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1.  Nicotine Vapor Inhalation Escalates Nicotine Self-Administration 
Addiction biology  2012;19(4):587-592.
Humans escalate their cigarette smoking over time, and a major obstacle in the field of pre-clinical nicotine addiction research has been the inability to produce escalated nicotine self-administration in rats. In Experiment 1, male Wistar rats were trained to respond for nicotine in 2-hr operant sessions, then exposed to chronic intermittent (12 hrs/day) nicotine vapor and repeatedly tested for nicotine self-administration at 8-12 hrs withdrawal. Rats were tested intermittently on days 1, 3 and 5 of the vapor exposure procedure, then tested on consecutive days 6-15 of nicotine vapor exposure. Rats exhibited transient increases in operant nicotine responding during intermittent testing, regardless of vapor condition, and this responding returned to baseline levels upon resumption of consecutive-days testing (i.e., nicotine deprivation effect). Nicotine vapor-exposed rats then escalated nicotine self-administration relative to both their own baseline (~200% increase) and non-dependent controls (~3x higher). In Experiment 2, rats were exposed or not exposed to chronic intermittent nicotine vapor, then tested for spontaneous and precipitated somatic signs of nicotine withdrawal. Eight hrs following removal from nicotine vapor, rats exhibited robust mecamylamine- precipitated somatic signs of withdrawal. There was a strong correlation between nicotine flow rate and air-nicotine concentration, and the air-nicotine concentrations used in Experiments 1 & 2 resemble concentrations experienced by human smokers. Collectively, these results suggest that chronic intermittent nicotine vapor inhalation produces somatic and motivational signs of nicotine dependence, the latter of which is evidenced by escalation of nicotine self-administration.
PMCID: PMC4516274  PMID: 23240929
Nicotine Dependence; Nicotine Self-Administration; Escalation; Withdrawal; Mecamylamine
2.  Time Course of Cocaine-Induced Behavioral and Neurochemical Plasticity 
Addiction biology  2012;19(4):529-538.
Factors that result in augmented reinstatement, including increased withdrawal period duration and high levels of cocaine consumption, may provide insight into relapse vulnerability. The neural basis of augmented reinstatement may arise from more pronounced changes in plasticity required for reinstatement and/or the emergence of plasticity expressed only during a specific withdrawal period or under specific intake conditions. In this study, we examined the impact of withdrawal period duration and cocaine intake on the magnitude of cocaine-primed reinstatement and extracellular glutamate in the nucleus accumbens, which has been shown to be required for cocaine-primed reinstatement. Rats were assigned to self-administer under conditions resulting in low (2 hr/day; 0.5 mg/kg/infusion, IV) or high (6 hr/day; 1.0 mg/kg/infusion, IV) levels of cocaine intake. After 1, 21, or 60 days of withdrawal, drug seeking and extracellular glutamate levels in the nucleus accumbens were measured before and after a cocaine injection. Cocaine reinstated lever pressing and elevated extracellular glutamate at every withdrawal time point tested, which is consistent with the conclusion that increased glutamatergic signaling in the nucleus accumbens is required for cocaine-induced reinstatement. Interestingly, high-intake rats exhibited augmented reinstatement at every time point tested, yet failed to exhibit higher levels of cocaine-induced increases in extracellular glutamate relative to low-intake rats. Our current data indicate that augmented reinstatement in high-intake rats is not due to relative differences in extracellular levels of glutamate in the nucleus accumbens, but rather may stem from intake-dependent plasticity.
PMCID: PMC3525779  PMID: 22970924
cocaine; glutamate; nucleus accumbens; reinstatement; relapse; self-administration
3.  Dissociable Brain Signatures of Choice Conflict and Immediate Reward Preferences in Alcohol Use Disorders 
Addiction biology  2012;19(4):743-753.
Impulsive delayed reward discounting (DRD) is an important behavioral process in alcohol use disorders (AUDs), reflecting incapacity to delay gratification. Recent work in neuroeconomics has begun to unravel the neural mechanisms supporting DRD, but applications of neuroeconomics in relation to AUDs have been limited. This study examined the neural mechanisms of DRD preferences in AUDs, with emphasis on dissociating activation patterns based on DRD choice type and level of cognitive conflict. Heavy drinking adult males with (n = 13) and without (n = 12) a diagnosis of an AUD completed a monetary DRD task during a functional magnetic resonance imaging scan. Participant responses were coded based on choice type (impulsive vs. restrained) and level of cognitive conflict (easy vs. hard). AUD+ participants exhibited significantly more impulsive DRD decision-making. Significant activation during DRD was found in several decision-making regions, including dorsolateral prefrontal cortex (DLPFC), insula, posterior parietal cortex (PPC), and posterior cingulate. An axis of cognitive conflict was also observed, with hard choices associated with anterior cingulate cortex and easy choices associated with activation in supplementary motor area. AUD+ individuals exhibited significant hyperactivity in regions associated with cognitive control (DLPFC) and prospective thought (PPC) and exhibited less task-related deactivation of areas associated with the brain's default network during DRD decisions. This study provides further clarification of the brain systems supporting DRD in general and in relation to AUDs.
PMCID: PMC3871988  PMID: 23231650
Alcohol use disorders; delay discounting; neuroeconomics
4.  Expression of HIV gp120 protein increases sensitivity to the rewarding properties of methamphetamine in mice 
Addiction biology  2012;19(4):593-605.
