Mu-opioid receptor knockout on Foxp2-expressing neurons leads to reduced aversion-resistant reward seeking.

Mu-opioid receptors (MORs) in the amygdala and striatum are important in addictive and rewarding behaviors. Foxp2 is a marker of intercalated (ITC) cells in the amygdala and a subset of striatal medium spiny neurons (MSNs), both of which express MORs in wild-type mice. For the current series of studies, we characterized the behavior of mice with genetic deletion of the MOR gene Oprm1 in Foxp2-expressing neurons (Foxp2-Cre/Oprm1 fl/fl ). Male and female Foxp2-Cre/Oprm1 fl/fl mice were generated and heterozygous Cre+ (knockout) and homozygous Cre-(control) animals were tested for aversion-resistant alcohol consumption using an intermittent access (IA) task, operant responding for a sucrose reward, conditioned place aversion (CPA) to morphine withdrawal, and locomotor sensitization to morphine. In IA, mice with the MOR-knockout were more sensitive to quinine-adulterated ethanol (EtOH) and less aversion-resistant, as they decreased EtOH consumption from baseline at all quinine concentrations, while control animals did not. In operant conditioning, Cre+ mice similarly exhibited less aversion-resistant reward seeking than Cre-mice when sucrose was adulterated with quinine. For CPA, both control and MOR-knockout mice demonstrated withdrawal-induced aversion. For locomotor sensitization, Cre+ mice demonstrated decreased locomotion following morphine injection compared to Cre-mice. The results of these studies suggest that MOR expression on Foxp2-expressing neurons is not necessary for rewarded behaviors or expression of opioid withdrawal but may be involved in aversion-resistance.

Cholinergic mu-opioid receptors participate in nicotine, but not ethanol, drinking behaviors.

Heavy alcohol use and binge drinking are important contributors to alcohol use disorder (AUD). The endogenous opioid system has been implicated in alcohol consumption and preference in both humans and animals. The mu opioid receptor (MOR) is expressed on multiple cells in the striatum, however little is known about the contributions of specific MOR populations to alcohol drinking behaviors. The current study used mice with a genetic deletion of MOR in cholinergic cells (ChAT-Cre/Oprm1 fl/fl ) to examine the role of MORs expressed in cholinergic interneurons (CINs) in home cage self-administration paradigms. Male and female ChAT-Cre/Oprm1 fl/fl mice were generated and heterozygous Cre+ (knockout) and Cre-(control) mice were tested for alcohol and nicotine consumption. In Experiment 1, binge-like and quinine-resistant drinking was tested using 15% ethanol (EtOH) in a two-bottle, limited-access Drinking in the Dark paradigm. Experiment 2 involved a six-week intermittent access paradigm in which mice received 20% EtOH, nicotine, and then a combination of the two drugs. Deleting MORs in cholinergic cells did not alter consumption of EtOH in Experiment 1 or 2. In Experiment 1, the MOR deletion resulted in greater consumption of quinine-adulterated EtOH in male Cre+ mice (vs. Cre-). In Experiment 2, Cre+ mice demonstrated a significantly lower preference for nicotine but did not differ from Cre-mice in nicotine or nicotine + EtOH consumption. These data suggest that cholinergic MORs are involved in nicotine, but not EtOH, drinking behaviors and may contribute to aversion resistant EtOH drinking in a sex-dependent manner.

Removal of the ovaries suppresses ethanol drinking and promotes aversion-resistance in C57BL/6J female mice.

RATIONALE: Female rodents consume more ethanol (EtOH) than males and exhibit greater aversion-resistant drinking in some paradigms. Ovarian hormones promote EtOH drinking but the contribution of ovarian hormones to aversion-resistant drinking has not been assessed. OBJECTIVES: We aimed to investigate the role of ovarian hormones to aversion-resistant drinking in female mice in a drinking in the dark (DID) task. METHODS: Female C57BL/6 J mice first underwent an ovariectomy (OVX, n = 16) or sham (SHAM, n = 16) surgery. Four weeks following surgery, mice underwent a DID paradigm where they were given access to water and 15% EtOH 3 h into the dark cycle for up to 4 h across 15 drinking sessions. To assess frontloading behavior, bottles were weighed at 30 min, 2 h, and 4 h. Aversion-resistance was tested by adding escalating concentrations of quinine (0, 100, 250, and 500 µM) to the 15% EtOH bottle on sessions 16 - 19. RESULTS: Removal of the ovaries reduced EtOH consumption in OVX subjects. When assessing aversion-resistant EtOH drinking, mice with ovarian hormones (SHAM) reduced consumption of 250 and 500 µM quinine in EtOH, while OVX subjects exhibited aversion-resistance at all quinine concentrations. OVX mice had greater frontloading for quinine + EtOH at higher concentrations of quinine. CONCLUSIONS: These results indicate that circulating ovarian hormones may be protective against the development of aversion-resistant EtOH drinking and call for further investigation of the role of ovarian hormones in models of addictive behavior.

