in attenuating ethanol intake, though it is not clear how activation of the HPA axis may promote ethanol dependence or contribute to the neuroadaptative changes that accompany the development of dependence and the severity of ethanol withdrawal. This review examines the role that glucocorticoids, in particular, have in promoting ethanol-associated plasticity of glutamatergic synapses by influencing expression of endogenous linear polyamines and polyamine-sensitive polypeptide subunits of N-methyl-d-aspartate (NMDA)-type glutamate receptors. We provide evidence that interactions among glucocorticoid systems, polyamines and NMDA receptors are highly relevant to both the development of ethanol dependence and to behavioral and neuropathological sequelae associated with ethanol withdrawal. Examination of these issues is likely to be of critical importance not only in further elucidating the neurobiology of HPA axis dysregulation in ethanol dependence, but also with regard to identification of novel therapeutic targets that may be exploited in the treatment of ethanol dependence.

Genetic research on cocaine dependence may help clarify our understanding of the disorder as well as provide insights for effective treatment. Since endocannabinoid signaling and dopamine neurotransmission have been shown to be involved with drug reward, genes related to these systems are plausible candidates for susceptibility to cocaine dependence. The cannabinoid receptor 1 (CB1) protein regulates both the endocannabinoid and dopaminergic neurobiological systems, and polymorphisms in the cannabinoid receptor gene, CNR1, have been previously been associated with substance dependence. In this study, we attempt to replicate a finding associating CNR1 with cocaine dependence in African Americans. Cocaine dependent individuals (n=883) and unaffected controls (n=334) of African descent were genotyped for two single nucleotide polymorphisms (SNPs) in the CB1 gene (rs6454674, rs806368). We observed a significant difference in genotype frequencies between cases and controls for both SNPs (p≤0.05). This study confirms the association between variants in the CNR1 and cocaine dependence. However, considering the substantial co-morbidity of cocaine dependence with other drugs of abuse, additional studies are necessary to determine whether polymorphisms in CNR1 induce a general susceptibility to substance dependence or are specific to cocaine addiction.

Voluntary oral ethanol consumption in rodents is generally limited by strong taste-aversion in these species. Historically, this has been overcome by combining ethanol with a sweetener, typically sucrose or saccharine, and then slowly ‘fading’ away the sweetener. While useful in most instances, this approach has not proven as successful for some inbred strains of mice (e.g. DBA/2J) despite consistent evidence in the literature that these same strains express strong conditioned place preference for intraperitoneal- or intragastric-administered ethanol. Importantly, DBA/2J mice express a polymorphism in a ‘sweet’ taste receptor subunit gene that reduces the potency of sweet substances in these mice. We hypothesized that the presence of this polymorphism might help explain the contrasting behavioral findings of weak voluntary oral ethanol consumption following sucrose-fade yet robust conditioned place-preference for ethanol in this strain. To test this, we compared ethanol consumption initiated by either a ‘traditional’ sucrose-fade or a fade from an alternative tastant, monosodium glutamate (MSG). We found that in both C57BL/6J and DBA/2J mice the MSG-fade produced robust increases in home-cage ethanol consumption relative to the traditional sucrose-fade. This increased ethanol intake following MSG-fade was evident across a range of ethanol concentrations. Our findings suggest the potential utility of the MSG-fade to establish stable voluntary oral ethanol consumption in mice, particularly ethanol ‘non-preferring’ strains like DBA/2J, and lend additional support to the notion that ethanol consumption in DBA/2J mice is limited by pronounced taste aversion.

Previous studies of the association of the C17T polymorphism of the mu opiate receptor gene with substance dependence compared cases with substance dependence to controls and usually found no significant association. However, the studies were limited by small sample size - no study had more than 12 subjects with the TT genotype, a genotype that is rare in white and Asian subjects. Moreover, drug use is not dichotomous but follows a spectrum from non-use to modest, intermittent use, to use several times daily. We asked whether the Kreek-McHugh-Schluger-Kellogg (KMSK) scales for alcohol, cocaine, opiates, and tobacco that quantify substance use during the time of a subject's maximal use might be more sensitive measures than dichotomous outcomes. We administered the KMSK scales and completed C17T genotyping on 1009 HIV-infected and 469 HIV-uninfected women in The Women's Interagency HIV Study (WIHS), an ongoing study of HIV in women. Forty-two of 697 African-American, 1 of 182 Hispanic, and none of 161 white women had the TT genotype. KMSK cocaine, alcohol, and tobacco scores were significantly higher in African-American women with the TT genotype (p =0.008, 0.0001, and 0.006 respectively) but opiate scores were not. Ordinal regression models controlling for HIV-serostatus, age, education, and income had odds ratios for the TT genotype for predicting alcohol, tobacco, cocaine, and opiates scores of 2.1 (p = 0.02), 2.4 (p = 0.0004), 2.0 (p = 0.03), and 1.9 (p = 0.07). We conclude that the TT genotype of OPRM1 may increase the risk of substance use and abuse.

