Assessment of Abuse-Related Discriminative Stimulus Effects of Nicotine Aerosol in Rodents.

The rapid increase in e-cigarette use highlights the importance of developing relevant, predictive animal models exploring their potential health implications. The goal of the present study was to examine the abuse-related effects of brief, repeated e-cigarette aerosol exposures in rodents modeling human e-cigarette user behavior. We evaluated the discriminative stimulus effects of brief, repeated puffs of inhaled nicotine in rats that had been trained to discriminate injected nicotine from saline. Locomotor activity measurement following exposure to injected and aerosolized nicotine was also assessed as an additional behavioral outcome. We hypothesized that the stimulus effects of nicotine aerosol were central nervous system (CNS)-mediated and comparable to that produced by an injected nicotine training stimulus. We further hypothesized that number of aerosol puffs and the e-liquid nicotine concentration which was aerosolized would impact the substitution of nicotine aerosol for injected nicotine. Both nicotine injections and exposures to nicotine aerosol produced a dose-dependent effect on locomotor activity. Nicotine aerosol under our puffing conditions produced e-liquid nicotine concentration-dependent and puff-number-dependent complete substitution for the injected nicotine training condition. The nicotinic antagonist, mecamylamine, completely blocked nicotine-appropriate responding produce by the training dose of 0.3 mg/kg injected nicotine as well as that resulting from exposure to aerosol puffs generated by e-liquid containing 3 mg/ml nicotine, demonstrating that the stimulus of inhaled nicotine was most likely CNS-mediated and not due to olfactory stimulus properties. Overall, the results support the hypothesis that an aerosol exposure drug discrimination model in rodents has applicability to studying the abuse-related effects of e-cigarettes. SIGNIFICANCE STATEMENT: Animal models of nicotine aerosol exposure using testing conditions resembling human e-cigarette use are lacking. In this study, we test a novel preclinical model of nicotine vaping in rodents which allows for the exploration of the abuse-related effects of e-cigarettes. This model has the potential to contribute both to our understanding of the abuse-related pharmacological effects of e-cigarettes as well as aid in the development of rationale, evidence-based e-cigarette regulatory policies.

Tyramine synthesis, vesicular packaging, and the SNARE complex function coordinately in astrocytes to regulate Drosophila alcohol sedation.

Identifying mechanisms underlying alcohol-related behaviors could provide important insights regarding the etiology of alcohol use disorder. To date, most genetic studies on alcohol-related behavior in model organisms have focused on neurons, leaving the causal roles of glial mechanisms less comprehensively investigated. Here, we report our studies on the role of Tyrosine decarboxylase 2 (Tdc2), which converts tyrosine to the catecholamine tyramine, in glial cells in Drosophila alcohol sedation. Using genetic approaches that drove transgene expression constitutively in all glia, constitutively in astrocytes and conditionally in glia during adulthood, we found that knockdown and overexpression of Tdc2, respectively, increased and decreased the sensitivity to alcohol sedation in flies. Manipulation of the genes tyramine ß-hydroxylase and tyrosine hydroxylase, which respectively synthesize octopamine and dopamine from tyramine and tyrosine, had no discernable effect on alcohol sedation, suggesting that Tdc2 affects alcohol sedation by regulating tyramine production. We also found that knockdown of the vesicular monoamine transporter (VMAT) and disruption of the SNARE complex in all glia or selectively in astrocytes increased sensitivity to alcohol sedation and that both VMAT and the SNARE complex functioned downstream of Tdc2. Our studies support a model in which the synthesis of tyramine and vesicle-mediated release of tyramine from adult astrocytes regulates alcohol sedation in Drosophila. Considering that tyramine is functionally orthologous to norepinephrine in mammals, our results raise the possibility that gliotransmitter synthesis release could be a conserved mechanism influencing behavioral responses to alcohol as well as alcohol use disorder.

Dietary yeast influences ethanol sedation in Drosophila via serotonergic neuron function.

