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Mu-opioid receptors (MORs) in the amygdala and striatum are important in addictive and rewarding behaviors. The transcription factor Foxp2 is a genetic 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 and are neuronal subpopulations of potential relevance to alcohol-drinking behaviors. 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. The results demonstrate that deletion of MOR on Foxp2-expressing neurons renders mice more sensitive to quinine-adulterated alcohol. Mice with the deletion (vs. Cre- controls) also consumed less alcohol during the final sessions of the IA task, were less active at baseline and following morphine injection, and there was a trend toward less responding for sucrose under an FR3 schedule. Foxp2-MOR deletion did not impair the ability to learn to respond for reward or develop a conditioned aversion to morphine withdrawal. Together, these investigations demonstrate that Foxp2-expressing neurons may be involved in escalation of alcohol consumption and the development of compulsive-like alcohol drinking., Competing Interests: Declaration of competing interest The authors declare no conflicting interests in this work., (Copyright © 2024. Published by Elsevier Inc.)

Background: In humans, early life stress (ELS) is associated with an increased risk for developing both alcohol use disorder (AUD) and posttraumatic stress disorder (PTSD). We previously used an infant footshock model in rats that produces stress-enhanced fear learning (SEFL) and increases aversion-resistant alcohol drinking to explore this shared predisposition. The goal of the current study was to test the viability of this procedure as a model of comorbid PTSD and AUD in male and female C57BL/6J mice., Methods: Acute ELS was induced using 15 footshocks on postnatal day (PND) 17. In adulthood, alcohol drinking behavior was tested in one of three two-bottle choice drinking paradigms. In continuous access, mice were given 24 h access to 5% and 10% ethanol and water for five consecutive drinking sessions each. In limited access drinking in the dark, mice were given 2 h of access to 15% ethanol and water across 15 sessions 3 h into the dark cycle. 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, 100, and 200 mg/L) were added to the ethanol to test aversion-resistant drinking. Intermittent access drinking was tested with and without a period of adolescent drinking (PND 35)., Results: Infant footshock did not alter drinking in the continuous or limited access tasks. In the intermittent access task, adult consumption and preference were lower in shocked mice when adolescent drinking was included. Aversion resistance was greater in females following infant footshock and adolescent drinking., Conclusions: Our results demonstrate that ELS, in the form of infant footshock on PND 17, must be followed by a period of adolescent drinking to affect adult alcohol consumption in mice., (© 2025 The Author(s). Alcohol, Clinical and Experimental Research published by Wiley Periodicals LLC on behalf of Research Society on Alcohol.)

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 consumption in two drinking paradigms: limited access "Drinking in the Dark" and intermittent access. Quinine was added to the drinking bottles in the DID experiment to test aversion-resistant, "compulsive" drinking. Nicotine and sucrose drinking were also assessed so comparisons could be made with other rewarding substances. Cholinergic MOR deletion did not influence consumption or preference for ethanol (EtOH) in either drinking task. Differences were observed in aversion-resistance in males with Cre + mice tolerating lower concentrations of quinine than Cre-. In contrast to EtOH, preference for nicotine was reduced following cholinergic MOR deletion while sucrose consumption and preference was increased in Cre+ (vs. Cre-) females. Locomotor activity was also greater in females following the deletion. These results suggest that cholinergic MORs participate in preference for rewarding substances. Further, while they are not required for consumption of alcohol alone, cholinergic MORs may influence the tendency to drink despite negative consequences., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: 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 previously used an infant footshock model that produces stress-enhanced fear learning (SEFL) in rats and mice and increases aversion-resistant alcohol drinking in rats to explore this shared predisposition. The goal of the current study was to extend this model of comorbid PTSD and AUD to male and female C57BL/6J mice., Methods: Acute ELS was induced using 15 footshocks on postnatal day (PND) 17. In adulthood, alcohol drinking behavior was tested in one of three two-bottle choice drinking paradigms. In continuous access, mice were given 24 h access to 5% or 10% ethanol and water for five consecutive drinking sessions each. In limited access drinking in the dark, mice were given 2 h of access to 15% ethanol and water across 15 sessions 3 h into the dark cycle. 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. Intermittent access drinking was tested with and without a period of adolescent drinking (PND 35)., Results: Infant footshock did not alter drinking in the continuous or limited access tasks. Adult consumption and preference were lower in the intermittent access task when adolescent drinking was included and there were ELS-induced differences in consumption of quinine-adulterated ethanol in females., Conclusions: Our results demonstrate that infant footshock followed by a period of adolescent drinking is a viable model of comorbid PTSD and AUD in rats and mice., Competing Interests: Conflict of interests: The authors declare no conflicting interests in this work.