Methamphetamine abuse and human immunodeficiency virus (HIV) infection induce neuropathological changes in corticolimbic brain areas involved in reward and cognitive function. Little is known about the combined effects of methamphetamine and HIV infection on cognitive and reward processes. The HIV/gp120 protein induces neurodegeneration in mice, similar to HIV-induced pathology in humans. We investigated the effects of gp120 expression on associative learning, preference for methamphetamine and non-drug reinforcers, and sensitivity to the conditioned rewarding properties of methamphetamine in transgenic (tg) mice expressing HIV/gp120 protein (gp120-tg). gp120-tg mice learned the operant response for food at the same rate as non-tg mice. In the two-bottle choice procedure with restricted access to drugs, gp120-tg mice exhibited greater preference for methamphetamine and saccharin than non-tg mice, whereas preference for quinine was similar between genotypes. Under conditions of unrestricted access to methamphetamine, the mice exhibited a decreased preference for increasing methamphetamine concentrations. However, male gp120-tg mice showed a decreased preference for methamphetamine at lower concentrations than non-tg male mice. gp120-tg mice developed methamphetamine-induced conditioned place preference at lower methamphetamine doses compared with non-tg mice. No differences in methamphetamine pharmacokinetics were found between genotypes. These results indicate that gp120-tg mice exhibit no deficits in associative learning or reward/motivational function for a natural reinforcer. Interestingly, gp120 expression resulted in increased preference for methamphetamine and a highly palatable non-drug reinforcer (saccharin) and increased sensitivity to methamphetamine-induced conditioned reward. These data suggest that HIV-positive individuals may have increased sensitivity to methamphetamine, leading to high methamphetamine abuse potential in this population.
PMCID: PMC3608835  PMID: 23252824
Conditioned place preference; food-maintained responding; oral self-administration; pharmacokinetics; quinine; saccharin
5.  Association of Smoking with Mu- Opioid Receptor Availability Before and During Naltrexone Blockade in Alcohol-Dependent Subjects 
Addiction biology  2012;19(4):733-742.
Persons with a history of alcohol dependence are more likely to use tobacco and to meet criteria for nicotine dependence compared to social drinkers or nondrinkers. The high levels of comorbidity of nicotine and alcohol use and dependence are thought to be related to interactions between nicotinic, opioid and dopamine receptors in mesolimbic regions. The current study examined whether individual differences in regional mu-opioid receptor (MOR) availability were associated with tobacco use, nicotine dependence, and level of nicotine craving in 25 alcohol dependent (AD) subjects. AD subjects completed an inpatient protocol, which included medically supervised alcohol withdrawal, monitored alcohol abstinence, transdermal nicotine maintenance (21 mg/day), and Positron Emission Tomography (PET) imaging using the MOR agonist [11C]-carfentanil (CFN) before (basal scan) and during treatment with 50 mg/day naltrexone (naltrexone scan). Subjects who had higher scores on the Fagerström Nicotine Dependence Test had significantly lower basal scan binding potential (BPND) across mesolimbic regions including the amygdala, cingulate, globus pallidus, thalamus and insula. Likewise, the number of cigarettes per day was negatively associated with basal scan BPND in mesolimbic regions Higher nicotine craving was significantly associated with lower BPND in amygdala, globus pallidus, putamen, thalamus and ventral striatum. Although blunted during naltrexone treatment, the negative association was maintained for nicotine dependence and cigarettes per day, but not for nicotine craving. These findings suggest that intensity of cigarette smoking and severity of nicotine dependence symptoms are systematically related to reduced BPND across multiple brain regions in AD subjects.
PMCID: PMC3638047  PMID: 23252742
mu opioid receptors; nicotine; alcoholism; dependence; PET imaging; humans
6.  Hippocampal neurogenesis protects against cocaine-primed relapse 
Addiction biology  2012;19(4):562-574.
Accumulating evidence demonstrates a functional role for the hippocampus in mediating relapse to cocaine-seeking behavior and extinction-induced inhibition of cocaine seeking, and dentate gyrus neurogenesis in the hippocampus may have a role. Here, we tested the hypothesis that disruption of normal hippocampal activity during extinction alters relapse to cocaine-seeking behavior as a function of dentate gyrus neurogenesis. Adult rats were trained to self-administer cocaine on a fixed-ratio schedule, followed by extinction and cocaine-primed reinstatement testing. Some rats received low frequency stimulation (LFS; 2 Hz for 25 min) after each extinction session in the dorsal or ventral hippocampal formation. All rats received an injection of the mitotic marker 5-bromo-2′-deoxyuridine (BrdU) to label developing dentate gyrus neurons during self-administration, as well as before or after extinction and LFS. We found that LFS during extinction did not alter extinction behavior, but enhanced cocaine-primed reinstatement. Cocaine self-administration reduced levels of twenty-four day old BrdU cells and dentate gyrus neurogenesis, which was normalized by extinction. LFS during extinction prevented extinction-induced normalization of dentate gyrus neurogenesis and potentiated cocaine-induced reinstatement of drug seeking. LFS inhibition of extinction-induced neurogenesis was not due to enhanced cell death, revealed by quantification of activated caspase3 labeled cells. These data suggest that LFS during extinction disrupts hippocampal networking via disrupting neurogenesis and also strengthens relapse-like behaviors. Thus, newly born dentate gyrus neurons during withdrawal and extinction learning facilitate hippocampal networking that mediates extinction-induced inhibition of cocaine seeking and may play a key role in preventing relapse.