Abuse of alcohol is a major clinical problem with far-reaching health consequences. Understanding the environmental and genetic factors that contribute to alcohol-related behaviors is a potential gateway for developing novel therapeutic approaches for patients that abuse the drug. To this end, we have used Drosophila melanogaster as a model to investigate the effect of diet, an environmental factor, on ethanol sedation. Providing flies with diets high in yeast, a routinely used component of fly media, increased their resistance to ethanol sedation. The yeast-induced resistance to ethanol sedation occurred in several different genetic backgrounds, was observed in males and females, was elicited by yeast from different sources, was readily reversible, and was associated with increased nutrient intake as well as decreased internal ethanol levels. Inhibition of serotonergic neuron function using multiple independent genetic manipulations blocked the effect of yeast supplementation on ethanol sedation, nutrient intake, and internal ethanol levels. Our results demonstrate that yeast is a critical dietary component that influences ethanol sedation in flies and that serotonergic signaling is required for the effect of dietary yeast on nutrient intake, ethanol uptake/elimination, and ethanol sedation. Our studies establish the fly as a model for diet-induced changes in ethanol sedation and raise the possibility that serotonin might mediate the effect of diet on alcohol-related behavior in other species.

Convergent Evidence From Humans and Drosophila melanogaster Implicates the Transcription Factor MEF2B/Mef2 in Alcohol Sensitivity.

BACKGROUND: Self-Rating of the Effects of Alcohol (SRE) measures level of response to ethanol (EtOH) in humans. Interestingly, there is a positive relationship between the SRE and risk for abusing alcohol, suggesting mechanistic connections between SRE and alcohol abuse. METHODS: To identify candidate genes with a role in SRE and alcohol-related behavior more generally, we coupled human genetic analyses with studies in Drosophila melanogaster. We first performed a gene-based analysis of Genomewide association studies (GWAS) summary statistics for SRE in the Avon Longitudinal Study of Parents and Children sample. Based on prior findings in humans, orthology to fly genes, and the availability of genetic reagents, we selected a subset of these genes for studies on EtOH behavior in Drosophila. RESULTS: We found 37 genes with nominal associations in our SRE GWAS. We explored the role of 6 orthologous genes in Drosophila EtOH sedation and rapid tolerance. We found that the transcription factor Mef2 is required for normal EtOH sedation in flies. Pan-neuronal expression of 2 independent Mef2 RNAi transgenes significantly reduced Mef2 expression and made flies resistant to EtOH sedation. Additionally, flies with multiple independent mutant alleles of Mef2 were also resistant to EtOH sedation, confirming a role for Mef2 in this behavior. Altered expression of Mef2 did not change EtOH rapid tolerance or cause a net change in internal EtOH concentrations. CONCLUSIONS: Our studies indicate that MEF2B influences SRE in humans and that Mef2 impacts EtOH sedation in Drosophila.

N-methyl-D-aspartate receptor channel blocker-like discriminative stimulus effects of nitrous oxide gas.

Nitrous oxide (N2O) gas is a widely used anesthetic adjunct in dentistry and medicine that is also commonly abused. Studies have shown that N2O alters the function of the N-methyl-d-aspartate (NMDA), GABAA, opioid, and serotonin receptors among others. However, the receptors systems underlying the abuse-related central nervous system effects of N2O are unclear. The present study explores the receptor systems responsible for producing the discriminative stimulus effects of N2O. B6SJLF1/J male mice trained to discriminate 10 minutes of exposure to 60% N2O + 40% oxygen versus 100% oxygen served as subjects. Both the high-affinity NMDA receptor channel blocker (+)-MK-801 maleate [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate] and the low-affinity blocker memantine partially mimicked the stimulus effects of N2O. Neither the competitive NMDA antagonist, CGS-19755 (cis-4-[phosphomethyl]-piperidine-2-carboxylic acid), nor the NMDA glycine-site antagonist, L701-324 [7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(1H)-quinolinone], produced N2O-like stimulus effects. A range of GABAA agonists and positive modulators, including midazolam, pentobarbital, muscimol, and gaboxadol (4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-ol), all failed to produce N2O-like stimulus effects. The µ-, κ-, and δ-opioid agonists, as well as 5-hydroxytryptamine (serotonin) 1B/2C (5-HT1B/2C) and 5-HT1A agonists, also failed to produce N2O-like stimulus effects. Ethanol partially substituted for N2O. Both (+)-MK-801 and ethanol but not midazolam pretreatment also significantly enhanced the discriminative stimulus effects of N2O. Our results support the hypothesis that the discriminative stimulus effects of N2O are at least partially mediated by NMDA antagonist effects similar to those produced by channel blockers. However, as none of the drugs tested fully mimicked the stimulus effects of N2O, other mechanisms may also be involved.