Opioid withdrawal: role in addiction and neural mechanisms.

Withdrawal from opioids involves a negative affective state that promotes maintenance of drug-seeking behavior and relapse. As such, understanding the neurobiological mechanisms underlying withdrawal from opioid drugs is critical as scientists and clinicians seek to develop new treatments and therapies. In this review, we focus on the neural systems known to mediate the affective and somatic signs and symptoms of opioid withdrawal, including the mesolimbic dopaminergic system, basolateral amygdala, extended amygdala, and brain and hormonal stress systems. Evidence from preclinical studies suggests that these systems are altered following opioid exposure and that these changes mediate behavioral signs of negative affect such as aversion and anxiety during withdrawal. Adaptations in these systems also parallel the behavioral and psychological features of opioid use disorder (OUD), highlighting the important role of withdrawal in the development of addictive behavior. Implications for relapse and treatment are discussed as well as promising avenues for future research, with the hope of promoting continued progress toward characterizing neural contributors to opioid withdrawal and compulsive opioid use.

Sex chromosome and gonadal hormone contributions to binge-like and aversion-resistant ethanol drinking behaviors in Four Core Genotypes mice.

Introduction: While substantial research has focused on the contribution of sex hormones to driving elevated levels of alcohol drinking in female rodents, fewer studies have investigated how genetic influences may underlie sex differences in this behavior. Methods: We used the Four Core Genotypes (FCG) mouse model to explore the contribution of sex chromosome complement (XX/XY) and gonad type [ovaries (Sry-)/testes (Sry+)] to ethanol (EtOH) consumption and quinine-resistant drinking across two voluntary self-administration tasks: limited access consumption in the home cage and an operant response task. Results: For limited access drinking in the dark, XY/Sry + (vs. XX/Sry +) mice consumed more 15% EtOH across sessions while preference for 15% EtOH vs. water was higher in XY vs. XX mice regardless of gonad type. XY chromosomes promoted quinine-resistant drinking in mice with ovaries (Sry-) and the estrous cycle did not affect the results. In the operant response task, responding for EtOH was concentration dependent in all genotypes except XX/Sry + mice, which maintained consistent response levels across all concentrations (5-20%) of EtOH. When increasing concentrations of quinine (100-500 µM) were added to the solution, FCG mice were insensitive to quinine-punished EtOH responding, regardless of sex chromosome complement. Sry + mice were further found to be insensitive to quinine when presented in water. Importantly, these effects were not influenced by sensitivity to EtOH's sedative effect, as no differences were observed in the time to lose the righting reflex or the time to regain the righting reflex between genotypes. Additionally, no differences in EtOH concentration in the blood were observed between any of the genotypes once the righting reflex was regained. Discussion: These results provide evidence that sex chromosome complement regulates EtOH consumption, preference, and aversion resistance and add to a growing body of literature suggesting that chromosomal sex may be an important contributor to alcohol drinking behaviors. Examination of sex-specific genetic differences may uncover promising new therapeutic targets for high-risk drinking.

Greater resistance to footshock punishment in female C57BL/6J mice responding for ethanol.