PMCID: PMC3620729  PMID: 23278919
Low-frequency stimulation; Self-administration; Dentate gyrus; BrdU; Ki-67; activated caspase3
7.  GDNF is a novel ethanol-responsive gene in the VTA: Implications for the development and persistence of excessive drinking 
Addiction biology  2013;19(4):623-633.
Glial cell line-derived neurotrophic factor (GDNF) is a potent inhibitor of ethanol consumption and relapse (Carnicella et al., 2008; Carnicella and Ron, 2009; Carnicella et al., 2009c; Barak et al., 2011a), and GDNF heterozygous knockout mice display increased reward sensitivity to ethanol, and consume more ethanol after a period of abstinence, than their wild-type littermates (Carnicella et al., 2009b). Here, we tested whether ethanol alters GDNF expression in the ventral tegmental area (VTA; GDNF’s site of action) and/or the nucleus accumbens (NAc; the main source of GDNF), and if so, determine the role of the endogenous growth factor in the regulation of ethanol consumption. Systemic administration of ethanol increased GDNF expression and protein levels in the VTA, but not the NAc. Additionally, GDNF levels were elevated after an ethanol-drinking session in rats that consumed ethanol in the intermittent-access two-bottle choice procedure for 1 week, but not 7 weeks. Deprivation following 7 weeks of excessive ethanol intake reduced GDNF levels, while a short ethanol binge drinking period following deprivation upregulated GDNF expression. Importantly, knockdown of GDNF within the VTA using adenovirus expressing short hairpin RNA facilitated the escalation of ethanol drinking by ethanol-naïve rats, but not by rats with a history of excessive ethanol consumption. These results suggest that during initial ethanol-drinking experiences, GDNF in the VTA is increased and protects against the development of excessive ethanol intake. However, the growth factor’s protective response to ethanol breaks down after protracted excessive ethanol intake and withdrawal, resulting in persistent, excessive ethanol consumption.
PMCID: PMC3626755  PMID: 23298382
Addiction; ethanol; GDNF; growth factor; VTA
Addiction biology  2013;19(4):687-699.
The adolescent hippocampus is highly vulnerable to alcohol-induced damage, which could contribute to their increased susceptibility to alcohol use disorders. Altered adult hippocampal neurogenesis represents one potential mechanism by which alcohol (ethanol) affects hippocampal function. Based on the vulnerability of the adolescent hippocampus to alcohol-induced damage, and prior reports of long-term alcohol-induced effects on adult neurogenesis, we predicted adverse effects on adult neurogenesis in the adolescent brain following abstinence from alcohol dependence. Thus, we examined neurogenesis in adolescent male rats during abstinence following a four-day binge model of alcohol dependence. Bromodeoxyuridine and Ki67 immunohistochemistry revealed a 2.2-fold increase in subgranular zone cell proliferation after 7 days of abstinence. Increased proliferation was followed by a 75% increase in doublecortin expression and a 56% increase in surviving bromodeoxyuridine-labeled cells 14 and 35 days post-ethanol exposure, respectively. The majority of newborn cells in ethanol and control groups co-localized with NeuN, indicating a neuronal phenotype and therefore a 1.6-fold increase in hippocampal neurogenesis during abstinence. Although these results mirror the magnitude of reactive neurogenesis described in adult rat studies, ectopic bromodeoxyuridine and doublecortin positive cells were detected in the molecular layer and hilus of adolescent rats displaying severe withdrawal symptoms, an effect that has not been described in adults. The presence of ectopic neuroblasts suggests that a potential defect exists in the functional incorporation of new neurons into the existing hippocampal circuitry for a subset of rats. Age-related differences in functional incorporation could contribute to the increased vulnerability of the adolescent hippocampus to ethanol.
PMCID: PMC3844012  PMID: 23844726
Alcoholism; Ethanol; Withdrawal
9.  Using genetic information from candidate gene and genome-wide association studies in risk prediction for alcohol dependence 
Addiction biology  2013;19(4):708-721.
Family-based and genome-wide association studies (GWAS) of alcohol dependence (AD) have reported numerous associated variants. The clinical validity of these variants for predicting AD compared to family history information has not been reported. Using the Collaborative Study on the Genetics of Alcoholism (COGA) and the Study of Addiction: Genes and Environment (SAGE) GWAS samples, we examined the aggregate impact of multiple single nucleotide polymorphisms (SNPs) on risk prediction. We created genetic sum scores by adding risk alleles associated in discovery samples, and then tested the scores for their ability to discriminate between cases and controls in validation samples. Genetic sum scores were assessed separately for SNPs associated with AD in candidate gene studies and SNPs from GWAS analyses that met varying p-value thresholds. Candidate gene sum scores did not exhibit significant predictive accuracy. Family history was a better classifier of case-control status, with a significant area under the receiver operating characteristic curve (AUC) of 0.686 in COGA and 0.614 in SAGE. SNPs that met less stringent p-value thresholds of 0.01 to 0.50 in GWAS analyses yielded significant AUC estimates, ranging from mean estimates of 0.549 for SNPs with p < 0.01 to 0.565 for SNPs with p < 0.50. This study suggests that SNPs currently have limited clinical utility, but there is potential for enhanced predictive ability with better understanding of the large number of variants that might contribute to risk.
