Aversion-associated drug and alcohol seeking in females.

Compulsive drug intake is characterized by the continuation of use regardless of negative consequences. This is modeled preclinically using procedures where a negative stimulus is delivered contingently with consumption of the reinforcer. In humans, women and men exhibit different drug taking behavior as it pertains to overall use, withdrawal symptoms, and rate of dependence. In substance use research, females have often been excluded from many studies due to concerns that circulating sex hormones may affect drug seeking behavior. However, the more recent inclusion of females in preclinical studies has identified interesting sex differences in aversion-resistant intake of drugs and alcohol. This review will serve to summarize key findings in aversion-related intake of alcohol, psychostimulants, and opioids in females by examining studies that have included female subjects. Further discussion will examine the effect of intake model, neuroanatomical pathways, and sex hormones in the expression of aversion-resistant drug and alcohol consumption.

Sex differences in neuronal activation in the cortex and midbrain during quinine-adulterated alcohol intake.

AIMS: Continued alcohol consumption despite negative consequences is a core symptom of alcohol use disorder. This is modeled in mice by pairing negative stimuli with alcohol, such as adulterating alcohol solution with quinine. Mice consuming alcohol under these conditions are considered to be engaging in aversion-resistant intake. Previously, we have observed sex differences in this behavior, with females more readily expressing aversion-resistant consumption. We also identified three brain regions that exhibited sex differences in neuronal activation during quinine-alcohol drinking: ventromedial prefrontal cortex (vmPFC), posterior insular cortex (PIC), and ventral tegmental area (VTA). Specifically, male mice showed increased activation in vmPFC and PIC, while females exhibited increased activation in VTA. In this study, we aimed to identify what specific type of neurons are activated in these regions during quinine-alcohol drinking. METHOD: We assessed quinine-adulterated alcohol intake using the two-bottle choice procedure. We also utilized RNAscope in situ hybridization in the three brain regions that previously exhibited a sex difference to examine colocalization of Fos, glutamate, GABA, and dopamine. RESULT: Females showed increased aversion-resistant alcohol consumption compared to males. We also found that males had higher colocalization of glutamate and Fos in vmPFC and PIC, while females had greater dopamine and Fos colocalization in the VTA. CONCLUSIONS: Collectively, these experiments suggest that glutamatergic output from the vmPFC and PIC may have a role in suppressing, and dopaminergic activity in the VTA may promote, aversion-resistant alcohol consumption. Future experiments will examine neuronal circuits that contribute to sex differences in aversion resistant consumption.

Vicarious defeat stress induces increased alcohol consumption in female mice: Role of neurokinin-1 receptor and interleukin-6.

There is a high frequency of comorbidity of alcohol use disorder (AUD) and depression in human populations. We have studied this relationship in our lab using the social defeat stress (SDS) model, which results in both depression-like behaviours and increased alcohol consumption in male mice. However, standard SDS procedures are difficult to use in female mice due to a lack of territorial aggression. In the experiments presented here, we used vicarious defeat stress (VDS) to assess social withdrawal and alcohol consumption in female C57BL6/J mice. We also assessed the expression of interleukin-6 (IL6), which is a proinflammatory cytokine that is associated with depression in humans and sensitivity to SDS in mice. In these experiments, C57BL/6 female mice underwent 10 days of VDS where they witnessed the physical defeat of a male conspecific by an aggressive CD1 mouse. After the end of VDS, mice were either given access to alcohol or sacrificed for the measurement of IL6 expression. We found that VDS increased alcohol consumption and IL6 expression in the frontal cortex and hippocampus. Given that the neurokinin-1 receptor (NK1R) can mediate both stress-induced alcohol consumption and IL6 expression, we tested the ability of NK1R antagonism to reduce VDS-induced alcohol consumption and found that this treatment reduced alcohol intake in both VDS-exposed mice and in unstressed controls. The observed increase in alcohol consumption suggests that VDS is a model that can be utilized to study stress-induced alcohol consumption in female mice, and that this is sensitive to NK1R antagonism.