Mu-opioid receptors (MORs) in the amygdala and striatum are important in addictive and rewarding behaviors. The transcription factor Foxp2 is a genetic 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 and are neuronal subpopulations of potential relevance to alcohol-drinking behaviors. 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/Oprm1fl/fl). Male and female Foxp2-Cre/Oprm1fl/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. The results demonstrate that deletion of MOR on Foxp2-expressing neurons renders mice more sensitive to quinine-adulterated ethanol (EtOH). Mice with the deletion (vs. Cre- controls) also consumed less alcohol during the final sessions of the IA task, responded less for sucrose under an FR3 schedule, and were less active at baseline and following morphine injection. Foxp2-MOR deletion did not impair the ability to learn to respond for reward or develop a conditioned aversion to morphine withdrawal. Together, these investigations demonstrate that Foxp2-expressing neurons may be involved in escalation of alcohol consumption and the development of compulsive-like alcohol drinking.

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/Oprm1fl/fl) to examine the role of MORs expressed in cholinergic interneurons (CINs) in home cage self-administration paradigms. Male and female ChAT-Cre/Oprm1fl/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. Experiment 3 assessed locomotor activity, sucrose preference, and quinine sensitivity. 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. Overall fluid consumption was also heightened in the Cre+ mice. In Experiment 3, Cre+ females were found to have greater locomotor activity and preference for sucrose vs. Cre- mice. These data suggest that cholinergic MORs are not required for EtOH, drinking behaviors but may contribute to aversion resistant EtOH drinking in a sex-dependent manner.

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (© 2023 Research Society on Alcohol.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sneddon, Masters, Ream, Fennell, DeMedio, Cash, Hollingsworth, Pandrangi, Thach, Shi and Radke.)

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., Competing Interests: Declaration of competing interest The authors declare no conflicting interests in this work., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (© 2022 Society for the Study of Addiction.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (© 2021 International Society for Neurochemistry.)

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.

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 D 1 -or D 2 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 D 1 -or D 2 receptor-expressing neurons did not alter compulsive-like EtOH intake. Control experiments showed that NAc core excitation and selective inhibition of D 1 -or D 2 -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 D 1 - CRE+ (but not the D 2 CRE+ ) group in a control experiment. Finally, pharmacological antagonism of D 1 and D 2 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 D 1 -and D 2 receptor-expressing medium spiny neurons participate in controlling this behavior., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (© 2021 Wiley Periodicals LLC.)

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.

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., (© 2021 Sneddon et al.; Published by Cold Spring Harbor Laboratory Press.)

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-HT 2B 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-HT 2B 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-HT 2B receptor, and provide further insight into the role of the 5-HT 2B receptor in cognition., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (© 2019 Society for the Study of Addiction.)

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., (© 2020 by the Research Society on Alcoholism.)

There is a compelling evidence that midbrain dopamine (DA) neurons and their projections to the ventral striatum provide a mechanism for motivating reward-seeking behavior, and for utilizing information about unexpected reward prediction errors (RPEs) to guide behavior based on current, rather than historical, outcomes. When this mechanism is compromised in addictions, it may produce patterns of maladaptive behavior that remain obdurate in the face of contrary information and even adverse consequences. Nonetheless, DAergic contributions to performance on behavioral tasks that rely on the ability to flexibly update stimulus-reward relationships remains incompletly understood. In the current study, we used a discrimination and reversal paradigm to monitor subsecond DA release in mouse NAc core (NAc) using in vivo fast-scan cyclic voltammetry (FSCV). We observed post-choice elevations in phasic NAc DA release; however, increased DA transients were only evident during early reversal when mice made responses at the newly rewarded stimulus. Based on this finding, we used in vivo optogenetic (eNpHR) photosilencing and (Channelrhodopsin2 [ChR2]) photostimulation to assess the effects of manipulating VTA-DAergic fibers in the NAc on reversal performance. Photosilencing the VTA → NAc DAergic pathway during early reversal increased errors, while photostimulation did not demonstrably affect behavior. Taken together, these data provide additional evidence of the importance of NAc DA release as a neural substrate supporting adjustments in learned behavior after a switch in expected stimulus-reward contingencies. These findings have possible implications for furthering understanding the role of DA in persistent, maladaptive decision-making characterizing addictions., (Published 2018. This article is a U.S. Government work and is in the public domain in the USA.)