Chronic cocaine exposure in both rodents and humans increases regional brain mu-opioid receptor (mOR) binding potential, suggesting that cocaine users might have an altered response to mOR agonists. We evaluated the response to IV carfentanil (a selective mOR agonist) in 23 cocaine users (mean [SD] age 33.8 [4.0] years, 83% men) who underwent PET scanning with [C-11]-carfentanil (44.7 [19.5] ng/kg) while housed on a closed research ward and 15 healthy non-drug-using controls (43.9 [14.2] years, 80% men) scanned (49.5 [12.6] ng/kg) as outpatients. Cocaine users had used for 8.7 [4.3] years and on 73 [22] % of days in the two weeks prior to PET scanning. Common adverse effects associated with mOR agonists (nausea, dizziness, headache, vomiting, itchiness) were assessed by self-report (5-point Likert scales) during and for 90 minutes after the scans. Cocaine users were significantly less likely than controls to report any symptom (30.4% vs. 60%) and had fewer total symptoms (0.43 [0.73] vs. 1.1 [1.0]) during scans, even after statistically controlling for age and carfentanil dose. These differences were also present after the scans and at repeat scans done after about one week or 12 weeks of monitored cocaine abstinence. In a larger group of cocaine users and separate controls, there was no significant group difference in carfentanil half-life, suggesting that the observed difference was pharmacodynamically, rather than pharmacokinetically, based. These findings suggest that cocaine users are less responsive than healthy controls to mOR agonist adverse effects, despite having increased regional brain mOR binding potential.

Analysis of mouse brain gene expression, using strains that differ in alcohol consumption, provided a number of novel candidate genes that potentially regulate alcohol consumption. We selected six genes [beta-2-microglobulin (B2m), cathepsin S (Ctss), cathepsin F (Ctsf), interleukin 1 receptor antagonist (Il1rn), CD14 molecule (Cd14) and interleukin 6 (Il6)] for behavioral validation using null mutant mice. These genes are known to be important for immune responses but were not specifically linked to alcohol consumption by previous research. Null mutant mice were tested for ethanol intake in three tests: 24 hr two-bottle choice, limited access two-bottle choice and limited access to one bottle of ethanol. Ethanol consumption and preference were reduced in all the null mutant mice in the 24 hr two-bottle choice test, the test that was the basis for selection of these genes. No major differences were observed in consumption of saccharin in the null mutant mice. Deletion of B2m, Ctss, Il1rn, Cd14 and Il6 also reduced ethanol consumption in the limited access two bottle choice test for ethanol intake; with the Il1rn and Ctss null mutants showing reduced intake in all three tests (with some variation between males and females). These results provide the most compelling evidence to date that global gene expression analysis can identify novel genetic determinants of complex behavioral traits. Specifically, they suggest a novel role for neuroimmune signaling in regulation of alcohol consumption.

Dual process models of addiction emphasize the importance of implicit (automatic) cognitive processes in the development and maintenance of substance use behavior. Although genetic influences are presumed to be relevant for dual process models, few studies have evaluated this possibility. The current study examined two polymorphsims with functional significance for alcohol use behavior (COMT Val158Met and ALDH2*2) in relation to automatic alcohol cognitions and tested additive and interactive effects of genotype and implicit cognitions on drinking behavior. Participants were college students (n = 69) who completed Implicit Association Tasks (IATs) designed to assess two classes of automatic drinking motives (enhancement motives and coping motives). Genetic factors did not show direct associations with IAT measures, however, COMT and ALDH2 moderated associations of implicit coping motives with drinking outcomes. Interaction effects indicated that associations of implicit motives with drinking outcomes were strongest in the context of genetic variants associated with relatively higher risk for alcohol use (COMT Met and ALDH2*1). Associations of genotype with drinking behavior were observed for ALDH2 but not COMT. These findings are consistent with the possibility that genetic risk or protective factors could potentiate or mitigate the influence of reflexive cognitive processes on drinking behavior, providing support for the evaluation of genetic influences in the context of dual process models of addiction.