An Adolescent Porcine Model of Voluntary Alcohol Consumption Exhibits Binge Drinking and Motor Deficits in a Two Bottle Choice Test.

AIMS: Alcohol is the most commonly abused substance leading to significant economic and medical burdens. Pigs are an attractive model for studying alcohol abuse disorder due to the comparable alcohol metabolism and consumption behavior, which are in stark contrast to rodent models. This study investigates the usage of a porcine model for voluntary binge drinking (BD) and a detailed analysis of gait changes due to motor function deficits during alcohol intoxication. METHODS: Adolescent pigs were trained to drink increasing concentration (0-8%) of alcohol mixed in a 0.2% saccharin solution for 1 h in a two bottle choice test for 2 weeks. The training period was followed by a 3-week alcohol testing period, where animals were given free access to 8% alcohol in 0.2% saccharin solution and 0.2% saccharin water solution. Blood alcohol levels were tested and gait analysis was performed pre-alcohol consumption, last day of training, and Day 5 of each testing period. RESULTS: Pigs voluntarily consumed alcohol to intoxication at all timepoints with blood alcohol concentration (BAL) ≥80 mg/dl. Spatiotemporal gait parameters including velocity, cadence, cycle time, swing time, stance time, step time, and stride length were perturbed as a result of intoxication. The stratification of the gait data based on BAL revealed that the gait parameters were affected in a dose-dependent manner. CONCLUSION: This novel adolescent BD porcine model with inherent anatomical and physiological similarities to humans display similar consumption and intoxication behavior that is likely to yield results that are translatable to human patients.

Intermittent Ethanol Access Increases Sensitivity to Social Defeat Stress.

BACKGROUND: Comorbidity between alcoholism and depression is extremely common. Recent evidence supports a relationship between alcohol exposure and stress sensitivity, an underlying factor in the development of depression. Our laboratory has recently shown that chronic alcohol gavage increases sensitivity to social defeat stress (SDS). However, the effects of voluntary alcohol consumption, resulting from protocols such as intermittent ethanol access (IEA), on defeat stress sensitivity have yet to be elucidated. METHODS: We first assessed the effects of 4 weeks of IEA to 20% alcohol on sensitivity to subthreshold SDS exposure. Next, to examine neuroinflammatory mechanisms, we analyzed gene expression of inhibitor of NFkB (IkB) following IEA or chronic alcohol exposure (10 days of 3.0 g/kg alcohol via intragastric gavage). Then, we quantified NFkB activation via ß-galactosidase immunohistochemistry following IEA or chronic alcohol gavage in NFkB-LacZ mice. RESULTS: IEA-exposed mice displayed an increase in sensitivity to subthreshold SDS compared to water-drinking controls. We also found that IkB gene expression was decreased in the nucleus accumbens (NAC) and amygdala (AMY) following IEA but was not altered following chronic alcohol gavage. Finally, we observed increased NFkB activity in the central amygdala (CEA), basolateral amygdala (BLA), and medial amygdala (MEA) after IEA, and increased NFkB activity solely in the CEA following chronic alcohol gavage. CONCLUSIONS: These findings further corroborate that prior alcohol exposure, in this case intermittent voluntary consumption, can impact development of depressive-like behavior by altering stress sensitivity. Furthermore, our results suggest the CEA as a potential mediator of alcohol's effects on stress sensitivity, as NFkB was activated in this region following both IEA and chronic alcohol gavage. Thus, this study provides novel insight on alterations in the NFkB pathway and identifies specific regions to target in future experiments assessing the functional role of NFkB in these processes.

Sex differences in oral oxycodone self-administration and stress-primed reinstatement in rats.