The original version of this Article contained an error in the spelling of the author Anna K Radke, which was incorrectly given as Anna R Radke. This has now been corrected in both the PDF and HTML versions of the Article.

Obsessive-compulsive disorder (OCD) is a severe, chronic neuropsychiatric disorder with a strong genetic component. The SLC1A1 gene encoding the neuronal glutamate transporter EAAT3 has been proposed as a candidate gene for this disorder. Gene variants affecting SLC1A1 expression in human brain tissue have been associated with OCD. Several mouse models fully or partially lacking EAAT3 have shown no alterations in baseline anxiety-like or repetitive behaviors. We generated a transgenic mouse model (EAAT3 glo ) to achieve conditional, Cre-dependent EAAT3 overexpression and evaluated the overall impact of increased EAAT3 expression at behavioral and synaptic levels. Mice with EAAT3 overexpression driven by CaMKIIα-promoter (EAAT3 glo /CMKII) displayed increased anxiety-like and repetitive behaviors that were both restored by chronic, but not acute, treatment with fluoxetine or clomipramine. EAAT3 glo /CMKII mice also displayed greater spontaneous recovery of conditioned fear. Electrophysiological and biochemical analyses at corticostriatal synapses of EAAT3 glo /CMKII mice revealed changes in NMDA receptor subunit composition and altered NMDA-dependent synaptic plasticity. By recapitulating relevant behavioral, neurophysiological, and psychopharmacological aspects, our results provide support for the glutamatergic hypothesis of OCD, particularly for the increased EAAT3 function, and provide a valuable animal model that may open novel therapeutic approaches to treat this devastating disorder.

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., (Published by Elsevier B.V.)

Background: Alcohol use disorder is characterized by compulsive alcohol intake, or drinking despite negative consequences. Previous studies have shown that female rodents have a heightened vulnerability to drug use across different stages of the addictive cycle, but no previous studies have studied females in a model of aversion-resistant alcohol intake. Here, we investigated sex differences in binge-like and aversion-resistant alcohol drinking in C57BL/6J mice using a modified drinking-in-the-dark (DID) paradigm., Methods: In Experiment 1, 24-hour aversion to quinine (0, 100, or 250 μM) was assessed. In Experiment 2, male and female adult C57BL/6J mice consumed 15% ethanol (EtOH) or water in a 2-bottle limited-access DID paradigm for 2 h/d for 15 days. The EtOH was next adulterated with quinine (0, 100, or 250 μM) over 3 consecutive drinking sessions to test aversion-resistant intake. In Experiment 3, intake of quinine-adulterated (100 μM) EtOH was assessed across all 15 drinking sessions., Results: Quinine was equally aversive to both sexes in Experiment 1. In Experiment 2, female mice consumed significantly more alcohol than male mice during the final 6 drinking sessions. Levels of aversion-resistant intake did not differ between the sexes. In Experiment 3, quinine suppressed consumption in all mice, though females drank significantly more on the final 2 sessions., Conclusions: The results of this study demonstrate that while female mice escalate and consume more EtOH than males, both sexes exhibit similar levels of aversion-resistant drinking. These results inform our understanding of how sex interacts with vulnerability for addiction and argue for the inclusion of females in more studies of aversion-resistant alcohol drinking., (© 2018 by the Research Society on Alcoholism.)

The N-methyl- D -aspartate receptor (NMDAR) is mechanistically involved in the behavioral and neurophysiological effects of alcohol, but the specific role of the GluN2A subunit remains unclear. Here, we exposed mice with constitutive GluN2A gene knockout (KO) to chronic intermittent ethanol vapor (CIE) and tested for EtOH consumption/preference using a two-bottle choice paradigm, as well as NMDAR-mediated transmission at basolateral amygdala synapses via ex vivo slice electrophysiology. Results showed that GluN2A KO mice attained comparable blood EtOH levels in response to CIE exposure, but did not exhibit the significant increase in EtOH drinking that was observed in CIE-exposed wildtypes. GluN2A KO mice also showed no alterations in BLA NMDAR-mediated synaptic transmission after CIE, relative to air-exposed, whereas C57BL/6 J mice showed an attenuated synaptic response to GluN2B antagonism. Taken together, these data add to mounting evidence supporting GluN2A-containing NMDARs as a mechanism underlying relative risk for developing EtOH dependence after repeated EtOH exposure., (Published by Elsevier B.V.)