PMCID: PMC3664249  PMID: 23362995
clinical validity; genetic risk prediction; polygenic risk score; psychiatric genetic counseling; receiver operating characteristic curve analysis
10.  Opioid system in the medial prefrontal cortex mediates binge-like eating 
Addiction biology  2013;19(4):652-662.
Binge eating disorder is an addiction-like disorder characterized by excessive food consumption within discrete periods of time. This study was aimed at understanding the role of the opioid system within the mPFC in the consummatory and motivational aspects of binge-like eating. For this purpose, we trained male rats to obtain either a sugary, highly palatable diet (Palatable rats) or a chow diet (Chow rats) for 1h a day. We then evaluated the effects of the opioid receptor antagonist, naltrexone, given either systemically or site-specifically into the NAcc or the mPFC on a fixed ratio 1 (FR1) and a progressive ratio schedule of reinforcement for food. Finally, we assessed the expression of the genes preOpioMelanoCortin (POMC), Pro-Dynorphin (PDyn), and Pro-Enkephalin (PEnk), coding for the opioids peptides in the NAcc and the mPFC in both groups. Palatable rats rapidly escalated their intake by 4 times. Naltrexone, when administered systemically and into the NAcc, reduced FR1 responding for food and motivation to eat under a progressive ratio in both Chow and Palatable rats; conversely, when administered into the mPFC, the effects were highly selective for binge eating rats. Furthermore, we found a two-fold increase in POMC and a ~50% reduction in PDyn gene expression in the mPFC of Palatable rats, when compared to control rats; however, no changes were observed in the NAcc. Our data suggest that neuroadaptations of the opioid system in the mPFC occur following intermittent access to highly palatable food, which may be responsible for the development of binge-like eating.
PMCID: PMC3664255  PMID: 23346966
addiction; binge eating disorder; nucleus accumbens; opioid; palatability; prefrontal cortex
11.  PTSD Risk Associated with a Functional DRD2 Polymorphism in Heroin Dependent Cases and Controls is Limited to Amphetamine Dependent Individuals 
Addiction biology  2013;19(4):700-707.
Posttraumatic stress disorder (PTSD), a pathologic response to severe stress, is a common comorbid disorder in substance dependent individuals. Evidence from twin studies suggests PTSD is moderately heritable. Genetic association studies to date have reported a limited number of replicated findings. We conducted a candidate gene association study in trauma-exposed individuals within the Comorbidity and Trauma Study’s sample (1343 heroin dependent cases and 406 controls from economically-disadvantaged neighborhoods). After data cleaning, the 1430 SNPs retained for analyses provided coverage of 72 candidate genes and included additional SNPs for which association was previously reported as well as 30 ancestry informative markers. We found a functional DRD2 promoter polymorphism (rs12364283) to be most highly associated with PTSD liability [OR 1.65 (1.27–2.15); p= 1.58 × 10−4]; however, this association was not significant with a stringent Bonferroni correction for multiple comparisons. The top hits include SNPs from other dopaminergic system genes: DRD2 DRD3, TH, and DBH. Additional analyses revealed the association involving rs12364283 is largely limited to amphetamine dependent individuals. Substantial risk is observed in amphetamine dependent individuals with at least one copy of this SNP [OR 2.86 (1.92–4.27); p=2.6 × 10−7]. Further analyses do not support extensive mediation of PTSD risk via self-reported impulsivity (BIS total score). These findings suggest roles for impairment in inhibitory control in the pathophysiology of PTSD and raise questions about stimulant use in certain populations (e.g., those in combat).
PMCID: PMC3883923  PMID: 23647975
amphetamine dependence; association study; DRD2; PTSD
12.  Repeated stress exposure causes strain-dependent shifts in the behavioral economics of cocaine in rats 
Addiction biology  2014;20(2):297-301.
Cocaine-experienced Wistar and Wistar Kyoto (WKY) rats received four daily repeated forced swim stress sessions (R-FSS), each of which preceded 4-hour cocaine self-administration sessions. Twenty-four hours after the last swim stress, cocaine valuation was assessed during a single-session threshold procedure. Prior exposure to R-FSS significantly altered cocaine responding in Wistar, but not WKY, rats. Behavioral economic analysis of responding revealed that the Wistar rats that had received R-FSS exhibited an increase in the maximum price that they were willing to pay for cocaine (Pmax). Pre-treatment with the long-lasting kappa opioid receptor (KOR) antagonist norbinaltorphimine prevented the stress-induced increase in Pmax. Thus, R-FSS exposure had strain-dependent effects on cocaine responding during the threshold procedure, and the stress effects on cocaine valuation exhibited by Wistar, but not WKY, required intact KOR signaling.
PMCID: PMC4448949  PMID: 24919534
Addiction; cocaine; kappa opioid receptor; self-administration; stress
13.  Glutamate transporter GLT-1 mediates N-acetylcysteine inhibition of cocaine reinstatement 
Addiction biology  2014;20(2):316-323.