The opioid epidemic has become a severe public health problem, with approximately 130 opioid-induced deaths occurring each day in the United States. Prescription opioids are responsible for approximately 40% of these deaths. Oxycodone is one of the most commonly abused prescription opioids, but despite its prevalent misuse, the number of preclinical studies investigating oxycodone-seeking behaviors is relatively limited. Furthermore, preclinical oxycodone studies that include female subjects are even more scarce, and it is critical that future work includes both sexes. Additionally, the oral route of administration is one of the most common routes for recreational users, especially in the early stages of drug experimentation. However, currently, only two studies have been published investigating operant oral oxycodone self-administration in rodents. Therefore, the primary goal of the present study was to establish an oral oxycodone operant self-administration model in adult male and female rats, as well as to examine a potential mechanism of stress-primed reinstatement. We found that females consumed significantly more oral oxycodone than males in operant self-administration sessions. We also found that active oxycodone self-administration was reduced by mu opioid receptor antagonism and by substitution of water for oxycodone solution. Lastly, we induced stress-primed reinstatement and found that this behavior was significantly attenuated by antagonism of the neurokinin-1 receptor, consistent with our prior work examining stress-induced reinstatement of alcohol- and cocaine-seeking.

Sex Differences in Aversion-Resistant Ethanol Intake in Mice.

AIMS: Compulsive ethanol intake, characterized by persistent consumption despite negative consequences, is an addictive behavior identified by the DSM-5 as a central criterion in diagnosing alcohol use disorders (AUD). Epidemiological data suggest that females transition from recreational alcohol use to AUD more rapidly than males. Because of this potential sex difference in the etiology of AUD, it is critical to assess addictive behaviors such as compulsive intake in both males and females in preclinical studies. METHODS: We used the model of aversion-resistant ethanol consumption to assess compulsive-like ethanol intake. In these experiments, C57BL6/J mice were first provided with continuous access two-bottle choice between water and ethanol to establish baseline intake. Ethanol solution was then adulterated with increasing concentrations of the bitter tastant quinine hydrochloride. Animals that consume ethanol solution despite its pairing with this negative stimulus are thought to be exhibiting compulsive-like behavior. RESULTS: We found that higher concentrations of quinine were required to suppress ethanol consumption in female mice relative to males. We found no effect of estrous cycle phase on baseline ethanol intake or on quinine-adulterated ethanol intake in females. CONCLUSIONS: Collectively, these data suggest that females exhibit a higher degree of aversion-resistance than male mice. Because we observed no effect of estrous cycle phase, it is likely that the presence of threshold levels of estradiol or progesterone, as opposed to their natural fluctuation across the estrous cycle, mediates increased aversion-resistance in females. Alternatively, or in combination, developmental effects of sex hormones could contribute to aversion-resistant ethanol intake.

Cellular and behavioral effects of lipopolysaccharide treatment are dependent upon neurokinin-1 receptor activation.

BACKGROUND: Several psychiatric conditions are affected by neuroinflammation and neuroimmune activation. The transcription factor nuclear factor kappa light-chain-enhancer of activated B cells (NFkB) plays a major role in inflammation and innate immunity. The neurokinin-1 receptor (NK1R) is the primary endogenous target of the neuroactive peptide substance P, and some data suggests that NK1R stimulation may influence NFkB activity. Both NK1R and NFkB have been shown to play a functional role in complex behaviors including stress responsivity, depression, and addiction. In this study, we test whether NFkB activity in the brain (stimulated by lipopolysaccharide administration) is dependent upon the NK1R. METHODS: Adult male Wistar rats were treated systemically with the NK1R antagonist L822429 followed by administration of systemic lipopolysaccharide (LPS, a strong activator of NFkB). Hippocampal extracts were used to assess expression of proinflammatory cytokines and NFkB-DNA-binding potential. For behavioral studies, rats were trained to consume 1% (w/v) sucrose solution in a continuous access two-bottle choice model. After establishment of baseline, animals were treated with L822429 and LPS and sucrose preference was measured 12 h post-treatment. RESULTS: Systemic LPS treatment causes a significant increase in proinflammatory cytokine expression and NFkB-DNA-binding activity within the hippocampus. These increases are attenuated by systemic pretreatment with the NK1R antagonist L822429. Systemic LPS treatment also led to the development of anhedonic-like behavior, evidenced by decreased sucrose intake in the sucrose preference test. This behavior was significantly attenuated by systemic pretreatment with the NK1R antagonist L822429. CONCLUSIONS: Systemic LPS treatment induced significant increases in NFkB activity, evidenced by increased NFkB-DNA binding and by increased proinflammatory cytokine expression in the hippocampus. LPS also induced anhedonic-like behavior. Both the molecular and behavioral effects of LPS treatment were significantly attenuated by systemic NK1R antagonism, suggesting that NK1R stimulation lies upstream of NFkB activation following systemic LPS administration and is at least in part responsible for NFkB activation.