Reversal learning paradigms are among the most widely used tests of cognitive flexibility and have been used as assays, across species, for altered cognitive processes in a host of neuropsychiatric conditions. Based on recent studies in humans, non-human primates, and rodents, the notion that reversal learning tasks primarily measure response inhibition, has been revised. In this review, we describe how cognitive flexibility is measured by reversal learning and discuss new definitions of the construct validity of the task that are serving as a heuristic to guide future research in this field. We also provide an update on the available evidence implicating certain cortical and subcortical brain regions in the mediation of reversal learning, and an overview of the principal neurotransmitter systems involved., Competing Interests: The authors report no conflicts of interest., (Copyright © 2016 IBRO. All rights reserved.)

Addictions, including alcohol use disorders, are characterized by the loss of control over drug seeking and consumption, but the neural circuits and signaling mechanisms responsible for the transition from controlled use to uncontrolled abuse remain incompletely understood. Prior studies have shown that 'compulsive-like' behaviors in rodents, for example, persistent responding for ethanol (EtOH) despite punishment, are increased after chronic exposure to EtOH. The main goal of the current study was to assess the effects of chronic intermittent EtOH (CIE) exposure on multiple, putative measures of compulsive-like EtOH seeking in C57BL/6 J mice. Mice were exposed to two or four weekly cycles of CIE and then, post-withdrawal, tested for progressive ratio responding for EtOH, sustained responding during signaled EtOH unavailability and (footshock) punished suppression of responding for EtOH. Results showed that mice exposed to CIE exhibited attenuated suppression of EtOH seeking during punishment, as compared with air-exposed controls. By contrast, CIE exposure affected neither punished food reward-seeking behavior, nor other putative measures of compulsive-like EtOH seeking. Ex vivo reverse transcription polymerase chain reaction analysis of brain tissue found reduced sensitivity to punished EtOH seeking after CIE exposure was accompanied by a significant increase in gene expression of the GluN1 and GluN2A subunits of the N-methyl-d-aspartate receptor, specifically in the medial orbitofrontal cortex. Moreover, slice electrophysiological analysis revealed increased N-methyl-d-aspartate receptor-mediated currents in the orbitofrontal cortex after CIE exposure in test-naïve mice. Collectively, the current findings add to the growing body of evidence demonstrating that chronic exposure to EtOH fosters resistance to punished EtOH seeking in association with adaptations in cortical glutamatergic transmission., (© 2015 Society for the Study of Addiction.)

N-Methyl-d-aspartate receptors (NMDAR) are involved in the regulation of alcohol drinking, but the contribution of NMDAR subunits located on specific neuronal populations remains incompletely understood. The current study examined the role of GluN2B-containing NMDARs expressed on cortical principal neurons and cortical interneurons in mouse ethanol drinking. Consumption of escalating concentrations of ethanol was measured in mice with GluN2B gene deletion in either cortical principal neurons (GluN2B CxNULL ) or interneurons (GluN2B InterNULL ), using a two-bottle choice paradigm. Results showed that GluN2B InterNULL , but not GluN2B CxNULL , mice consumed significantly less ethanol, at relatively high concentrations, than non-mutant controls. In a second paradigm in which mice were offered a 15% ethanol concentration, without escalation, GluN2B CxNULL mice were again no different from controls. These findings provide novel evidence for a contribution of interneuronal GluN2B-containing NMDARs in the regulation of ethanol drinking., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Rats selectively bred for high (HiS) or low (LoS) saccharin intake are a well-established model of drug-abuse vulnerability, with HiS rats being more likely to consume sweets and cocaine than LoS rats. Still, the nature of these differences is poorly understood. This study examined whether the motivational consequences of cocaine exposure are differentially expressed in HiS and LoS rats by measuring intracranial self-stimulation (ICSS) thresholds following acute injections of cocaine (10 mg/kg). Reductions in ICSS thresholds following cocaine injection were greater in HiS rats than in LoS rats, suggesting that the reward-enhancing effects of cocaine are greater in the drug-vulnerable HiS than LoS rats. Higher cocaine-induced reward, indicated by lower ICSS thresholds, may explain the higher rates of drug consumption in sweet-preferring animal models, providing a clue to the etiology of cocaine addiction in vulnerable populations.