BACKGROUND: One characteristic of alcohol use disorder is compulsive drinking or drinking despite negative consequences. When quinine is used to model such aversion-resistant drinking, female rodents typically are more resistant to punishment than males. Using an operant response task where C57BL/6J responded for ethanol mixed with quinine, we previously demonstrated that female mice tolerate higher concentrations of quinine in ethanol than males. Here, we aimed to determine whether this female vulnerability to aversion-resistant drinking behavior is similarly observed with footshock punishment. METHODS: Male and female C57BL/6J mice were trained to respond for 10% ethanol in an operant task on a fixed-ratio three schedule. After consistent responding, mice were tested in a punishment session using either a 0.25 mA or 0.35 milliamp (mA) footshock. To assess footshock sensitivity, a subset of mice underwent a flinch, jump, and vocalize test in which behavioral responses to increasing amplitudes of footshock (0.05 to 0.95 mA) were assessed. In a separate cohort of mice, males and females were trained to respond for 2.5% sucrose and responses were punished using a 0.25 mA footshock. RESULTS: Males and females continued to respond for 10% ethanol when paired with a 0.25 mA footshock. Females alone continued to respond for ethanol when a 0.35 mA footshock was delivered. Both males and females reduced responding for 2.5% sucrose when punished with a 0.25 mA footshock. Footshock sensitivity in the flinch, jump, and vocalize test did not differ by sex. CONCLUSIONS: Females continue to respond for 10% ethanol despite a 0.35 mA footshock, and this behavior is not due to differences in footshock sensitivity between males and females. These results show that female C57BL/6J mice are generally more resistant to punishment in an operant self-administration paradigm. The findings add to the literature characterizing aversion-resistant alcohol-drinking behaviors in females.

Sex, but not early life stress, effects on two-bottle choice alcohol drinking behaviors in mice.

In humans, early life stress (ELS) is associated with an increased risk for developing both alcohol use disorder (AUD) and post-traumatic stress disorder (PTSD). We have previously used an infant footshock model to explore this shared predisposition. Infant footshock produces stress-enhanced fear learning (SEFL) in rats and mice and increases aversion-resistant alcohol drinking in rats. The goal of the current study was to extend this model of comorbid PTSD and AUD to male and female C57BL/6J mice. Acute ELS was induced using 15 foot-shocks on postnatal day 17. In adulthood, after PND 90, ethanol drinking behavior was tested in one of three two-bottle choice drinking paradigms: continuous access, limited access drinking in the dark, or intermittent access. In continuous access, mice were given 24 h access to 5% or 10% ethanol and water. Each ethanol concentration was provided for five consecutive drinking sessions. In limited access drinking in the dark, mice were given 2 h of access to 15% ethanol and water across 15 sessions. Ethanol was provided 3 h into the dark cycle to maximize task engagement when mice are most active. In intermittent access, mice were presented with 20% ethanol and water Monday, Wednesday, and Friday, for four consecutive weeks. In a fifth week of intermittent access drinking, increasing concentrations of quinine (10 mg/L, 100 mg/L, and 200 mg/L) were added to the ethanol to test aversion-resistant drinking. Our results indicate that infant footshock does not influence adult ethanol consumption in mice. Infant footshock did not affect ethanol-only consumption or preference in any of the three drinking paradigms. Further, and in contrast to our previous results in rats, infant footshock did not appear to influence consumption of quinine-adulterated ethanol. The biological sex of the mice did affect ethanol-only consumption in all three drinking paradigms, with females consuming more ethanol than males. Preference for ethanol vs. water was higher in females only under continuous access conditions. Our results suggest that infant footshock alone may not be sufficient to increase drinking levels in mice. We hypothesize that infant footshock may require a secondary, adolescent stress exposure to influence ethanol drinking behavior. Further research is needed to create a valid model of PTSD-AUD comorbidity in male and female mice.

Crossed high alcohol preferring mice exhibit aversion-resistant responding for alcohol with quinine but not footshock punishment.

A symptom of alcohol use disorder (AUD) is compulsive drinking, or drinking that persists despite negative consequences. In mice, aversion-resistant models are used to model compulsive-like drinking by pairing the response for alcohol with a footshock or by adding quinine, a bitter tastant, to the alcohol solution. crossed High Alcohol Preferring (cHAP) mice, a selectively bred line of mice that consumes pharmacologically relevant levels of alcohol, demonstrate a high level of aversion-resistance to quinine-adulterated alcohol. The current study investigated quinine-resistant and footshock-resistant responding for 10% ethanol in male and female cHAP mice with vs. without a history of alcohol exposure. cHAP mice were first trained to respond for 10% ethanol in an operant-response task. Next, mice were exposed to water or 10% ethanol for twelve 24-h sessions using a two-bottle choice procedure. Footshock-resistant ethanol responding was then tested in the operant chamber by pairing a footshock (0.35 mA) with the nose-poke response during one session. Quinine-resistant responding for alcohol was tested over five sessions (500-2500 µM quinine). Finally, footshock sensitivity was assessed using a flinch, jump, vocalize test. Alcohol exposure history did not influence responses for 10% ethanol or either measure of aversion-resistance. Further, cHAP mice were sensitive to footshock punishment but continued to respond for alcohol at all quinine concentrations. No sex differences were observed in any measure of alcohol responding, but female cHAP mice were less sensitive to footshock than males. These results replicate and extend the previous demonstration of a robust, innate resistance to quinine aversion in cHAP mice and further suggest that this tendency is not observed when footshock is used to punish drinking.