Discriminative stimulus effects of nitrous oxide in mice: comparison with volatile hydrocarbons and vapor anesthetics.

The abuse-related behavioral effects produced by nitrous oxide (N2O) gas have been suggested as being unique compared with other abused inhalants. The drug discrimination paradigm in animals can be used to study subjective effects of drugs in humans and to test this hypothesis. The goals of the present experiment were to establish N2O discrimination in mice and to compare its discriminative stimulus effects with those of abused volatile vapors and vapor anesthetics. Sixteen B6SJLF1/J mice were trained to discriminate between 10 min of exposure to 60% N2O+40% oxygen (O2) and 10 min of exposure to 100% O2. The time course of N2O discrimination was examined, followed by cross-substitution testing with abused vapors, volatile anesthetics, ethanol, D-amphetamine, and 2-butanol. Mice acquired the ability to discriminate between N2O and O2 in 40 days. N2O fully substituted for 10 min of exposure to 60% N2O in a concentration-dependent manner. Full substitution required 7 min of 60% N2O exposure, but the offset of stimulus effects following the cessation of exposure was more rapid. The aromatic hydrocarbon toluene almost fully substituted for N2O. 1,1,1-Trichloroethane, methoxyflurane, isoflurane, and ethanol showed lesser degrees of substitution. D-amphetamine and the odorant 2-butanol did not substitute for N2O. Given the varying degrees of incomplete substitution by test compounds, the discriminative stimulus properties of N2O and, perhaps, its subjective effects in humans are probably not unique. As none of the inhalants tested fully mimicked N2O, its overall effects may include one or more novel stimulus components.

Stimulus mediation, specificity and impact of menthol in rats trained to discriminate puffs of nicotine e-cigarette aerosol from nicotine-free aerosol.

RATIONALE: It is unclear if e-cigarettes have reduced abuse liability relative to traditional cigarettes, especially when considering advanced devices which deliver nicotine more efficiently. Translatable and predictive animal models are needed to addresses this question. OBJECTIVES: Our goal was to explore the subjective stimulus effects of e-cigarettes by training rats to discriminate puffs of nicotine aerosol from vehicle aerosol using an aerosol delivery system designed to model e-cigarette use patterns in humans. METHODS: Rats were trained to discriminate between ten, 10 s puffs of aerosol generated from 3 mg/ml nicotine e-liquid and nicotine-free e-liquid using a food-reinforced operant procedure. Following acquisition, tests were conducted to determine the specificity of the nicotine aerosol stimulus as well as the impact to the stimulus effects of nicotine resulting from the addition of menthol to e-liquid. RESULTS: Rats learned the nicotine aerosol puff vs vehicle puff discrimination in a mean of 25 training sessions. Injected nicotine fully substituted for the stimulus effects of nicotine aerosol. The stimulus effects of nicotine aerosol were blocked by the nicotinic receptor antagonist mecamylamine. The nicotinic receptor partial agonist, varenicline as well as the stimulant d-amphetamine substituted more robustly for nicotine aerosol puffs than did the NMDA antagonist, ketamine. Menthol enhanced the stimulus effects of nicotine aerosol without altering nicotine blood plasma levels. CONCLUSIONS: Nicotine aerosol puffs can function as a training stimulus in rats. The stimulus effects were CNS-mediated and receptor specific. Menthol appears to enhance the stimulus effects of nicotine aerosol through a pharmacodynamic rather than pharmacokinetic mechanism.

Role of adrenal glucocorticoid signaling in prefrontal cortex gene expression and acute behavioral responses to ethanol.