Rationale: Compulsive behavior, which is a hallmark of psychiatric disorders such as addiction and obsessive-compulsive disorder, engages corticostriatal circuits. Previous studies indicate a role for corticostriatal N-methyl-D-aspartate receptors (NMDARs) in mediating compulsive-like responding for drugs of abuse, but the specific receptor subunits controlling reward-seeking in the face of punishment remain unclear., Objectives: The current study assessed the involvement of corticostriatal GluN2B-containing NMDARs in measures of persistent and punished food reward-seeking., Methods: Mice with genetic deletion of GluN2B in one of three distinct neuronal populations, cortical principal neurons, forebrain interneurons, or striatal medium spiny neurons, were tested for (1) sustained food reward-seeking when reward was absent, (2) reward-seeking under a progressive ratio schedule of reinforcement, and (3) persistent reward-seeking after a footshock punishment., Results: Mutant mice with genetic deletion of GluN2B in cortical principal neurons demonstrated attenuated suppression of reward-seeking during punishment. These mice performed normally on other behavioral measures, including an assay for pain sensitivity. Mutants with interneuronal or striatal GluN2B deletions were normal on all behavioral assays., Conclusions: Current findings offer novel evidence that loss of GluN2B-containing NMDARs expressed on principal neurons in the cortex results in reduced punished food reward-seeking. These data support the involvement of GluN2B subunit in cortical circuits regulating cognitive flexibility in a variety of settings, with implications for understanding the basis of inflexible behavior in neuropsychiatric disorders including obsessive-compulsive disorders (OCD) and addictions.

Investigations into animal models of drug withdrawal have largely found that emotional signs of withdrawal (e.g. anxiety, anhedonia, and aversion) in adolescents are experienced earlier and less severely than in their adult counterparts. The majority of these reports have examined withdrawal from ethanol or nicotine. To expand our knowledge about the emotional withdrawal state in adolescent rats, we used potentiation of the acoustic startle reflex after an acute dose of morphine (10 mg/kg, subcutaneously) as a measure of opiate withdrawal. Startle was measured at four time points after morphine injection (2, 3, 4, and 5 h) in 28-day-old and 90-day-old male and female rats. The results of this experiment revealed that peak potentiation of the startle reflex occurred at 3 h in the adolescent rats and at 5 h in the adult rats, and that the magnitude of withdrawal was larger in the adults. No sex differences were observed. Overall, these results affirm that, similar to withdrawal from ethanol and nicotine, opiate withdrawal signs are less severe in adolescent than in adult rats.

Rationale: Sweet preference is a marker of vulnerability to substance use disorders, and rats selectively bred for high (HiS) vs. low saccharin (LoS) intake display potentiated drug-seeking behaviors. Recent work indicated that LoS rats were more responsive to the negative effects of drugs in several assays., Objective: The current study used the intracranial self-stimulation (ICSS) procedure to investigate the anhedonic component of morphine withdrawal in male HiS and LoS rats., Methods: Rats were administered morphine (10mg/kg) or saline for 8 days. To evaluate withdrawal effects, reward thresholds were measured 24 and 28h following the 8th morphine injection (spontaneous withdrawal) and again for 4 days following daily acute morphine and naloxone (1mg/kg) administration (precipitated withdrawal)., Results: 24h following the final morphine injection, reward thresholds in LoS rats were significantly elevated compared to reward thresholds in LoS controls, indicating spontaneous withdrawal. This effect was not observed in HiS rats. LoS rats also showed greater elevations of reward thresholds on several days during naloxone-precipitated withdrawal compared to their HiS counterparts., Conclusions: LoS rats were more sensitive to morphine withdrawal-mediated elevations in ICSS thresholds than HiS rats. While these differences were generally modest, our data suggest that severity of the negative affective component of opiate withdrawal may be influenced by genotypes related to addiction vulnerability., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cocaine use results in anhedonia during withdrawal, but it is not clear how this emotional state interacts with an individual's vulnerability for addiction. Rats selectively bred for high (HiS) or low (LoS) saccharin intake are a well-established model of drug abuse vulnerability, with HiS rats being more likely to consume sweets and drugs of abuse such as cocaine and heroin (Carroll et al., 2002) than LoS rats. This study examined whether the motivational consequences of cocaine withdrawal are differentially expressed in HiS and LoS rats. HiS and LoS rats were trained to respond for a sucrose reward on a progressive ratio (PR) schedule of reinforcement and breakpoints were measured during and after chronic, continuous exposure to cocaine (30 mg/kg/day). Cocaine, but not saline, treatment resulted in lower breakpoints for sucrose during withdrawal in LoS rats only. These results suggest anhedonia during withdrawal is more pronounced in the less vulnerable LoS rats. Fewer motivational deficits during withdrawal may contribute to greater drug vulnerability in the HiS line., (Published by Elsevier Inc.)