The present study was undertaken to examine whether genetically predetermined differences in components of the endocannabinoid system were present in the brain of Sardinian alcohol-preferring (sP) and Sardinian alcohol-non preferring (sNP) rats, a pair of rat lines selectively bred for opposite alcohol preference. The effects of acquisition and maintenance of alcohol drinking, alcohol withdrawal, and alcohol re-exposure on the endocannabinoid system was also assessed in the striatum of sP rats. The findings revealed significantly higher density of the CB1 receptors and levels of CB1 receptor mRNA, CB1 receptor-mediated G-protein coupling, and endocannabinoids in the cerebral cortex, hippocampus and striatum of alcohol-naive sP rats than sNP rats. A significantly lower expression of mFAAH enzyme was evident in the hippocampus of alcohol-naive sP rats. Alcohol drinking (during both acquisition and maintenance phases) in sP rats resulted in a significant reduction in striatal CB1 receptor-mediated G-protein coupling whereas alcohol withdrawal attenuated this effect. Alcohol consumption was also associated with markedly increased levels of endocannabinoids in the striatum. Co-administration of the CB1 receptor antagonist, rimonabant (SR141716A) reduced alcohol intake, and reversed alcohol-induced changes in CB1 receptor-mediated G-protein activation. These findings provided a new insight into a potential genetic basis of excessive alcohol consumption, suggesting innate differences in the endocannabinoid system might be associated with higher alcohol preference in sP rats. The data also indicate a modulation of CB1 receptor-mediated signaling following alcohol consumption, and further strengthen the potential of the endocannabinoid system as a target for the treatment of alcohol related behaviors.

Alcoholism is a devastating condition that represents a progression from initial alcohol use to dependence. Although most individuals are capable of consuming alcohol in a limited fashion, the development of alcohol dependence in a subset of individuals is often associated with negative emotional states (including anxiety and depression). Since the alleviation of this negative motivational state via excessive alcohol consumption often becomes a central goal of alcoholics, the transition from initial use to dependence is postulated to be associated with a transition from positive to negative reinforcement mechanisms. Vasopressin is a neuropeptide known to potentiate the effects of CRF on the HPA axis, and emerging evidence also suggests a role for centrally located vasopressin acting on V1b receptors in the regulation of stress- and anxiety-like behaviors in rodents. The present study determined state-dependent alterations in vasopressin/V1bR signaling in an animal model of ethanol dependence. The V1bR antagonist SSR149415 dose-dependently reduced excessive levels of ethanol self-administration observed in dependent animals without affecting the limited levels of ethanol drinking in non-dependent animals. Ethanol self-administration reduced V1b receptor levels in the basolateral amygdala of non-dependent animals, a neuroadaptation that could theoretically facilitate the positive reinforcing effects of alcohol. In contrast, V1bR levels were seemingly restored in ethanol-dependent rats, a switch that may in part underlie a transition from positive to negative reinforcement mechanisms with dependence. Together, our data suggest a key role for vasopressin/V1bR signaling in the transition to ethanol dependence.

Alcohol dependence (AD) is an important contributory factor to the global burden of disease. The etiology of AD involves both environmental and genetic factors, and the disorder has a heritability of around 50%. The aim of the present study was to identify susceptibility genes for AD by performing a genome-wide association study (GWAS). The sample comprised 1,333 male in-patients with severe DSM-IV AD and 2,168 controls. These included 487 patients and 1,358 controls from a previous GWAS study by our group. All individuals were of German descent. Single marker tests and a polygenic score based analysis to assess the combined contribution of multiple markers with small effects were performed. The SNP rs1789891, which is located between the ADH1B and ADH1C genes, achieved genome-wide significance (p=1.27E–8; OR=1.46). Other markers from this region were also associated with AD, and conditional analyses indicated that these made a partially independent contribution. The SNP rs1789891 is in complete linkage disequilibrium with the functional Arg272Gln variant (p=1.24E–7, OR=1.31) of the ADH1C gene, which has been reported to modify the rate of ethanol oxidation to acetaldehyde in vitro. A polygenic score based approach produced a significant result (p=9.66E–9). This is the first GWAS of AD to provide genome-wide significant support for the role of the ADH gene cluster and to suggest a polygenic component to the etiology of AD. The latter result suggests that many more AD susceptibility genes still await identification.