BACKGROUND: Glucocorticoid hormones modulate acute and chronic behavioral and molecular responses to drugs of abuse including psychostimulants and opioids. There is growing evidence that glucocorticoids might also modulate behavioral responses to ethanol ( EtOH ). Acute EtOH activates the hypothalamic-pituitary-adrenal axis, causing the release of adrenal glucocorticoid hormones. Our prior genomic studies suggest that glucocorticoids play a role in regulating gene expression in the prefrontal cortex (PFC) of DBA2/J (D2) mice following acute EtOH administration. However, few studies have analyzed the role of glucocorticoid signaling in behavioral responses to acute EtOH . Such work could be significant, given the predictive value for the level of response to acute EtOH in the risk for alcoholism. METHODS: We studied whether the glucocorticoid receptor (GR) antagonist, RU-486, or adrenalectomy (ADX) altered male D2 mouse behavioral responses to acute (locomotor activation, anxiolysis, or loss-of-righting reflex [LORR]) or repeated (sensitization) EtOH treatment. Whole-genome microarray analysis and bioinformatics approaches were used to identify PFC candidate genes possibly responsible for altered behavioral responses to EtOH following ADX. RESULTS: ADX and RU-486 both impaired acute EtOH (2 g/kg)-induced locomotor activation in D2 mice without affecting basal locomotor activity. However, neither ADX nor RU-486 altered the initiation of EtOH sensitization (locomotor activation or jump counts), EtOH -induced anxiolysis, or LORR. ADX mice showed microarray gene expression changes in PFC that significantly overlapped with acute EtOH -responsive gene sets derived by our prior microarray studies. Q-rtPCR analysis verified that ADX decreased PFC expression of Fkbp5 while significantly increasing Gpr6 expression. In addition, high-dose RU-486 pretreatment blunted EtOH -induced Fkbp5 expression. CONCLUSIONS: Our studies suggest that EtOH 's activation of adrenal glucocorticoid release and subsequent GR activation may partially modulate EtOH 's acute locomotor activation in male D2 mice. Furthermore, because adrenal glucocorticoid basal tone regulated PFC gene expression, including a significant set of acute EtOH -responsive genes, this suggests that glucocorticoid-regulated PFC gene expression may be an important factor modulating acute behavioral responses to EtOH .

Comparison of three DREADD agonists acting on Gq-DREADDs in the ventral tegmental area to alter locomotor activity in tyrosine hydroxylase:Cre male and female rats.

RATIONALE: Despite the increasingly pervasive use of chemogenetic tools in preclinical neuroscience research, the in vivo pharmacology of DREADD agonists remains poorly understood. The pharmacological effects of any ligand acting at receptors, engineered or endogenous, are influenced by numerous factors including potency, time course, and receptor selectivity. Thus, rigorous comparison of the potency and time course of available DREADD ligands may provide an empirical foundation for ligand selection. OBJECTIVES: Compare the behavioral pharmacology of three different DREADD ligands clozapine-N-oxide (CNO), compound 21 (C21), and deschloroclozapine (DCZ) in a locomotor activity assay in tyrosine hydroxylase:cre recombinase (TH:Cre) male and female rats. METHODS: Locomotor activity in nine adult TH:Cre Sprague-Dawley rats (5 female, 4 male) was monitored for two hours following administration of d-amphetamine (vehicle, 0.1-3.2 mg/kg, IP), DCZ (vehicle, 0.32-320 µg/kg, IP), CNO (vehicle, 0.32-10 mg/kg), and C21 (vehicle, 0.1-3.2 mg/kg, IP). Behavioral sessions were conducted twice per week prior to and starting three weeks after bilateral intra-VTA hM3Dq DREADD virus injection. RESULTS: d-Amphetamine significantly increased locomotor activity pre- and post-DREADD virus injection. DCZ, CNO, and C21 did not alter locomotor activity pre-DREADD virus injection. There was no significant effect of DCZ, CNO, and C21 on locomotor activity post-DREADD virus injection; however, large individual differences in both behavioral response and receptor expression were observed. CONCLUSIONS: Large individual variability was observed in both DREADD agonist behavioral effects and receptor expression. These results suggest further basic research would facilitate the utility of these chemogenetic tools for behavioral neuroscience research.

Ethanol metabolism and osmolarity modify behavioral responses to ethanol in C. elegans.