Bidirectional relationship between alcohol intake and sensitivity to social defeat: association with Tacr1 and Avp expression.

While epidemiological studies show that alcohol abuse is often co-morbid with affective disorders, the causal direction of this association is unclear. We examined this relationship using mouse models including social defeat stress (SDS), social interaction (SI) and voluntary alcohol consumption. C57BL6/J mice exposed to SDS segregate into two subpopulations, those that express depressive-like phenotypes ('susceptible') and those that do not ('resilient'). First, we stratified SDS-exposed mice and measured their voluntary alcohol consumption. Next, we determined whether SI behavior in alcohol-naïve mice could predict alcohol intake. Finally, we assessed the effect of binge-like alcohol exposure on sensitivity to SDS. We quantified Tacr1 (neurokinin-1 receptor gene) and Avp (vasopressin peptide gene) mRNA in brain regions involved in depression, addiction and social behavior. We found that susceptible mice consumed more alcohol compared with resilient mice, suggesting that depression-like phenotypes associate with increased alcohol intake. Interestingly, we observed a negative correlation between SI and alcohol intake in stress- and alcohol-naïve mice, suggesting that individual differences in SI associate with alcohol preference. Finally, alcohol pre-treatment increased sensitivity to SDS, indicating that alcohol exposure alters sensitivity to social stress. Quantification of mRNA revealed that increased expression of Tacr1 and Avp generally associated with decreased SI and increased alcohol intake. C57BL6/J mice are an inbred strain; thus, it is likely that individual differences in behavior and gene expression are driven by epigenetic factors. Collectively, these results support a bidirectional relationship between alcohol exposure and susceptibility to stress that is associated with variations in neuropeptide expression.

Proinflammatory signaling regulates voluntary alcohol intake and stress-induced consumption after exposure to social defeat stress in mice.

Proinflammatory activity has been postulated to play a role in addictive processes and stress responses, but the underlying mechanisms remain largely unknown. Here, we examined the role of interleukin 1 (IL-1) and tumor necrosis factor-α (TNF-α) in regulation of voluntary alcohol consumption, alcohol reward and stress-induced drinking. Mice with a deletion of the IL-1 receptor I gene (IL-1RI KO) exhibited modestly decreased alcohol consumption. However, IL-1RI deletion affected neither the rewarding properties of alcohol, measured by conditioned place preference (CPP), nor stress-induced drinking induced by social defeat stress. TNF-α signaling can compensate for phenotypic consequences of IL1-RI deletion. We therefore hypothesized that double deletion of both IL-1RI and TNF-1 receptors (TNF-1R) may reveal the role of these pathways in regulation of alcohol intake. Double KOs consumed significantly less alcohol than control mice over a range of alcohol concentrations. The combined deletion of TNF-1R and IL-1RI did not influence alcohol reward, but did prevent increased alcohol consumption resulting from exposure to repeated bouts of social defeat stress. Taken together, these data indicate that IL-1RI and TNF-1R contribute to regulation of stress-induced, negatively reinforced drinking perhaps through overlapping signaling events downstream of these receptors, while leaving rewarding properties of alcohol largely unaffected.