Gonadal hormones and sex chromosome complement differentially contribute to ethanol intake, preference, and relapse-like behaviour in four core genotypes mice.

Alcohol use and high-risk alcohol drinking behaviours among women are rapidly rising. In rodent models, females typically consume more ethanol (EtOH) than males. Here, we used the four core genotypes (FCG) mouse model to investigate the influence of gonadal hormones and sex chromosome complement on EtOH drinking behaviours. FCG mice were given access to escalating concentrations of EtOH in a two-bottle, 24-h continuous access drinking paradigm to assess consumption and preference. Relapse-like behaviour was measured by assessing escalated intake following repeated cycles of deprivation and re-exposure. Twenty-four-hour EtOH consumption was greater in mice with ovaries (Sry-), relative to those with testes, and in mice with the XX chromosome complement, relative to those with XY sex chromosomes. EtOH preference was higher in XX versus XY mice. For both consumption and preference, the influences of the Sry gene and sex chromosomes were concentration dependent. Escalated intake following repeated cycles of deprivation and re-exposure emerged only in XX mice (vs. XY). Mice with ovaries (Sry- FCG mice and C57BL/6J females) were also found to consume more water than mice with testes. These results demonstrate that aspects of EtOH drinking behaviour may be independently regulated by sex hormones and chromosomes and inform our understanding of the neurobiological mechanisms which contribute to EtOH dependence in male and female mice. Future investigation of the contribution of sex chromosomes to EtOH drinking behaviours is warranted. We used the FCG mouse model to investigate the influence of gonadal hormones and sex chromosome complement on EtOH drinking behaviours, including the alcohol deprivation effect. Escalated intake following repeated cycles of deprivation and re-exposure emerged only in XX mice (vs. XY). These results demonstrate that aspects of EtOH drinking behaviour may be independently regulated by sex hormones and chromosomes.

Orbitofrontal cortex subregion inhibition during binge-like and aversion-resistant alcohol drinking.

Two important contributors to alcohol-related problems and alcohol use disorder (AUD) are binge- and compulsive-like drinking. The orbitofrontal cortex (OFC), a brain region implicated in outcome valuation and behavioral flexibility, is functionally altered by alcohol exposure. Data from animal models also suggest that both the medial (mOFC) and lateral (lOFC) subregions of the OFC regulate alcohol-related behaviors. The current study was designed to examine the contributions of mOFC and lOFC using a model of binge-like and aversion-resistant ethanol drinking in C57BL/6J male and female mice. The inhibitory Designer Receptor Exclusively Activated by Designer Drugs (DREADD) hM4Di were used to inhibit neurons in either the mOFC or the lOFC in mice drinking 15% ethanol in a two-bottle, limited-access, modified drinking in the dark paradigm. The effects of chemogenetic inhibition on consumption of quinine-adulterated ethanol, water, and water + quinine were also assessed. Inhibiting the mOFC did not alter consumption of ethanol or aversion-resistant drinking of ethanol + quinine. In contrast, inhibition of neurons in the lOFC increased consumption, but not preference, of ethanol alone. mOFC and lOFC inhibition did not alter water or quinine-adulterated water intake, indicating the effects shown here are specific to ethanol drinking. These data support the role of the lOFC in regulating alcohol consumption but fail to find a similar role for mOFC.

Recent Perspectives on Sex Differences in Compulsion-Like and Binge Alcohol Drinking.