BACKGROUND: Ethanol (EtOH) is metabolized by a 2-step process in which alcohol dehydrogenase (ADH) oxidizes EtOH to acetaldehyde, which is further oxidized to acetate by aldehyde dehydrogenase (ALDH). Although variation in EtOH metabolism in humans strongly influences the propensity to chronically abuse alcohol, few data exist on the behavioral effects of altered EtOH metabolism. Here, we used the nematode Caenorhabditis elegans to directly examine how changes in EtOH metabolism alter behavioral responses to alcohol during an acute exposure. Additionally, we investigated EtOH solution osmolarity as a potential explanation for contrasting published data on C. elegans EtOH sensitivity. METHODS: We developed a gas chromatography assay and validated a spectrophotometric method to measure internal EtOH in EtOH-exposed worms. Further, we tested the effects of mutations in ADH and ALDH genes on EtOH tissue accumulation and behavioral sensitivity to the drug. Finally, we tested the effects of EtOH solution osmolarity on behavioral responses and tissue EtOH accumulation. RESULTS: Only a small amount of exogenously applied EtOH accumulated in the tissues of C. elegans and consequently their tissue concentrations were similar to those that intoxicate humans. Independent inactivation of an ADH-encoding gene (sodh-1) or an ALDH-encoding gene (alh-6 or alh-13) increased the EtOH concentration in worms and caused hypersensitivity to the acute sedative effects of EtOH on locomotion. We also found that the sensitivity to the depressive effects of EtOH on locomotion is strongly influenced by the osmolarity of the exogenous EtOH solution. CONCLUSIONS: Our results indicate that EtOH metabolism via ADH and ALDH has a statistically discernable but surprisingly minor influence on EtOH sedation and internal EtOH accumulation in worms. In contrast, the osmolarity of the medium in which EtOH is delivered to the animals has a more substantial effect on the observed sensitivity to EtOH.

Reinforcing effects of fentanyl and sufentanil aerosol puffs in rats.

RATIONALE: Rapidly evolving e-cigarette technology developed for self-administering nicotine aerosol has the potential to be utilized to self-administer other aerosolized drugs of abuse. Rodent models which mirror characteristics of human e-cigarette use are necessary to explore the degree to which this may be a public health concern. OBJECTIVES: Our goal was to develop a highly translational model of discrete nose-only aerosol puff drug delivery to explore the reinforcing effects of fentanyl and sufentanil aerosols in rats. METHODS: Male and female Sprague-Dawley rats were trained to perform a multiple schedule FR1 lever-press, 4-s (second) nose hold operant during which the subject's orofacial areas were exposed to drug-free glycerol/propylene glycol aerosol produced by a commercial e-cigarette at a power setting of 18 watts. Each completed 4-s drug-free vehicle aerosol exposure resulted in a 3-s presentation of a 0.1-ml dipper of sweetened milk solution. After training, rats were then allowed to self-administer 4-s nose-only puffs of fentanyl (100-6000 µg/ml) or sufentanil (30-500 µg/ml) aerosol in the absence of paired milk dipper reinforcers. RESULTS: All 31 rats learned the lever-press/nose-poke multiple schedule for milk dippers alone and 25 accepted exposure to 4 s of 18 watts of drug-free vehicle aerosol when paired with milk dipper presentations. In the absence of paired milk dipper presentations, fentanyl aerosol puffs at concentrations of 1000 and 3000 µg/ml as well as 100 µg/ml puffs of sufentanil served as reinforcers compared to both air puffs and drug-free vehicle aerosol puffs. There were no significant differences between males and females in number of fentanyl or sufentanil puffs self-administered. CONCLUSIONS: Discrete nose-only puffs of two potent opioids under exposure conditions comparable to puff durations in human e-cigarette users serve as reinforcers in rats. This outcome suggests that under appropriate conditions e-cigarettes might be a potential alternative delivery mechanism for illicit opioids.

Pharmacological characterization of the discriminative stimulus of inhaled 1,1,1-trichloroethane.

The present study examined the involvement of the GABAA, N-methyl-D-aspartate (NMDA), nicotinic acetylcholine, and mu-opioid receptor systems in the transduction of the discriminative stimulus effects of the abused inhalant 1,1,1-trichloroethane (TCE). Sixteen B6SJLF1/J mice were trained to discriminate 10 min of exposure to 12,000-ppm inhaled TCE vapor from air. Substitution and antagonism tests and TCE blood concentration analysis were subsequently conducted. TCE blood concentrations decreased rapidly after cessation of exposure, falling by 66% within 5 min. TCE vapor concentration-dependently substituted for the 12,000-ppm training stimulus. The volatile anesthetic halothane concentration-dependently and fully substituted for TCE. The benzodiazepine midazolam partially substituted for TCE, producing a maximum of 68% TCE-lever selection. The benzodiazepine antagonist flumazenil attenuated midazolam substitution for TCE, but not the discriminative stimulus effects of TCE itself. The noncompetitive NDMA receptor antagonists phencyclidine and dizocilpine failed to substitute for TCE. Nicotine and the central nicotinic receptor antagonist mecamylamine also failed to produce any TCE-lever selection, nor did they antagonize the discriminative stimulus of TCE. The mu-opioid receptor agonist morphine did not substitute for TCE. The opioid antagonist naltrexone failed to antagonize the discriminative stimulus of TCE. Overall, the present results, combined with previous studies, suggest that the discriminative stimulus effects of TCE are mediated primarily by positive GABAA receptor modulatory effects though a mechanism distinct from the benzodiazepine binding site.