Three experiments used the Intragastric Alcohol Consumption (IGAC) procedure to examine effects of variations in passive ethanol exposure on withdrawal and voluntary ethanol intake in two inbred mouse strains, C57BL/6J (B6) and DBA/2J (D2). Experimental treatments were selected to induce quantitative differences in ethanol dependence and withdrawal severity by: (a) varying the periodicity of passive ethanol exposure (3, 6 or 9 infusions/day), (b) varying the dose per infusion (Low, Medium or High), and (c) varying the duration of passive exposure (3, 5 or 10 days). All experiments included control groups passively exposed to water. B6 mice generally self-infused more ethanol than D2 mice, but passive ethanol exposure increased IGAC in both strains, with D2 mice showing larger relative increases during the first few days of ethanol access. Bout data supported the characterization of B6 mice as sippers and D2 mice as gulpers. Three larger infusions per day produced a stronger effect on IGAC than six or nine smaller infusions, especially in D2 mice. Increased IGAC was strongly predicted by cumulative ethanol dose and intoxication during passive exposure in both strains. Withdrawal during the passive exposure phase was also a strong predictor of increased IGAC in D2 mice. However, B6 mice showed little withdrawal, precluding analysis of its potential role. Overall, these data support the hypothesis that dependence-induced increases in IGAC are jointly determined by two processes that might vary across genotypes: (a) tolerance to aversive post-absorptive ethanol effects, and (b) negative reinforcement (i.e., alleviation of withdrawal by self-administered ethanol).

The mammalian target of rapamycin (mTOR) is a serine-threonine kinase that controls global protein synthesis, in part, by modulating translation initiation, a rate-limiting step for many mRNAs. Previous studies implicate mTOR in regulating stimulant-induced sensitization and antidepressive-like behavior in rodents, as well as drug craving in abstinent heroin addicts. To determine if signaling downstream of mTOR is affected by repeated cocaine administration in reward-associated brain regions, and if inhibition of mTOR alters cocaine-induced behavioral plasticity, C57BL/6J mice received 4 intraperitoneal (IP) injections of 15 mg/kg cocaine and levels of phosphorylated P70S6 kinase and ribosomal S6 protein - two translational regulators directly downstream of mTOR - were analyzed by immunoblotting across several brain regions. Cocaine place-preference and locomotor sensitization were elicited by 4 pairings of cocaine with a distinct environment and the effects of mTOR inhibition were assessed by pretreating the mice with 10 mg/kg rapamycin, 1 hr prior to (a) each saline/cocaine conditioning session, (b) a post-conditioning test or (c) a test for locomotor sensitization conducted at 3 weeks withdrawal. While systemic pretreatment with 10 mg/kg rapamycin during conditioning failed to alter the development of a cocaine place-preference or locomotor sensitization, pretreatment prior to the post-conditioning test attenuated the expression of the place-preference. Additionally, rapamycin pretreatment prior to a cocaine challenge 3 weeks post-conditioning blocked the expression of the sensitized locomotor response. These ndings suggest a role for mTOR activity, and perhaps translational control, in the expression of cocaine-induced place preference and locomotor sensitization.

Alcohol is thought to contribute to an increase in risk-taking behavior, but the neural correlates underlying this effect are not well understood. In this study, participants were given intravenous alcohol or placebo while undergoing functional magnetic resonance imaging (fMRI) and playing a risk-taking game. The game allowed us to examine the neural response to choosing a safe or risky option, anticipating outcome and receiving feedback. We found that alcohol increased risk-taking behavior, particularly among participants who experienced more stimulating effects of alcohol. fMRI scans demonstrated that alcohol increased activation in the striatum to risky compared with safe choices and dampened the neural response to notification of both winning and losing throughout the caudate, thalamus and insula. This study suggests that alcohol may increase risk-taking behavior by both activating brain regions involved in reward when a decision is made, and dampening the response to negative and positive feedback.