Alcohol use disorder remains a substantial social, health, and economic problem and problem drinking levels in women have been increasing in recent years. Understanding whether and how the underlying mechanisms that drive drinking vary by sex is critical and could provide novel, more targeted therapeutic treatments. Here, we examine recent results from our laboratories and others which we believe provide useful insights into similarities and differences in alcohol drinking patterns across the sexes. Findings for binge intake and aversion-resistant, compulsion-like alcohol drinking are considered, since both are likely significant contributors to alcohol problems in humans. We also describe studies regarding mechanisms that may underlie sex differences in maladaptive alcohol drinking, with some focus on the importance of nucleus accumbens (NAcb) core and shell regions, several receptor types (dopamine, orexin, AMPA-type glutamate), and possible contributions of sex hormones. Finally, we discuss how stressors such as early life stress and anxiety-like states may interact with sex differences to contribute to alcohol drinking. Together, these findings underscore the importance and critical relevance of studying female and male mechanisms for alcohol and co-morbid conditions to gain a true and clinically useful understanding of addiction and neuropsychiatric mechanisms and treatment.

Studying Sex Differences in Rodent Models of Addictive Behavior.

Animal models of addictive behaviors are useful for uncovering neural mechanisms involved in the development of dependence and for identifying risk factors for drug abuse. One such risk factor is biological sex, which strongly moderates drug self-administration behavior in rodents. Female rodents are more likely to acquire drug self-administration behaviors, consume higher amounts of drug, and reinstate drug-seeking behavior more readily. Despite this female vulnerability, preclinical addiction research has largely been done in male animals. The study of sex differences in rodent models of addictive behavior is increasing, however, as more investigators are choosing to include both male and female animals in experiments. This commentary is meant to serve as an introductory guide for preclinical investigators new to the study of sex differences in addiction. We provide an overview of self-administration models, a broad view of female versus male self-administration behaviors, and suggestions for study design and implementation. Inclusion of female subjects in preclinical addiction research is timely, as problem drug and alcohol use in women is increasing. With proper attention, design, and analysis, the study of sex differences in addiction has the potential to uncover novel neural mechanisms and lead to greater translational success for addiction research. © 2021 Wiley Periodicals LLC.

Advances in understanding meso-cortico-limbic-striatal systems mediating risky reward seeking.

The risk of an aversive consequence occurring as the result of a reward-seeking action can have a profound effect on subsequent behavior. Such aversive events can be described as punishers, as they decrease the probability that the same action will be produced again in the future and increase the exploration of less risky alternatives. Punishment can involve the omission of an expected rewarding event ("negative" punishment) or the addition of an unpleasant event ("positive" punishment). Although many individuals adaptively navigate situations associated with the risk of negative or positive punishment, those suffering from substance use disorders or behavioral addictions tend to be less able to curtail addictive behaviors despite the aversive consequences associated with them. Here, we discuss the psychological processes underpinning reward seeking despite the risk of negative and positive punishment and consider how behavioral assays in animals have been employed to provide insights into the neural mechanisms underlying addictive disorders. We then review the critical contributions of dopamine signaling to punishment learning and risky reward seeking, and address the roles of interconnected ventral striatal, cortical, and amygdala regions to these processes. We conclude by discussing the ample opportunities for future study to clarify critical gaps in the literature, particularly as related to delineating neural contributions to distinct phases of the risky decision-making process.

The contribution of medium spiny neuron subtypes in the nucleus accumbens core to compulsive-like ethanol drinking.

Compulsive alcohol use, or drinking that persists despite negative or aversive consequences, is a defining characteristic of alcohol use disorder. Here, chemogenetic technology (i.e. Designer Receptors Exclusively Activated by Designer Drugs; DREADDs) was used to inhibit or excite the NAc core or selectively inhibit D1-or D2 receptor-expressing neurons in the NAc core to understand the role of the NAc core and how these subpopulations of neurons may influence compulsive-like ethanol (EtOH) drinking using C57BL/6J, Drd1-cre, and Drd2-cre male and female mice. Compulsive-like EtOH drinking was modeled with a two-bottle choice, drinking in the dark paradigm. The major finding of this study was that mice decreased compulsive-like EtOH intake when the NAc core was inhibited and there was no change of EtOH + quinine intake when the NAc core was excited. Interestingly, inhibition of D1-or D2 receptor-expressing neurons did not alter compulsive-like EtOH intake. Control experiments showed that NAc core excitation and selective inhibition of D1-or D2-receptor-expressing neurons had no effect on baseline EtOH drinking, intake of water, or intake of quinine-adulterated water. CNO reduced amphetamine-induced locomotion in the D1-CRE+ (but not the D2CRE+) group in a control experiment. Finally, pharmacological antagonism of D1 and D2 receptors together, but not separately, reduced quinine-resistant EtOH drinking. These results suggest that the NAc core is a critical region involved in compulsive-like EtOH consumption, and that both D1-and D2 receptor-expressing medium spiny neurons participate in controlling this behavior.

