Cholinergic mu-opioid receptor deletion alters reward preference and aversion-resistance.

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 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.

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.

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.