Both pre-clinical and clinical studies indicate that N-acetylcysteine (NAC) may be useful in treating relapse to addictive drug use. Cocaine self-administration in rats reduces both cystine-glutamate exchange and glutamate transport via GLT-1 in the nucleus accumbens, and NAC treatment normalizes these two glial processes critical for maintaining glutamate homeostasis. However, it is not known if one or both of these actions by NAC is needed to inhibit relapse to cocaine seeking. To determine whether the restoration of GLT-1 and/or cystine-glutamate exchange is required for NAC to inhibit cue-induced reinstatement of cocaine seeking, we utilized the rat self-administration/extinction/reinstatement model of cocaine relapse. Rats were pre-treated in the nucleus accumbens with vivo-morpholino anti-sense oligomers targeting either GLT-1 or xCT (catalytic subunit of the cystine-glutamate exchanger) overlapping with daily NAC administration during extinction (100 mg/kg, i.p. for the last 5 days). Rats then underwent cue-induced reinstatement of active lever pressing in the absence of NAC, to determine if preventing NAC-induced restoration of one or the other protein was sufficient to block the capacity of chronic NAC to inhibit reinstatement. The vivo-morpholino suppression of xCT reduced cystine-glutamate exchange but did not affect NAC-induced reduction of reinstated cocaine seeking. In contrast, suppressing NAC-induced restoration of GLT-1 not only prevented NAC from inhibiting reinstatement, but augmented the capacity of cues to reinstate cocaine seeking. We hypothesized that the increased reinstatement after inhibiting NAC induction of GLT-1 resulted from increased extracellular glutamate, and show that augmented reinstatement is prevented by blocking mGluR5. Restoring GLT-1, not cystine-glutamate exchange, is a key mechanism whereby daily NAC reduces cue-induced cocaine reinstatement.
PMCID: PMC4437505  PMID: 24612076
Cocaine; cystine-glutamate exchange; glutamate; glutamate transport; mGluR5; nucleus accumbens; N-acetylcysteine; reinstatement
14.  Relation between corticosterone and fear-related behavior in mice selectively bred for high or low alcohol preference 
Addiction biology  2013;19(4):663-675.
Blunted cortisol responses to stress or trauma have been linked with genetic (familial) risk for both alcoholism and post-traumatic stress disorder (PTSD). Mouse lines selectively bred for high (HAP) or low (LAP) alcohol preference may be a relevant model of genetic risk for co-morbid alcoholism and PTSD in humans. HAP mice show greater fear-potentiated startle (FPS), a model used to study PTSD, than LAP mice. The relation between corticosterone (CORT) and FPS behavior was explored in four experiments. Naïve male and female HAP2 and LAP2 mice received fear-conditioning or control treatments, and CORT levels were measured before and immediately after fear-conditioning or FPS testing. In two other experiments, HAP2 mice received CORT (1.0, 5.0 or 10.0 mg/kg) or a glucocorticoid receptor antagonist (mifepristone; 25.0 and 50.0 mg/kg) 30 minutes before fear conditioning. HAP2 mice exposed to fear conditioning and to control foot shock exposures showed lower CORT after the fear-conditioning and FPS testing sessions than LAP2 mice. A trend toward higher FPS was seen in HAP2 mice pretreated with 10.0 mg/kg CORT, and CORT levels were the lowest in this group, suggesting negative feedback inhibition of CORT release. Mifepristone did not alter FPS. Overall, these results are consistent with data in humans and rodents indicating that lower cortisol/CORT levels after stress are associated with PTSD/PTSD-like behavior. These findings in HAP2 and LAP2 mice suggest that a blunted CORT response to stress may be a biological marker for greater susceptibility to develop PTSD in individuals with increased genetic risk for alcoholism.
PMCID: PMC4426868  PMID: 23331637
Alcohol; corticosterone; fear-potentiated startle; genetic correlation; mifepristone; PTSD
15.  NrCAM regulating neural systems and addiction related behaviors 
Addiction biology  2012;19(3):343-353.
We have previously shown that a haplotype associated with decreased NrCAM expression in brain is protective against addiction vulnerability for polysubstance abuse in humans and that Nrcam knockout mice do not develop conditioned place preferences for morphine, cocaine, or amphetamine. In order to gain insight into NrCAM involvement in addiction vulnerability, which may involve specific neural circuits underlying behavioral characteristics relevant to addiction, we evaluated several behavioral phenotypes in Nrcam knockout mice. Consistent with a potential general reduction in motivational function, Nrcam knockout mice demonstrated less curiosity for novel objects and for an unfamiliar conspecific, showed also less anxiety in the zero maze. Nrcam heterozygote knockout mice reduced alcohol preference and buried fewer marbles in home cage. These observations provide further support for a role of NrCAM in substance abuse including alcoholism vulnerability, possibly through its effects on behavioral traits that may affect addiction vulnerability, including novelty seeking, obsessive compulsion and responses to aversive or anxiety-provoking stimuli. Additionally, in order to prove glutamate homeostasis hypothesis of addiction, we analyzed glutamatergic molecules regulated by NRCAM. Glutaminase appears to be involved in NrCAM-related molecular pathway in two different tissues from human and mouse. An inhibitor of the enzyme, PLG, treatment produced, at least, some of the phenotypes of mice shown in alcohol preference and in anxiety-like behavior. Thus, NrCAM could affect addiction-related behaviors via at least partial modulation of some glutamatargic pathways and neural function in brain.