Effect of drug-paired exteroceptive stimulus presentations on methamphetamine reinstatement in rats.

The purpose of the present study was to examine the impact of drug-paired cues on methamphetamine reinstatement. Three groups of rats were trained to self-administer 0.1 mg/kg/infusion methamphetamine. Each methamphetamine infusion was accompanied by a 6 s flashing light+tone stimulus (cues). After training, the groups were then given 12, daily extinction sessions either with or without response-contingent drug-paired cues and then tested for 1 mg/kg i.p. methamphetamine priming-induced reinstatement either with or without cues. Methamphetamine priming significantly reinstated drug-appropriate responding regardless of whether response-contingent cues were omitted during both extinction and testing, presented during both extinction and testing, or omitted during extinction but presented during reinstatement testing. The group in which cues were omitted during extinction and presented during reinstatement exhibited significantly greater reinstatement than did the other two groups. A separate group of rats was also tested demonstrating that response-contingent presentation of previously methamphetamine-paired cues alone, without methamphetamine priming, significantly reinstated drug-appropriate responding. These data show that methamphetamine priming produces a robust reinstatement effect which can be influenced by drug-paired cues.

Employee relations.

This review highlights some of the more important employee relation aspects involved in starting, establishing, or expanding an existing dental practice. Despite a competitive compensation package, staff-related conflicts can sometimes hamper the progress of a dental practice. Such conflicts can be reduced by having policies and procedures in place for each employee that set expectations concerning the hours of operation, professional manner, dress code, job tasks, performance evaluations, disciplinary actions, and termination if violations occur. Understanding the legal requirements set by various governmental agencies such as OSHA can help ensure that the rights and well-being of every employee are protected.

Discriminative Stimulus Effects of Abused Inhalants.

Inhalants are a loosely organized category of abused compounds defined entirely by their common route of administration. Inhalants include volatile solvents, fuels, volatile anesthetics, gasses, and liquefied refrigerants, among others. They are ubiquitous in modern society as ingredients in a wide variety of household, commercial, and medical products. Persons of all ages abuse inhalants but the highest prevalence of abuse is in younger adolescents. Although inhalants have been shown to act upon a host of neurotransmitter receptors, the stimulus effects of the few inhalants which have been trained or tested in drug discrimination procedures suggest that their discriminative stimulus properties are mediated by a few key neurotransmitter receptor systems. Abused volatile solvent inhalants have stimulus effects that are similar to a select group of GABAA positive modulators comprised of benzodiazepines and barbiturates. In contrast the stimulus effects of nitrous oxide gas appear to be at least partially mediated by uncompetitive antagonism of NMDA receptors. Finally, volatile anesthetic inhalants have stimulus effects in common with both GABAA positive modulators as well as competitive NMDA antagonists. In addition to a review of the pharmacology underlying the stimulus effects of inhalants, the chapter also discusses the scientific value of utilizing drug discrimination as a means of functionally grouping inhalants according to their abuse-related pharmacological properties.

Characterization of the ethanol-deprivation effect in substrains of C57BL/6 mice.

