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.

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.

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.

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.

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.

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.

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.

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.

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.

Pharmacological classification of the abuse-related discriminative stimulus effects of trichloroethylene vapor.

Inhalants are distinguished as a class primarily based upon a shared route of administration. Grouping inhalants according to their abuse-related in vivo pharmacological effects using the drug discrimination procedure has the potential to provide a more relevant classification scheme to the research and treatment community. Mice were trained to differentiate the introceptive effects of the trichloroethylene vapor from air using an operant procedure. Trichloroethylene is a chlorinated hydrocarbon solvent once used as an anesthetic as well as in glues and other consumer products. It is now primarily employed as a metal degreaser. We found that the stimulus effects of trichloroethylene were similar to those of other chlorinated hydrocarbon vapors, the aromatic hydrocarbon toluene and the vapor anesthetics methoxyflurane and isoflurane. The stimulus effects of trichloroethylene overlapped with those of the barbiturate methohexital, to a lesser extent the benzodiazepine midazolam and to ethanol. NMDA antagonists, the kappa opioid agonist U50,488 and the mixed 5-HT agonist mCPP largely failed to substitute for trichloroethylene. These data suggest that stimulus effects of chlorinated hydrocarbon vapors are mediated at least partially by GABAA receptor positive modulatory effects.

Rat nucleus accumbens core astrocytes modulate reward and the motivation to self-administer ethanol after abstinence.

Our understanding of the active role that astrocytes play in modulating neuronal function and behavior is rapidly expanding, but little is known about the role that astrocytes may play in drug-seeking behavior for commonly abused substances. Given that the nucleus accumbens is critically involved in substance abuse and motivation, we sought to determine whether nucleus accumbens astrocytes influence the motivation to self-administer ethanol following abstinence. We found that the packing density of astrocytes that were expressing glial fibrillary acidic protein increased in the nucleus accumbens core (NAcore) during abstinence from EtOH self-administration. No change was observed in the nucleus accumbens shell. This increased NAcore astrocyte density positively correlated with the motivation for ethanol. Astrocytes can communicate with one another and influence neuronal activity through gap-junction hemichannels. Because of this, the effect of blocking gap-junction hemichannels on the motivation for ethanol was examined. The motivation to self-administer ethanol after 3 weeks abstinence was increased following microinjection of gap-junction hemichannel blockers into the NAcore at doses that block both neuronal and astrocytic channels. In contrast, no effect was observed following microinjection of doses that are not thought to block astrocytic channels or following microinjection of either dose into the nucleus accumbens shell. Additionally, the motivation for sucrose after 3 weeks abstinence was unaffected by NAcore gap-junction hemichannel blockers. Next, Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) were selectively expressed in NAcore astrocytes to test the effect of astrocyte stimulation. DREADD activation increased cytosolic calcium in primary astrocytes, facilitated responding for rewarding brain stimulation, and reduced the motivation for ethanol after 3 weeks abstinence. This is the first work to modulate drug-seeking behavior with astrocyte-specific DREADDs. Taken together, our findings demonstrate that NAcore astrocytes can shape the motivation to self-administer ethanol; suggesting that the development of ligands which selectively stimulate astrocytes may be a successful strategy to abate ethanol-seeking behavior.

Assessment of reinforcement enhancing effects of toluene vapor and nitrous oxide in intracranial self-stimulation.

RATIONALE: Despite widespread abuse, there are few validated methods to study the rewarding effects of inhalants. One model that may have utility for this purpose is intracranial self-stimulation (ICSS). OBJECTIVES: This study aims to compare and contrast the ICSS reward-facilitating effects of abused inhalants to other classes of abused drugs. Compounds were examined using two different ICSS procedures in mice to determine the generality of each drug's effects on ICSS and the sensitivity of the procedures. METHODS: Male C57BL/6J mice with electrodes implanted in the medial forebrain bundle were trained under a three-component rate-frequency as well as a progressive ratio (PR) ICSS procedure. The effects of nitrous oxide, toluene vapor, cocaine, and diazepam on ICSS were then examined. RESULTS: Concentrations of 1,360-2,900 parts per million (ppm) inhaled toluene vapor significantly facilitated ICSS in the rate-frequency procedure and 1,360 ppm increased PR breakpoint. A concentration of 40 % nitrous oxide facilitated ICSS in the rate-frequency procedure but reduced PR breakpoint. Doses of 3-18 mg/kg cocaine facilitated ICSS in the rate-frequency procedure, and 10 and 18 mg/kg increased PR breakpoint. Doses of 1 and 3 mg/kg diazepam facilitated ICSS in the rate-frequency procedure, and 3 mg/kg increased PR breakpoint. CONCLUSIONS: The reinforcement-facilitating effect of toluene in ICSS is at least as great as diazepam. By contrast, nitrous oxide weakly enhances ICSS in only the rate-frequency procedure. The data suggest that the rate-frequency procedure may be more sensitive than the PR schedule to the reward-facilitating effects of abused inhalants.