Craving is a significant factor which can lead to relapse during smoking quit attempts. Attempts to resist urges to smoke during cue-elicited craving have been shown to activate regions in the brain associated with decision-making, anxiety regulation, and visual processing. In this study, 32 treatment-seeking, nicotine-dependent smokers viewed blocks of smoking and neutral cues alternating with rest periods during MRI scanning in a 3T Siemens scanner. While viewing cues or control images, participants were instructed either to “allow yourself to crave” or “resist craving.” Data were analyzed with FSL 4.1.5, focused on the smoking cues versus neutral cues contrast, using cluster thresholding (Z>2.3 and corrected cluster threshold of p=0.05) at the individual and group levels. During the Crave Condition activation was seen the left anterior cingulated cortex (LACC), medial prefrontal cortex, left middle cingulate gyrus, bilateral posterior cingulated gyrus and bilateral precuneus, areas associated with attention, decision-making and episodic memory. The LACC and areas of the prefrontal cortex associated with higher executive functioning were activated during the Resist Condition. No clear distinctions between group crave and resist analyses as a whole were seen without taking into account specific strategies used to resist the urge to smoke, supporting the idea that craving is associated with some degree of resisting the urge to smoke, and trying to resist is almost always accompanied by some degree of craving. Different strategies for resisting, such as distraction, activated different regions. Understanding the underlying neurobiology of resisting craving to smoke may identify new foci for treatments.

We investigated possible alterations of pharmacologically-isolated, evoked GABAA inhibitory postsynaptic potentials (eIPSPs) and miniature GABAA inhibitory postsynaptic currents (mIPSCs) in the rat central amygdala (CeA) elicited by acute application of μ-opioid receptor (MOR) agonists (DAMGO and morphine; 1 μM) and by chronic morphine treatment with morphine pellets. The acute activation of MORs decreased the amplitudes of eIPSPs, increased paired-pulse facilitation (PPF) of eIPSPs and decreased the frequency (but not the amplitude) of mIPSCs in a majority of CeA neurons, suggesting that acute MOR-dependent modulation of this GABAergic transmission is mediated predominantly via presynaptic inhibition of GABA release. We observed no significant changes in the membrane properties, eIPSPs, PPF or mIPSCs of CeA neurons during chronic morphine treatment compared to CeA of naïve or sham rats. Superfusion of the MOR antagonist CTOP (1 μM) increased the mean amplitude of eIPSPs in a majority of CeA neurons to the same degree in both naïve/sham and morphine-treated rats, suggesting a tonic activation of MORs in both conditions. Superfusion of DAMGO decreased eIPSP amplitudes and the frequency of mIPSCs equally in both naïve/sham and morphine-treated rats but decreased the amplitude of mIPSCs only in morphine treated rats, an apparent postsynaptic action. Our combined findings suggest the development of tolerance of the CeA GABAergic system to inhibitory effects of acute activation of MORs on presynaptic GABA release and possible alteration of MOR-dependent postsynaptic mechanisms that may represent important neuroadaptations of the GABAergic and MOR systems during chronic morphine treatment.

Alcoholism is characterized by a progressive loss of control over ethanol intake. The purpose of this study was to identify transcriptional changes selectively associated with excessive ethanol drinking in dependent mice, as opposed to non-dependent mice maintaining a stable voluntary consumption or mice solely undergoing forced intoxication. We measured expression levels of 106 candidate genes in the extended amygdala, a key brain structure for the development of drug addiction. Cluster analysis identified 17 and 15 genes selectively induced or repressed, respectively, under conditions of excessive drinking. These genes belong to signaling pathways involved in neurotransmission and transcriptional regulation.

Onset of alcohol use at an early age increases the risk for later alcohol dependence. We investigated the role of the glucocorticoid receptor (GR) gene (NR3C1) in onset of alcohol use and abuse in 14-year-old adolescents (n = 4534). Several NR3C1 polymorphisms were associated with onset of alcohol drinking or drunkenness at this age. Strongest associations were observed in females, with one marker (rs244465) remaining significant after correction for multiple testing (Padj = 0.0067; odds ratio = 1.7, for drunkenness). Our data provide the first evidence that GR modulates initiation of alcohol abuse and reveal a polymorphism that might contribute to susceptibility to addiction.

The genetic, epigenetic and environmental factors may influence the risk for neuropsychiatric disease through their effects on gene transcription. Mechanistically, these effects may be integrated through regulation of methylation of CpG dinucleotides overlapping with single-nucleotide polymorphisms (SNPs) associated with a disorder. We addressed this hypothesis by analyzing methylation of prodynorphin (PDYN) CpG-SNPs associated with alcohol dependence, in human alcoholics. Postmortem specimens of the dorsolateral prefrontal cortex (dl-PFC) involved in cognitive control of addictive behavior were obtained from 14 alcohol-dependent and 14 control subjects. Methylation was measured by pyrosequencing after bisulfite treatment of DNA. DNA binding proteins were analyzed by electromobility shift assay. Three PDYN CpG-SNPs associated with alcoholism were found to be differently methylated in the human brain. In the dl-PFC of alcoholics, methylation levels of the C, non-risk variant of 3′-untranslated region (3′-UTR) SNP (rs2235749; C > T) were increased, and positively correlated with dynorphins. A DNA-binding factor that differentially targeted the T, risk allele and methylated and unmethylated C allele of this SNP was identified in the brain. The findings suggest a causal link between alcoholism-associated PDYN 3′-UTR CpG-SNP methylation, activation of PDYN transcription and vulnerability of individuals with the C, non-risk allele(s) to develop alcohol dependence.