Selective enhancement of fear learning and resistance to extinction in a mouse model of acute early life trauma.

Early life stress (ELS) experiences can cause changes in cognitive and affective functioning. This study examined the persistent effects of a single traumatic event in infancy on several adult behavioral outcomes in male and female C57BL/6J mice. Mice received 15 footshocks in infancy and were tested for stress-enhanced fear learning, extinction learning, discrimination and reversal learning, and novel object recognition. Infant trauma potentiated fear learning in adulthood and produced resistance to extinction but did not influence other behaviors, suggesting restricted effects of infant trauma on behaviors reliant on cortico-amygdala circuitry.

Aversion-resistant fentanyl self-administration in mice.

RATIONALE: Animal models of compulsive drug use that continues despite negative consequences can be used to investigate the neural mechanisms of addiction. However, models of punished or aversion-resistant opioid self-administration are notably lacking. OBJECTIVES: We sought to develop an aversion-resistant, oral fentanyl self-administration paradigm. METHODS: In Experiment 1, C57BL/6J male and female, adult mice consumed fentanyl (10 µg/mL) in a two-bottle drinking in the dark task and escalating concentrations of quinine were added to the bottles. In Experiment 2, mice were trained to administer oral fentanyl (10 µg/mL) in an operant response task. Quinine was next added to the fentanyl solution in escalating concentrations. In Experiment 3, mice were trained to respond for oral fentanyl or fentanyl adulterated with 500 µM quinine on every session. In Experiment 4, mice were trained to respond for a 1% sucrose solution before introduction of quinine. RESULTS: Quinine reduced two-bottle choice consumption in males but not in females. Both sexes demonstrated the ability to detect the selected concentrations of quinine in fentanyl. In the operant chamber, mice responded robustly for oral fentanyl but introduction of quinine at any stage of training was insufficient to reduce responding. In contrast, quinine reduced responding for sucrose at concentrations above 250 µM. CONCLUSIONS: Mice will respond for and consume oral fentanyl in both a two-bottle choice and an operant response task. Quinine is detectable in fentanyl but mice will continue to respond for and consume fentanyl with quinine in both paradigms. These data support the use of these models in behavioral studies of compulsive-like opioid use.

Improved visual discrimination learning in mice with partial 5-HT2B gene deletion.

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) has been linked to multiple aspects of cognition. For example, in rodents, discrimination and reversal learning are altered by experimentally induced changes in brain serotonin levels, and reduced expression of the 5-HT2B receptor subtype in mice and humans is associated with decreased serotonergic tone and increased behavioral impulsivity. Serotonin modulates cognitive flexibility as well as fear and anxiety, but the specific contributions of 5-HT2B receptors to these behaviors is unknown. The current study assessed mice with partial Htr2b deletion for performance on a touchscreen-based pairwise visual discrimination and reversal learning task followed by a test of cued fear learning. Male Htr2b heterozygous mice (+/-) and littermate controls (+/+) were trained to discriminate between two visual stimuli presented on a touch-sensitive screen, one which predicted delivery of a 14-mg food pellet and the other which was not rewarded. Once discrimination performance criterion was attained, the stimulus-reward contingencies were reversed. Htr2b +/- mice were faster to reach discrimination criterion than +/+ controls, and made fewer errors. Htr2b +/- mice were also slower to make responses and collect rewards. Conversely, measures of reversal learning were not different between genotypes. Pavlovian cued fear conditioning was also normal in Htr2b +/-mice. These data demonstrate a selective improvement in touchscreen-based discrimination learning in mice with partial deletion of the 5-HT2B receptor, and provide further insight into the role of the 5-HT2B receptor in cognition.

Increased Responding for Alcohol and Resistance to Aversion in Female Mice.