DNA methylation in the medial prefrontal cortex regulates alcohol-induced behavior and plasticity.

Recent studies have suggested an association between alcoholism and DNA methylation, a mechanism that can mediate long-lasting changes in gene transcription. Here, we examined the contribution of DNA methylation to the long-term behavioral and molecular changes induced by a history of alcohol dependence. In search of mechanisms underlying persistent rather than acute dependence-induced neuroadaptations, we studied the role of DNA methylation regulating medial prefrontal cortex (mPFC) gene expression and alcohol-related behaviors in rats 3 weeks into abstinence following alcohol dependence. Postdependent rats showed escalated alcohol intake, which was associated with increased DNA methylation as well as decreased expression of genes encoding synaptic proteins involved in neurotransmitter release in the mPFC. Infusion of the DNA methyltransferase inhibitor RG108 prevented both escalation of alcohol consumption and dependence-induced downregulation of 4 of the 7 transcripts modified in postdependent rats. Specifically, RG108 treatment directly reversed both downregulation of synaptotagmin 2 (Syt2) gene expression and hypermethylation on CpG#5 of its first exon. Lentiviral inhibition of Syt2 expression in the mPFC increased aversion-resistant alcohol drinking, supporting a mechanistic role of Syt2 in compulsive-like behavior. Our findings identified a functional role of DNA methylation in alcohol dependence-like behavioral phenotypes and a candidate gene network that may mediate its effects. Together, these data provide novel evidence for DNA methyltransferases as potential therapeutic targets in alcoholism.

microRNA-206 in rat medial prefrontal cortex regulates BDNF expression and alcohol drinking.

Escalation of voluntary alcohol consumption is a hallmark of alcoholism, but its neural substrates remain unknown. In rats, escalation occurs following prolonged exposure to cycles of alcohol intoxication, and is associated with persistent, wide-ranging changes in gene expression within the medial prefrontal cortex (mPFC). Here, we examined whether induction of microRNA (miR) 206 in mPFC contributes to escalated alcohol consumption. Following up on a microarray screen, quantitative real-time reverse transcription PCR (qPCR) confirmed that a history of dependence results in persistent (>3weeks) up-regulation of miR-206 expression in the mPFC, but not in the ventral tegmental area, amygdala, or nucleus accumbens. Viral-mediated overexpression of miR-206 in the mPFC of nondependent rats reproduced the escalation of alcohol self-administration seen following a history of dependence and significantly inhibited BDNF expression. Bioinformatic analysis identified three conserved target sites for miR-206 in the 3'-UTR of the rat BDNF transcript. Accordingly, BDNF was downregulated in post-dependent rats on microarray analysis, and this was confirmed by qPCR. In vitro, BDNF expression was repressed by miR-206 but not miR-9 in a 3'-UTR reporter assay, confirming BDNF as a functional target of miR-206. Mutation analysis showed that repression was dependent on the presence of all three miR-206 target sites in the BDNF 3'-UTR. Inhibition of miR-206 expression in differentiated rat cortical primary neurons significantly increased secreted levels of BDNF. In conclusion, recruitment of miR-206 in the mPFC contributes to escalated alcohol consumption following a history of dependence, with BDNF as a possible mediator of its action.

The neurokinin-1 receptor in addictive processes.