Rationale: Rats bred for high (HiS) and low (LoS) saccharin intake exhibit divergent behavioral responses to multiple drugs of abuse, with HiS rats displaying greater vulnerability to drug taking. Previous research indicates that this effect may be due to increased sensitivity to reward in HiS rats and to the aversive effects of acute drug administration in LoS rats., Objective: The current study investigated whether HiS and LoS rats also exhibit different behavioral signs of withdrawal following one or repeated opiate exposures., Methods: Emotional signs of opiate withdrawal were assessed with potentiation of the acoustic startle reflex and conditioned place aversion (CPA) in male and female HiS and LoS rats. Startle was measured before and 4 h after a 10-mg/kg injection of morphine on days 1, 2, and 7 of opiate exposure. CPA was induced with a 2-day, naloxone-precipitated conditioning paradigm. Somatic signs of withdrawal and weight loss were also measured., Results: Male and female LoS rats exhibited lower startle potentiation than HiS rats on the seventh day of morphine exposure. LoS male rats also failed to develop a CPA to morphine withdrawal. No differences in physical withdrawal signs were observed between HiS and LoS rats, but males of both lines had more physical signs of withdrawal than females., Conclusions: These results suggest that LoS rats are less vulnerable to the negative emotional effects of morphine withdrawal than HiS rats. A less severe withdrawal syndrome may contribute to decreased levels of drug taking in the LoS line.

A number of lines of evidence suggest that negative emotional symptoms of withdrawal involve reduced activity in the mesolimbic dopamine system. This study examined the contribution of dopaminergic signaling in structures downstream of the ventral tegmental area to withdrawal from acute morphine exposure, measured as potentiation of the acoustic startle reflex. Systemic administration of the general dopamine receptor agonist apomorphine or a cocktail of the D1-like receptor agonist SKF82958 and the D2-like receptor agonist quinpirole attenuated potentiated startle during morphine withdrawal. This effect was replicated by apomorphine infusion into the nucleus accumbens shell. Finally, apomorphine injection was shown to relieve startle potentiation during nicotine withdrawal and conditioned place aversion to morphine withdrawal. These results suggest that transient activation of the ventral tegmental area mesolimbic dopamine system triggers the expression of anxiety and aversion during withdrawal from multiple classes of abused drugs.

Opponent process theory predicts that the first step in the induction of drug withdrawal is the activation of reward-related circuitry. Using the acoustic startle reflex as a model of anxiety-like behavior in rats, we show the emergence of a negative affective state during withdrawal after direct infusion of morphine into the ventral tegmental area (VTA), the origin of the mesolimbic dopamine system. Potentiation of startle during withdrawal from systemic morphine exposure requires a decrease in opiate receptor stimulation in the VTA and can be relieved by administration of the dopamine receptor agonist apomorphine. Together, our results suggest that the emergence of anxiety during withdrawal from acute opiate exposure begins with activation of VTA mesolimbic dopamine circuitry, providing a mechanism for the opponent process view of withdrawal.

Rationale: Elevated acoustic startle amplitude has been used to measure anxiety-like effects of drug withdrawal in humans and animals. Withdrawal from a single opiate administration has been shown to produce robust elevations in startle amplitude ("withdrawal-potentiated startle") that escalate in severity with repeated exposure. Although anxiety is a clinical symptom of nicotine dependence, it is currently unknown whether anxiety-like behavior is elicited during the early stages of nicotine dependence in rodents., Objective: The objective of this study is to examine whether, as is the case with opiates, single or repeated exposure to nicotine can produce withdrawal-potentiated startle., Methods: Rats received daily nicotine injections for 14 days, and startle amplitude was tested during spontaneous withdrawal on injection days 1, 7, and 14., Results: Elevated startle responding was observed during nicotine withdrawal on days 7 and 14 but not on day 1, was greater at higher nicotine doses, and was reduced by a nicotine replacement injection given during an additional test session on day 15. Additional experiments demonstrated that nicotine withdrawal-potentiated startle was reduced by the alpha(2)-adrenergic agonist clonidine and that precipitated withdrawal-potentiated startle could not be induced by injection of the nicotinic acetylcholine receptor antagonist mecamylamine., Conclusions: These results suggest that nicotine withdrawal escalates in severity across days, similar to the previously reported escalation of opiate withdrawal-potentiated startle. Potentiated startle may be a reliable measure of withdrawal from different classes of abused drugs and may be useful in the study of the early stages of drug dependence.