Alcohol and nicotine use disorders are often treated as separate diseases, despite evidence that approximately 80–90% of alcohol dependent individuals are also heavy smokers. Both nicotine and ethanol have been shown to interact with neuronal nicotinic acetylcholine receptors (nAChRs), suggesting these receptors are a common biological target for the effects of nicotine and ethanol in the brain. There are few studies that have examined the effects of co-administered nicotine and ethanol on the activity of nAChRs in rodents. In the present study, we show that Sprague-Dawley rats, a strain often used for nicotine studies but not as often for voluntary ethanol intake studies, will consume 20% ethanol using both the intermittent-access two-bottle-choice and operant self-administration models without the need for sucrose fading. We show that nicotine (0.2mg/kg and 0.8mg/kg, s.c.) significantly increases operant 20% ethanol self-administration and varenicline (2mg/kg, s.c), a partial agonist at nAChRs, significantly decreases operant ethanol self-administration and nicotine-induced increases in ethanol self-administration. This suggests that nAChRs play an important role in increasing ethanol self-administration and that varenicline may be an efficacious treatment for alcohol and nicotine co-dependencies.

The identification of genes that contribute to polygenic (complex) behavioral phenotypes is a key goal of current genetic research. One approach to this goal is to combine gene expression information with genetic information, i.e., to map chromosomal regions that regulate gene expression levels. This approach has been termed “genetical genomics”, and, when used in conjunction with the identification of genomic regions (QTLs) that regulate the complex physiological trait under investigation, provides a strong basis for candidate gene discovery. In this paper, we describe the implementation of the genetical genomic/phenotypic approach to identify candidate genes for sensitivity to the analgesic effect of morphine in BXD recombinant inbred mice. Our analysis was performed “in silico”, using an online interactive resource called PhenoGen (http://phenogen.ucdenver.edu). We describe in detail the use of this resource, which identified a set of candidate genes, some of whose products regulate the cellular localization and activity of the mu opiate receptor. The results demonstrate how PhenoGen can be used to identify a novel set of genes that can be further investigated for their potential role in pain, morphine analgesia and/or morphine tolerance.

Several recent studies suggest functional and molecular interactions between striatal adenosine A2A and cannabinoid CB1 receptors. Here we demonstrate that A2A receptors selectively modulate reinforcing effects of cannabinoids. We studied effects of A2A receptor blockade on the reinforcing effects of delta-9-tetrahydrocannabinol (THC) and the endogenous CB1 receptor ligand anandamide under a fixed-ratio (FR) schedule of intravenous drug injection in squirrel monkeys. A low dose of the selective adenosine A2A receptor antagonist MSX-3 (1 mg/kg) caused downward shifts of THC and anandamide dose-response curves. In contrast, a higher dose of MSX-3 (3 mg/kg) shifted THC and anandamide dose-response curves to the left. MSX-3 did not modify cocaine or food-pellet self-administration. Also, MSX-3 neither promoted reinstatement of extinguished drug-seeking behavior nor altered reinstatement of drug-seeking behavior by non-contingent priming injections of THC. Finally, using in-vivo microdialysis in freely-moving rats, a behaviorally active dose of MSX-3 significantly counteracted THC-induced, but not cocaine-induced, increases in extracellular dopamine levels in the nucleus accumbens shell. The significant and selective results obtained with the lower dose of MSX-3 suggest that adenosine A2A antagonists acting preferentially at presynaptic A2A receptors might selectively reduce reinforcing effects of cannabinoids that lead to their abuse. However, the appearance of potentiating rather than suppressing effects on cannabinoid reinforcement at the higher dose of MSX-3 would likely preclude the use of such a compound as a medication for cannabis abuse. Adenosine A2A antagonists with more selectivity for presynaptic versus postsynaptic receptors could be potential medications for treatment of cannabis abuse.