Stress can trigger drug-seeking behavior, increase self-administration rates, and enhance drug reward. A number of stress-related neuropeptides have been shown to mediate these behavioral processes. The most studied peptide in this category is corticotropin-releasing hormone (CRH), which has been shown to mediate stress-induced reinstatement of drug seeking, escalated self-administration, and drug withdrawal, but it does not seem to be involved in baseline drug self-administration or cue-induced reinstatement. This pattern of effects holds for many classes of drugs, including alcohol, opiates, and psychostimulants. The neurokinin-1 receptor (NK1R) is the preferred receptor for the endogenous stress-related neuropeptide substance P (SP). The SP/NK1R system is a major mediator of stress and anxiety, and over the last several years, it has been demonstrated that the SP/NK1R system can have effects similar to those of CRH on drug taking and drug seeking. Specifically, NK1R inhibition attenuates escalated self-administration of alcohol as well as stress-induced reinstatement of alcohol and cocaine seeking; however, in contrast to other stress systems, the NK1R also appears to have a role in primary reward and reinforcement for opiates. This review outlines the role of NK1R in drug-seeking behaviors and highlights recent results from clinical studies that suggest that the NK1R may be a promising drug target going forward.

Sex differences in neuronal activation during aversion-resistant alcohol consumption.

BACKGROUND: One of the DSM-5 criteria for Alcohol Use Disorder is continued alcohol consumption despite negative consequences. This has been modeled in mice using adulteration of alcohol solution with the bitter tastant quinine. Mice that continue to consume alcohol despite this adulteration are considered aversion resistant. The limited number of studies dissecting the underlying neuronal mechanisms of aversion-resistant drinking behaviors used only male subjects. We have previously shown that female mice are more resistant to quinine adulteration of alcohol than males. Our aim here is to identify potential sex differences in neuronal activation that may underlie this behavior. METHODS: Male and female C57BL/6J mice were allowed continuous access to 20% alcohol in a two-bottle choice procedure. To test aversion-resistance, the alcohol was adulterated with increasing concentrations (0.03, 0.1, and 0.2 mM) of quinine hydrochloride. After consumption rates were calculated, brains were extracted to examine neuronal activation using Fos immunohistochemistry. RESULTS: We found that female mice suppressed their intake to a lesser extent than males when the alcohol solution was adulterated with quinine. Our Fos staining revealed three regions of interest that exhibit a sex difference during quinine-adulterated alcohol drinking: the ventromedial prefrontal cortex (vmPFC), the posterior insular cortex (PIC), and the ventral tegmental area (VTA). Both the vmPFC and the PIC exhibited higher neuronal activation in males during quinine-adulterated alcohol consumption. However, females showed higher Fos activation in the VTA during quinine-adulterated alcohol consumption. CONCLUSIONS: Females more readily exhibit aversion-resistant alcohol intake than their male counterparts and exhibit some differences in neuronal activation patterns. We conclude that there are sex differences in neurocircuitry that may underlie compulsive drinking behaviors.

Estradiol mediates sex differences in aversion-resistant alcohol intake.

Introduction: Alcohol consumption despite negative consequences is a core symptom of alcohol use disorder. This can be modeled in mice by pairing aversive stimuli with alcohol consumption, such as adding the bitter tastant quinine to the alcohol solution. If an animal continues to drink alcohol despite such negative stimuli, this is typically considered aversion-resistant, or inflexible, drinking behavior. Previous studies in our lab have found that females are more aversion-resistant than males in that they tolerate higher concentrations of quinine before they suppress their alcohol intake. Interestingly, we did not observe any differences in intake across the estrous cycle. In regards to neuronal activation patterns during quinine-alcohol intake, we have found that male mice show higher levels of activation in the ventromedial prefrontal cortex and posterior insular cortex, while females show higher levels of activation in the ventral tegmental area. Methods: In the experiments presented here, we conducted ovariectomies to further examine the role of circulating sex hormones in aversion-resistant alcohol intake and neuronal activation patterns. Furthermore, we used hormonal addback of estradiol or progesterone to determine which ovarian sex hormone mediates aversion-resistant consumption. Results: We found that ovariectomy reduced quinine-adulterated alcohol intake, demonstrating that circulating sex hormones play a role in this behavior. We also observed reduced neuronal activation in the VTA of ovariectomized mice compared to sham females, and that estradiol supplementation reversed the effect of ovariectomy on quinine-alcohol intake. Discussion: Taken together with our prior data, these findings suggest that circulating estradiol contributes to the expression of aversion-resistant alcohol intake and neuronal activity in the VTA. However, since this behavior is not affected by the estrous cycle, we believe this is due to a threshold level of this hormone, as opposed to fluctuations that occur across the estrous cycle.