PMCID: PMC3470748  PMID: 22780223
behavior; cell adhesion molecule; glutamate
16.  Brain region- and sex-specific alterations in DAMGO-stimulated [35S]GTPγS binding in mice with Oprm1 A112G 
Addiction biology  2012;19(3):354-361.
The A118G single nucleotide polymorphism (SNP) of the human µ fopioid receptor (MOPR) gene (OPRM1) was associated with heightened dopamine release by alcohol intake, better treatment outcome for nicotine and alcohol addiction and reduced analgesic responses to morphine. A mouse model that possesses the equivalent substitution (A112G) in the oprm1 gene was generated to delineate the mechanisms of the impact of the SNP. Mice homozygous for the G allele (G/G) displayed lower morphine-induced antinociception than A/A mice, similar to humans. In this study, we examined whether A112G SNP affected MOPR-mediated G protein activation in the mouse model. We compared A/A and G/G mice in the MOPR selective agonist DAMGO-stimulated [35S]GTPγS binding in brain regions by autoradiography. When the data of males and females were combined, G/G mice exhibited lower DAMGO-stimulated [35S]GTPγS binding in the VTA than A/A mice, in accord with previously reported reduced morphine-induced hyperactivity and locomotor sensitization in G/G mice. In the NAc core, female G/G mice displayed lower DAMGO-stimulated [35S]GTPγS binding than female A/A mice, which is consistent with previously reported deficiency in morphine-induced conditioned place preference in female G/G mice. In G/G mice, males showed higher DAMGO-stimulated [35S]GTPγS binding than females in the cingulate cortex, CPu, NAc core, thalamus and amygdala. Thus A112G SNP affects DAMGO-stimulated [35S]GTPγS binding in region- and sex-specific manners.
PMCID: PMC3864546  PMID: 22862850
A118G; mu opioid receptor; single nucleotide polymorphism; [35S]GTPγS binding autoradiography
17.  Reduced Expression of α-Synuclein in Alcoholic Brain: Influence of SNCA-Rep1 Genotype 
Addiction biology  2012;19(3):509-515.
α-Synuclein has recently been implicated in the pathophysiology of alcohol abuse due to its role in dopaminergic neurotransmission. In these studies, genetic variability in the α-synuclein gene influences its expression which may contribute to susceptibility to chronic alcohol abuse. Real-time PCR was used to quantify α-synuclein mRNA expression in autopsy samples of human dorsolateral prefrontal cortex. Because of the association between length of the α-synuclein-repeat 1 microsatellite marker and expression levels of the gene, this marker was genotyped in a Caucasian sample of 126 controls and 117 alcoholics using capillary gel electrophoresis. The allele and genotype frequencies of α-synuclein-repeat 1 marker differed significantly between alcoholics and controls. Alcoholics had greater frequencies of the shortest allele found (267 bp). The shortest allele of the α-synuclein-repeat 1 marker was associated with decreased expression of α-synuclein in prefrontal cortex. Individuals with at least one copy of the 267 bp allele were more likely to exhibit an alcohol abuse phenotype. These results suggest that individuals with the 267 bp allele may be at increased risk of developing alcoholism and that genetic variation at the α-synuclein-repeat 1 locus may influence α-synuclein expression in the prefrontal cortex.
PMCID: PMC3543514  PMID: 22974310
human; mRNA; post-mortem; prefrontal cortex; SNCA-Rep1
18.  Role of ventral subiculum in context-induced reinstatement of heroin seeking in rats 
Addiction biology  2012;19(3):338-342.
In rats, re-exposure to heroin-paired contexts after extinction of lever-responding in a different context reinstates heroin seeking. Previous reports indicate that ventral hippocampus/Ca1 region plays a critical role in cocaine-, cue-, and context-induced reinstatement of cocaine seeking. Here, we examined whether ventral subiculum, the output region of ventral hippocampus, is involved in context-induced reinstatement of heroin seeking. We found that reversible inactivation of ventral, but not posterior Ca1, with the GABAergic agonists muscimol+baclofen decreased context-induced reinstatement of heroin seeking. Our findings, together with previous studies on cocaine seeking, indicate a critical role of ventral subiculum in context-induced relapse across drug classes.
PMCID: PMC3622847  PMID: 23231571
Conditioned cues; drug environment; heroin self-administration; hippocampus; muscimol+baclofen; relapse; reinstatement
19.  Opioid sensitivity in mice selectively bred to consume or not consume methamphetamine 
Addiction biology  2012;19(3):370-379.
There has been little investigation of genetic factors and associated mechanisms that influence risk for development of methamphetamine (MA) dependence. Selectively bred mouse lines that exhibit high (MAHDR) or low (MALDR) levels of MA intake in a two-bottle choice MA drinking (MADR) procedure provide a genetic tool for this purpose. These lines were used to determine whether opioid sensitivity and MA intake are genetically associated, since opioid mediated pathways influence some effects of MA. . Sensitivity to the analgesic effects of the μ-opioid receptor (MOP-r) agonist fentanyl (0.05, 0.1, 0.2, 0.4 mg/kg) was examined using two acute thermal tests (hot plate and tail flick) and one chronic pain test (magnesium sulfate abdominal constriction). Locomotor stimulant responses to fentanyl (0.05, 0.1, 0.2, 0.4 mg/kg) and morphine (10, 20, 30 mg/kg) were also examined. In addition, MADR was measured in the progenitor strains (C57BL/6J (B6), DBA/2J (D2)) of the F2 population from which the selected lines were generated. The MADR lines did not differ in sensitivity to the analgesic effects of fentanyl; however, MALDR mice exhibited greater locomotor activation than MAHDR mice to both fentanyl and morphine. D2 mice consumed more MA than B6 mice. The line differences for MA consumption and morphine activation recapitulated B6 and D2 strain differences for these two traits, but not strain differences previously found for opioid analgesic responses. These results support a negative genetic correlation between MA consumption and sensitivity to the stimulant effects of opioids and suggest the involvement of MOP-r regulated systems in MA intake.