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.

Efficacy of buspirone for attenuating cocaine and methamphetamine reinstatement in rats.

BACKGROUND: There are no approved pharmacotherapies for preventing psychomotor stimulant relapse. The operant reinstatement model has been suggested as a screen for identifying candidate medications. The present study examined if the anxiolytic buspirone could attenuate reinstatement of extinguished responding in Long-Evans rats that previously self-administered intravenous cocaine or methamphetamine. METHODS: Rats were trained in 2-h daily sessions to self-administer 0.5mg/kg cocaine or 0.1mg/kg methamphetamine infusions followed by 12 days of instrumental extinction. Reinstatement was evoked by 17mg/kg i.p. cocaine primes or response-contingent cocaine-paired cues in cocaine-reinforced rats, and by 1mg/kg i.p. methamphetamine primes or response-contingent methamphetamine-paired cues in methamphetamine-reinforced rats. RESULTS: Buspirone (1 and 3mg/kg) significantly (p<0.05) attenuated cocaine cue but not cocaine prime reinstatement. Buspirone (1 and 3mg/kg) also significantly attenuated methamphetamine cue reinstatement. Buspirone (3mg/kg) significantly attenuated methamphetamine prime reinstatement. During all reinstatement tests, 3mg/kg buspirone reduced levels of inactive lever pressing relative to those of vehicle, significantly so during the cocaine cue-induced reinstatement tests. CONCLUSIONS: Given the complexity of buspirone's neuropharmacology consisting of serotonin 5-HT1A receptor partial agonist activity, and dopamine D2, D3 and D4 receptor antagonist effects, it is uncertain which of these activities or their combination is responsible for the present results. Overall, these results suggest that buspirone may reduce the likelihood of relapse to cocaine and methamphetamine use under some conditions, although this speculation must be interpreted with caution given buspirone's similar potency to attenuate inactive-lever responding.

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 .

Benzodiazepine-like discriminative stimulus effects of toluene vapor.

In vitro studies show that the abused inhalant toluene affects a number of ligand-gated ion channels.The two most consistently implicated of these are γ-aminobutyric acid type A(GABAA) receptors which are positively modulated by toluene and N-methyl-D-aspartate(NMDA) receptors which are negatively modulated by toluene. Behavioral studies also suggest an interaction of toluene with GABAA and/or NMDA receptors but it is unclear if these receptors underlie the abuse-related intoxicating effects of toluene. Seventeen B6SJLF1/J mice were trained using a two-choice operant drug discrimination procedure to discriminate 10 min of exposure to 2000 ppm toluene vapor from 10 min of exposure to air. The discrimination was acquired in a mean of 65 training sessions. The stimulus effects of 2000 ppm toluene vapor were exposure concentration-dependent but rapidly diminished following the cessation of vapor exposure. The stimulus effects of toluene generalized to the chlorinated hydrocarbon vapor perchloroethylene but not 1,1,2-trichloroethane nor the volatile anesthetic isoflurane. The competitive NMDA antagonist CGS-19755, the uncompetitive antagonist dizocilpine and the glycine-site antagonist L701,324 all failed to substitute for toluene. The classical nonselective benzodiazepines midazolam and chlordiazepoxide produced toluene-like stimulus effects but the alpha 1 subunit preferring positive GABAA modulator zaleplon failed to substitute for toluene. The barbiturates pentobarbital and methohexital and the GABAA positive modulator neurosteroid allopregnanolone did not substitute for toluene. These data suggest that the stimulus effects of toluene may be at least partially mediated by benzodiazepine-like positive allosteric modulation of GABAA receptors containing alpha 2, 3 or 5 subunits.

Prime-, stress-, and cue-induced reinstatement of extinguished drug-reinforced responding in rats: cocaine as the prototypical drug of abuse.

This unit describes the testing of rats in prime-, footshock-, and cue-induced reinstatement procedures. Evaluating rats in these procedures enables the assessment of treatments on behavior thought to model drug relapse precipitated by re-contact with an abused drug (prime-induced), induced by stress (footshock-induced), or by stimuli previously associated with drug administration (cue-induced). For instance, levels of reinstatement under the effects of test compound administration could be compared to levels under vehicle administration to help identify potential treatments for drug relapse, or reinstatement levels of different rat strains could be compared to identify potential genetic determinants of perseverative drug-seeking behavior. Cocaine is used as a prototypical drug of abuse, and relapse to its use serves as the model in this unit, but other self-administered drugs could readily be substituted with little modification to the procedures.

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.