The kappa opioid receptor antagonist JDTic attenuates alcohol seeking and withdrawal anxiety.

The role of kappa-opioid receptors (KOR) in the regulation of alcohol-related behaviors is not completely understood. For example, alcohol consumption has been reported to increase following treatment with KOR antagonists in rats, but was decreased in mice with genetic deletion of KOR. Recent studies have further suggested that KOR antagonists may selectively decrease alcohol self-administration in rats following a history of dependence. We assessed the effects of the KOR antagonist JDTic on alcohol self-administration, reinstatement of alcohol seeking induced by alcohol-associated cues or stress, and acute alcohol withdrawal-induced anxiety ('hangover anxiety'). JDTic dose-dependently reversed hangover anxiety when given 48 hours prior to testing, a time interval corresponding to the previously demonstrated anxiolytic efficacy of this drug. In contrast, JDTic decreased alcohol self-administration and cue-induced reinstatement of alcohol seeking when administered 2 hours prior to testing, but not at longer pre-treatment times. For comparison, we determined that the prototypical KOR antagonist nor-binaltorphimine can suppress self-administration of alcohol at 2 hours pre-treatment time, mimicking our observations with JDTic. The effects of JDTic were behaviorally specific, as it had no effect on stress-induced reinstatement of alcohol seeking, self-administration of sucrose, or locomotor activity. Further, we demonstrate that at a 2 hours pre-treatment time JDTic antagonized the antinociceptive effects of the KOR agonist U50,488H but had no effect on morphine-induced behaviors. Our results provide additional evidence for the involvement of KOR in regulation of alcohol-related behaviors and provide support for KOR antagonists, including JDTic, to be evaluated as medications for alcoholism.

The effect of concurrent access to alcohol and oxycodone on self-administration and reinstatement in rats.

RATIONALE: Although polysubstance use is highly prevalent, preclinical studies that assess voluntary consumption of multiple substances at the same time are rather uncommon. Overlooking drug taking patterns commonly observed in humans may limit the translational value of preclinical models. OBJECTIVES: Here, we aimed to develop a model of polysubstance use that could be used to assess oral operant self-administration patterns under concurrent access to alcohol and the prescription opioid oxycodone. METHODS: After a training period where animals associated specific cues and levers with each drug, rats self-administered alcohol and oxycodone solutions concurrently in daily sessions. Oxycodone was then removed to assess potential changes in alcohol consumption. The role of cues and stress on alcohol consumption and oxycodone seeking was also examined under reinstatement conditions. RESULTS: We found that females consumed more alcohol and oxycodone than males when given access to both drugs, and this effect on alcohol intake persisted when oxycodone was removed. Additionally, re-exposure to oxycodone cues in combination with the administration of the pharmacological stressor yohimbine drove reinstatement of oxycodone seeking in females but did not have a strong effect in males, possibly due to low levels of oxycodone intake during active self-administration in male rats. Additionally, yohimbine drove increased alcohol consumption, in line with prior findings from our group and others. CONCLUSIONS: Taken together, this study demonstrates that rats will concurrently self-administer both oxycodone and alcohol in operant chambers, and this procedure can serve as a platform for future investigations in polysubstance use and relapse-like behavior.

Neurokinin receptors in drug and alcohol addiction.