PMCID: PMC3796126  PMID: 23145527
amphetamine; μ-opioid receptor; selective breeding
20.  Neural network activation during a stop-signal task discriminates cocaine-dependent from non-drug-abusing men 
Addiction biology  2012;19(3):427-438.
Cocaine dependence is defined by a loss of inhibitory control over drug use behaviors, mirrored by measurable impairments in laboratory tasks of inhibitory control. The current study tested the hypothesis that deficits in multiple sub-processes of behavioral control are associated with reliable neural processing alterations that define cocaine addiction. While undergoing fMRI, 38 cocaine-dependent men and 27 healthy control men performed a stop-signal task of motor inhibition. An independent component analysis (ICA) on fMRI time courses identified task-related neural networks attributed to motor, visual, cognitive and affective processes. The statistical associations of these components with five different stop-signal task conditions were selected for use in a linear discriminant analysis to define a classifier for cocaine addiction from a subsample of 26 cocaine-dependent men and 18 controls. Leave-one-out cross validation accurately classified 89.5% (39/44; chance accuracy = 26/44 = 59.1%) of subjects (with 84.6% (22/26) sensitivity and 94.4% (17/18) specificity. The remaining 12 cocaine-dependent and 9 control men formed an independent test sample, for which accuracy of the classifier was 81.9% (17/21; chance accuracy = 12/21 = 57.1%) with 75% (9/12) sensitivity and 88.9% (8/9) specificity. The cocaine addiction classification score was significantly correlated with a measure of impulsiveness as well as the duration of cocaine use for cocaine-dependent men. The results of this study support the ability of a pattern of multiple neural network alterations associated with inhibitory motor control to define a binary classifier for cocaine addiction.
PMCID: PMC3726576  PMID: 23231419
classification; fMRI; cocaine dependence; inhibitory motor control; stop-signal task
21.  Long-lasting, experience-dependent alcohol preference in Drosophila 
Addiction biology  2013;19(3):392-401.
To understand the molecular and neural mechanisms underlying alcohol addiction, many models ranging from vertebrates to invertebrates have been developed. In Drosophila melanogaster, behavioral paradigms from assaying acute responses to alcohol, to behaviors more closely modeling addiction, have emerged in recent years. However, both the CAFÉ assay, similar to a 2-bottle choice consumption assay, as well as conditioned odor preference, where ethanol is used as the reinforcer, are labor intensive and have low throughput. To address this limitation, we have established a novel ethanol consumption preference assay, called FRAPPÉ, which allows for fast, high throughput measurement of consumption in individual flies, using a fluorescence plate reader. We show that naïve flies do not prefer to consume ethanol, but various pre-exposures, such as ethanol vapor or voluntary ethanol consumption, induce ethanol preference. This ethanol-primed preference is long lasting and is not driven by calories contained in ethanol during the consumption choice. Our novel experience-dependent model of ethanol preference in Drosophila – a highly genetically tractable organism – therefore recapitulates salient features of human alcohol abuse and will facilitate the molecular understanding of the development of alcohol preference.
PMCID: PMC3991769  PMID: 24164972
Addiction; alcohol; Drosophila; genetics; model organism; self-administration
22.  The Cerebellum and Addiction: Insights Gained from Neuroimaging Research 
Addiction biology  2014;19(3):317-331.
Although cerebellar alterations have been consistently noted in the addiction literature, the pathophysiology of this link remains unclear. The cerebellum is commonly classified as a motor structure, but human functional neuroimaging along with clinical observations in cerebellar stroke patients and anatomical tract tracing in non-human primates suggest its involvement in cognitive and affective processing. A comprehensive literature search on the role of the cerebellum in addiction was performed. This review article (1) considers the potential role of the cerebellum in addiction, (2) summarizes the cerebellar structural alterations linked to addiction, (3) presents the functional neuroimaging evidence linking the cerebellum with addiction, and (4) proposes a model for addiction that underscores the role of the cerebellum. The data implicate the cerebellum as an intermediary between motor and reward, motivation and cognitive control systems, as all are relevant etiologic factors in addiction. Furthermore, consideration of these findings could contribute to deeper and more sophisticated insights into normal reward and motivational function. The goal of this review is to spread awareness of cerebellar involvement in addictive processes, and to suggest a preliminary model for its potential role.
PMCID: PMC4031616  PMID: 24851284
PET; MRI; craving; opioids; cocaine; marijuana; alcohol
23.  Functional imaging of an alcohol-Implicit Association Test (IAT) 
Addiction biology  2013;19(3):467-481.