BACKGROUND: More women are being diagnosed with alcohol use disorder (AUD), are increasing the amount of alcohol they are drinking, and are partaking in risky drinking behaviors. Compulsive drinking which persists despite negative consequences is a hallmark of AUD. Preclinical aversion-resistant models suggest that females may be more vulnerable to the rewarding effects of alcohol such that they show increased compulsivity when drinking is punished with quinine, a bitter tastant. METHODS: Male and female C57BL/6J mice were trained in an operant response task on a first-order fixed ratio schedule. Experiment 1 tested responding for escalating concentrations (10 to 25%) of ethanol (EtOH). Experiment 2 assessed the effects of increasing concentrations of quinine (100, 250, or 500 µM) on responding for 10% EtOH followed by a 48-hour 2-bottle choice quinine preference test. Experiment 3 investigated the effects of increasing concentrations of quinine (100, 250, or 500 µM) on responding for 2.5% sucrose. RESULTS: Experiment 1 revealed that females respond more than males for 15% EtOH. Experiment 2 showed that females tolerate higher concentrations of quinine in EtOH than males. Males reduced responding for 10% EtOH when adulterated with 250 or 500 µM of quinine, while females did not reduce responding at any concentration of quinine. Males and females also exhibited similar preference for quinine in a 2-bottle drinking task. Experiment 3 demonstrated that both males and females reduced responding for 2.5% sucrose when quinine (100, 250, or 500 µM) was added. CONCLUSIONS: Females respond more for EtOH at higher concentrations and continue to respond for 10% EtOH at all concentrations of quinine, suggesting that female mice are more motivated to respond for EtOH in an operant self-administration paradigm than males. Understanding behavioral and mechanistic sex differences in responding for alcohol will allow for the advancement of treatments for women with AUD.

Additive influences of acute early life stress and sex on vulnerability for aversion-resistant alcohol drinking.

Acute early life stress (ELS) alters stress system functioning in adulthood and increases susceptibility to posttraumatic stress disorder (PTSD) and alcohol use disorder (AUD). The current study assessed the effects of acute, infant ELS on alcohol drinking, including aversion-resistant drinking, in male and female Long Evans rats. Acute ELS was induced using a stress-enhanced fear learning (SEFL) protocol that consisted of 15 footshocks delivered on postnatal day (PND) 17. Alcohol drinking during adolescence and adulthood was measured with a two-bottle choice intermittent alcohol access paradigm. Aversion-resistant drinking was assessed in adulthood by adding quinine (0.01, 0.1, and 1.0 g/L) to the alcohol bottle after 5 to 6 weeks and 11 to 12 weeks of drinking. ELS had minimal influences on adolescent and adult alcohol consumption and preference. However, ELS, sex, and alcohol exposure history all influenced aversion-resistant alcohol drinking in an additive fashion. Higher concentrations of quinine were tolerated in females, ELS-exposed rats, and after 11 to 12 weeks of drinking. Tests of quinine sensitivity in a separate cohort of animals found that rats can detect concentrations of quinine as low as 0.001 g/L in water and that quinine sensitivity is not influenced by sex or ELS exposure. These results agree with reports of sex differences in aversion-resistant drinking and are the first to demonstrate an influence of ELS on this behavior. Our results also suggest that a single traumatic stress exposure in infancy may be a promising model of comorbid PTSD and AUD and useful in studying the interactions between ELS, sex, and alcohol dependence.

NMDA receptor deletion on dopamine neurons disrupts visual discrimination and reversal learning.

The dopamine (DA) system is critical for various forms of learning about salient environmental stimuli. Prior work has shown that deletion of the obligatory NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor on neurons expressing the DA transporter (DAT) in mice results in reduced phasic release from DA-containing neurons. To further investigate the contribution of phasic DA release to reward-related learning and cognitive flexibility, the current study evaluated DAT-NR1 null mutant mice in a touchscreen-based pairwise visual discrimination and reversal learning paradigm. Results showed that these mutants were slower to attain a high level of choice accuracy on the discrimination task, but showed improved late reversal performance on sessions where correct choice was above chance. A number of possible interpretations are offered for this pattern of effects, including the opposing possibilities that discrimination memory was either stronger by the completion of training (overtraining effect) or weaker (learning deficit), both of which could potentially produce faster reversal. These data add to the extensive literature ascribing a critical role for DAergic neurotransmission in cognitive functions and the regulation of reward-related behaviors of relevance to addictions.