Ethanol craving plays a major role in relapse drinking behavior. Relapse and ethanol craving are an important focus for the treatment of alcoholism. The ethanol-deprivation effect (EDE) is a widely used animal model of alcohol craving. While the EDE is widely studied in rats, the molecular mechanisms underlying EDE are not clearly understood. The C57BL/6 inbred mouse strain is widely used for behavioral and molecular analyses of ethanol drinking but studies on the EDE have not been reported in this strain. In the present study, we characterized a simple behavioral protocol that rapidly and reliably induced EDE in C57BL/6 mice. Briefly, single-housed adult male C57BL/6NCrl and C57BL/6J mice were presented at the beginning of dark phase with two-bottle choice drinking containing either 10% wt/vol ethanol or tap water for 18 h/day, as well as food ad libitum. Following ethanol drinking for 4 days or 14 days, mice were deprived of ethanol for a period of 4 days. To study EDE, mice were reinstated with two bottles containing either ethanol (10% wt/vol) or water. Mice were exposed to single or multiple ethanol-deprivation cycles. Ethanol consumption (g/kg/18 h) and percent ethanol preference (% preference/18 hrs) was recorded for individual mice. C57BL/6NCrl mice consumed moderate amounts (4.78+/-0.63 g/kg) of ethanol but showed robust EDE after ethanol-drinking episodes (4 days or 14 days) as evidenced by increased ethanol consumption and ethanol preference following reinstatement of ethanol. While repeated ethanol deprivation in C57BL/6NCrl mice transiently increased ethanol consumption and ethanol preference, the magnitude of these behaviors was reduced as compared to the first deprivation cycle. In contrast, the C57BL/6J substrain consumed substantially higher levels (9.65+/-0.90 g/kg) of ethanol but did not show a clear EDE after single or multiple ethanol-deprivation cycles. In conclusion, we established a simple and reliable behavioral model to study EDE in C57BL/6NCrl mice. A reliable behavioral model to study EDE in inbred C57BL/6NCrl mice could greatly facilitate further studies on molecular mechanisms of ethanol craving behavior.

Negative allosteric modulation of GABAA receptors inhibits facilitation of brain stimulation reward by drugs of abuse in C57BL6/J mice.

RATIONALE: There is an emerging body of evidence that implicates a crucial role of γ-aminobutyric acid subtype A (GABAA) receptors in modulating the rewarding effects of a number of abused drugs. Modulation of GABAA receptors may therefore represent a novel drug-class independent mechanism for the development of abuse treatment pharmacotherapeutics. OBJECTIVES: We tested the hypothesis that the GABAA receptor benzodiazepine-site (BDZ) negative modulator Ro15-4513 would reduce the reward-related effects of three pharmacologically dissimilar drugs; toluene vapor, d-methamphetamine, and diazepam using intracranial self-stimulation (ICSS) in mice. We also examined whether Ro15-4513 attenuated dopamine release produced by d-methamphetamine in an in vivo microdialysis procedure. RESULTS: Ro15-4513 abolished ICSS reward facilitation produced by all three abused drugs at Ro15-4513 doses which had no effect on ICSS when administered alone. In contrast, the BDZ antagonist flumazenil only attenuated the ICSS-facilitating effects of diazepam. Administration of the same dose of Ro15-4513 which abolished drug-facilitated ICSS produced a 58 % decrease in d-methamphetamine-stimulated dopamine in the nucleus accumbens of mice relative to d-methamphetamine alone. CONCLUSIONS: These results demonstrate that negative modulation of GABAA receptors can produce profound reductions in reward-related effects of a diverse group of drugs that activate the mesolimbic reward pathway through different mechanisms. These data suggest that pharmacological modulation of GABAA receptors may represent a viable pathway for the development of drug abuse pharmacotherapies.

Differential effects of the novel kappa opioid receptor antagonist, JDTic, on reinstatement of cocaine-seeking induced by footshock stressors vs cocaine primes and its antidepressant-like effects in rats.