This research assessed activation in neural substrates involved in implicit associative processes through the imaging (functional magnetic resonance imaging) of an alcohol-Implicit Association Test (IAT) focused on positive outcomes of alcohol use. Comparisons involved 17 heavy and 19 light drinkers, ranging in age from 18 to 22, during compatible and incompatible association task trials. Behaviorally, a significant IAT effect was found with heavy drinkers showing stronger positive implicit associations toward alcohol use than light drinkers. Imaging data revealed heavy drinkers showed greater activity during compatible trials relative to incompatible trials in the left putamen and insula while no significant difference in activity between conditions was found in the light drinkers. Light drinkers showed significantly more activity in the left orbital frontal cortex during both compatible and incompatible trials than heavy drinkers, and the dorsolateral prefrontal cortex was engaged more in both light and heavy drinkers during incompatible trials relative to compatible trials. Further, within-group analyses showed significant amygdala activity along with the putamen and insula among heavy drinkers during compatible trials relative to incompatible trials. These results are consistent with a dual process framework of appetitive behaviors proposing that (1) implicit associations underlying habit are mediated through neural circuitry dependent on the striatum, and (2) controlled behaviors are mediated through neural circuitry more dependent on the prefrontal cortex. This is the first study to evaluate the neural mechanisms elicited by an alcohol-IAT, providing an additional step toward increasing understanding of associative habit processes and their regulatory influence over addictive behaviors.
PMCID: PMC4116809  PMID: 23822813
alcohol-IAT; associative processes; dorsal striatum; fMRI; habit; implicit associations
24.  Subregion-specific Role of Glutamate Receptors in the Nucleus Accumbens on Drug Context-induced Reinstatement of Cocaine-seeking Behavior in Rats 
Addiction biology  2011;17(2):287-299.
The functional integrity of the nucleus accumbens (NAC) core and shell is necessary for contextual cocaine-seeking behavior in the reinstatement animal model of drug relapse; however, the neuropharmacological mechanisms underlying this phenomenon are poorly understood. The present study evaluated the contribution of metabotropic glutamate receptor subtype 1 (mGluR1) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor populations to drug context-induced reinstatement of cocaine-seeking behavior. Rats were trained to lever press for un-signaled cocaine infusions in a distinct context followed by extinction training in a different context. Cocaine-seeking behavior (non-reinforced active lever pressing) was then assessed in the previously cocaine-paired and extinction contexts after JNJ16259685 (mGluR1 antagonist: 0.0, 0.6, or 30 pg/0.3 μl/hemisphere) or CNQX (AMPA/kainate receptor antagonist: 0.0, 0.03, or 0.3 μg/0.3 μl/hemisphere) administration into the NAC core, medial or lateral NAC shell, or the ventral caudate-putamen (vCPu, anatomical control). JNJ16259685 or CNQX in the NAC core dose-dependently impaired contextual cocaine-seeking behavior relative to vehicle. Conversely, CNQX, but not JNJ16259685, in the lateral or medial NAC shell attenuated, whereas CNQX or JNJ16259685 in vCPu failed to inhibit, this behavior. The manipulations failed to alter instrumental behavior in the extinction context, general motor activity, or food-reinforced instrumental behavior in control experiments. Thus, glutamate-mediated changes in drug context-induced motivation for cocaine involve distinct neuropharmacological mechanisms within the core and shell subregions of the NAC, with the stimulation of mGlu1 and AMPA/kainate receptors in the NAC core and the stimulation of AMPA/kainate, but not mGlu1, receptors in the NAC shell being necessary for this phenomenon.
PMCID: PMC4384648  PMID: 21521425
Cocaine; Context; Ionotropic glutamate receptor; Metabotropic glutamate receptor; Nucleus accumbens; Reinstatement
25.  Group I metabotropic glutamate receptor-mediated activation of PKC gamma in the nucleus accumbens core promotes the reinstatement of cocaine seeking 
Addiction biology  2014;20(2):285-296.
Emerging evidence indicates that type I metabotropic glutamate receptors (mGluRs) in the nucleus accumbens play a critical role in cocaine seeking. The present study sought to determine the role of accumbens core mGluR1, mGluR5 and protein kinase C (PKC) in cocaine priming-induced reinstatement of drug seeking. Here, we show that intra-accumbens core administration of the mGluR1/5 agonist DHPG (250 μM) promoted cocaine seeking in rats. Consistent with these results, administration of an mGluR1 (50.0 μM YM 298198) or mGluR5 (9.0 μM MPEP) antagonist directly into the accumbens core prior to a priming injection of cocaine (10 mg/kg) attenuated the reinstatement of drug seeking. mGluR1/5 stimulation activates a signaling cascade including PKC. Intracore microinjection of PKC inhibitors (10 μM Ro 31–8220 or 30.0 μM chelerythrine) also blunted cocaine seeking. In addition, cocaine priming-induced reinstatement of drug seeking was associated with increased phosphorylation of PKCγ, but not PKCα or PKCβII, in the core. There were no effects of pharmacological inhibition of mGluR1, mGluR5 or PKC in the accumbens core on sucrose seeking. Together, these findings indicate that mGluR1 and mGluR5 activation in the accumbens core promotes cocaine seeking and that these effects are reinforcer specific. Furthermore, stimulation of mGluR1 and mGluR5 in the accumbens core may regulate cocaine seeking, in part, through activation of PKCγ.
PMCID: PMC4380181  PMID: 24506432
Addiction; glutamate; psychostimulant; relapse; self-administration; striatum

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