The neurokinins are a class of peptide signaling molecules that mediate a range of central and peripheral functions including pain processing, gastrointestinal function, stress responses, and anxiety. Recent data have linked these neuropeptides with drug-related behaviors. Specifically, substance P (SP) and neurokinin B (NKB), have been shown to influence responses to alcohol, cocaine, and/or opiate drugs. SP and NKB preferentially bind to the neurokinin-1 receptor (NK1R) and neurokinin-3 receptor (NK3R), respectively, but do have some affinity for all classes of neurokinin receptor at high concentrations. NK1R activity has been shown to influence reward and reinforcement for opiate drugs, stimulatory and neurochemical responses to cocaine, and escalated and stress-induced alcohol seeking. In reinstatement models of relapse-like behavior, NK1R antagonism attenuates stress-induced reinstatement for all classes of drugs tested to date. The NK3R also influences alcohol intake and behavioral/neurochemical responses to cocaine, but less research has been performed in regard to this particular receptor in preclinical models of addiction. Clinically, agents targeting these receptors have shown some promise, but have produced mixed results. Here, the preclinical findings for the NK1R and NK3R are reviewed, and discussion is provided to interpret clinical findings. Additionally, important factors to consider in regards to future clinical work are suggested.

Increased Alcohol Consumption in Mice Lacking Sodium Bicarbonate Transporter NBCn1.

The previous reports on an addiction vulnerability marker in the human SLC4A7 gene encoding the Na/HCO3 transporter NBCn1 suggest that this pH-regulating protein may affect alcohol-related behavior and response. Here, we examined alcohol consumption and sensitivity to the sedative effects of alcohol in male NBCn1 knockout mice. These mice displayed lower pH in neurons than wildtype controls, determined by intracellular pH in hippocampal neuronal cultures. Neurons from knockout mice had a higher action potential threshold and a more depolarized membrane potential, thus reducing membrane excitability. In a two-bottle free choice procedure, knockout mice consumed more alcohol than controls and consistently increased alcohol consumption after repeated alcohol deprivation periods. Quinine and sucrose preference was similar between genotypes. Knockout mice showed increased propensity for alcohol-induced conditioned place preference. In loss of righting reflex assessment, knockout mice revealed increased sensitivity to alcohol-induced sedation and developed tolerance to the sedation after repeated alcohol administrations. Furthermore, chronic alcohol consumption caused NBCn1 downregulation in the hippocampus and striatum of mice and humans. These results demonstrate an important role of NBCn1 in regulation of alcohol consumption and sensitivity to alcohol-induced sedation.

Escalated Alcohol Self-Administration and Sensitivity to Yohimbine-Induced Reinstatement in Alcohol Preferring Rats: Potential Role of Neurokinin-1 Receptors in the Amygdala.

Genetic factors significantly contribute to the risk for developing alcoholism. To study these factors and other associated phenotypes, rodent lines have been developed using selective breeding for high alcohol preference. One of these models, the alcohol preferring (P) rat, has been used in hundreds of preclinical studies over the last few decades. However, very few studies have examined relapse-like behavior in this rat strain. In this study, we used operant self-administration and yohimbine-induced reinstatement models to examine relapse-like behavior in P rats. Our previous work has demonstrated that P rats show increased expression of the neurokinin-1 receptor (NK1R) in the central nucleus of the amygdala (CeA), and this functionally contributes to escalated alcohol consumption in this strain. We hypothesized that P rats would show increased sensitivity to yohimbine-induced reinstatement that is also mediated by NK1R in the CeA. Using Fos staining, site-specific infusion of NK1R antagonist, and viral vector overexpression, we examined the influence of NK1R on the sensitivity to yohimbine-induced reinstatement of alcohol seeking. We found that P rats displayed increased sensitivity to yohimbine-induced reinstatement as well as increased neuronal activation in the CeA after yohimbine injection compared to the control Wistar strain. Intra-CeA infusion of NK1R antagonist attenuates yohimbine-induced reinstatement in P rats. Conversely, upregulation of NK1R within the CeA of Wistar rats increases alcohol consumption and sensitivity to yohimbine-induced reinstatement. These findings suggest that NK1R upregulation in the CeA contributes to multiple alcohol-related phenotypes in the P rat, including alcohol consumption and sensitivity to relapse.