RATIONALE: Stress and depression have been linked to relapse of cocaine abuse. Antagonism of the kappa opioid receptor (KOR) has been reported to attenuate some effects of stressors, and antagonism of the KOR has been reported to have antidepressant-like properties. OBJECTIVES: Our objective was to determine whether the potent and selective KOR antagonist, (3R)-7-hydroxy-N-{(1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl}-1,2,3,4-tetrahydro-3-isoquinoline-carboxamide (JDTic), can reduce the ability of a stressor (intermittent footshock) to reinstate cocaine-seeking behavior and to have antidepressant-like effects in the forced swim test (FST). METHODS: Male Long-Evans hooded rats were trained to lever-press, reinforced with 0.5 mg/kg i.v. infusion of cocaine, according to fixed ratio 1 reinforcement schedules during daily 2-h experimental sessions. After performance had stabilized, lever pressing was extinguished for 12 consecutive sessions, and doses of 0 (vehicle), 3, 10, and 30 mg/kg JDTic were then administered i.g. to separate groups of 12 rats. Twenty four hours later, the rats were given 15 min of intermittent footshock (0.87 mA, 0.5 s activation time, average inter-activation interval of 40 s) or a 17-mg/kg i.p. administration of cocaine prime followed by a 2-h reinstatement test session. JDTic was also evaluated for its ability to block diuresis induced by the KOR agonist, U50,488H (10 mg/kg, s.c.), during 5-h test sessions beginning 1 h after footshock reinstatement tests to verify its KOR antagonist activity. In the FST, male Sprague-Dawley rats were treated with either nor-binaltorphimine (nor-BNI) or JDTic (both at 0.3, 1, 3, or 10 mg/kg, injected s.c. 23 h before), or desipramine (5.6, 10, or 17 mg/kg, injected i.p. 23, 5, and 1 h before) and placed in a cylinder of water, during which the predominance of immobility, swimming, and climbing were scored during 5-s intervals for 5 min. RESULTS: The 10- and 30-mg/kg doses of JDTic significantly reduced footshock-induced reinstatement of responding previously reinforced by cocaine and significantly attenuated U50,488H-induced diuresis. In contrast, JDTic did not affect cocaine-prime-induced reinstatement. Both nor-BNI and JDTic decreased immobility and increased swimming time in the FST, similar to the antidepressant desipramine. CONCLUSIONS: Depression and stress are two states during cocaine abstinence which users identify as precipitating relapse, and JDTic may have properties which attenuate both.

Oral operant ethanol self-administration in the absence of explicit cues, food restriction, water restriction and ethanol fading in C57BL/6J mice.

RATIONALE: Mouse models of ethanol (EtOH) self-administration are useful to identify genetic and biological underpinnings of alcohol use disorder. OBJECTIVES: These experiments developed a novel method of oral operant EtOH self-administration in mice without explicitly paired cues, food/water restriction, or EtOH fading. METHODS: Following magazine and lever training for 0.2 % saccharin (SAC), mice underwent nine weekly overnight sessions with lever pressing maintained by dipper presentation of 0, 3, 10, or 15 % EtOH in SAC or water vehicle. Ad libitum water was available from a bottle. RESULTS: Water vehicle mice ingested most fluid from the water bottle in contrast to SAC vehicle mice, which despite lever pressing demands, drank most of their fluid from the liquid dipper. Although EtOH in SAC vehicle mice showed concentration-dependent increases of g/kg EtOH intake, lever pressing decreased with increasing EtOH concentration and did not exceed that of SAC vehicle alone at any EtOH concentration. Mice reinforced with EtOH in water ingested less EtOH than mice reinforced with EtOH in SAC. EtOH in water mice, however, showed concentration-dependent increases in g/kg EtOH intake and lever presses. Fifteen percent EtOH in water mice showed significantly greater levels of lever pressing than water vehicle mice and a significant escalation of responding across weeks of exposure. Naltrexone pretreatment reduced EtOH self-administration and intake in these mice without altering responding in the vehicle control condition during the first hour of the session. CONCLUSIONS: SAC facilitated EtOH intake but prevented observation of EtOH reinforcement. Water vehicle unmasked EtOH's reinforcing effects.

Chronic ethanol consumption transiently reduces adult neural progenitor cell proliferation.

Adult neural stem/progenitor cells proliferate throughout the life of the animal in the subependymal zone and the subgranular zone of the dentate gyrus (DG). Treatments such as enriched environment, dietary restriction, running and anti-depressants increase proliferation, however, stress and opiates have been shown to decrease proliferation. While models of binge ethanol drinking decreases proliferation, few studies have characterized the effect chronic ethanol usage has on progenitor cell proliferation. In this study, we have examined changes in the progenitor cell proliferation rate following chronic ethanol consumption. Animals were given a nutritionally balanced liquid diet containing 6.5% v/v ethanol or an isocalorically balanced liquid diet. Bromodeoxyuridine (BrdU) was administered (150 mg/kg x 3) and the animals sacrificed 2 h after the last injection on days 3, 10 or 30 of the ethanol diet. Coronal brain blocks were paraffin embedded and 6 microm sections sliced and immunohistochemically stained for BrdU. Quantitation of the number of BrdU-labeled cells in the subgranular zone of the DG revealed a significant decrease only at the 3-day time-point, with recovery by the 10- and 30-day time-points. Thus, the progenitor cell proliferation rate is transiently decreased by chronic ethanol usage. This data suggests that chronic alcohol use results in a compensatory response that restores the progenitor cell proliferation rate.