BACKGROUND:Predator stress (PS) is used to model trauma leading to post-traumatic stress disorder, and it increases ethanol drinking in a proportion of male and female rodents. The goals of the present studies were to identify male and female mice with prior binge drinking experience that exhibited sensitivity and resilience to PS-enhanced drinking and then to test two target molecules (corticotropin releasing factor receptor 1 [CRF-R1] antagonist NBI-27914 [NBI] and G-protein coupled receptor 39 [GPR39] agonist TC-G 1008 [TC-G]) for their ability to selectively reduce PS-enhanced drinking. METHODS:Adult male and female C57BL/6J mice received seven binge ethanol sessions, a period of abstinence, and acclimation to lickometer chambers to examine the effects of NBI or TC-G on stress-associated drinking. Following establishment of stable baseline (BL) drinking and four intermittent PS exposures, mice were classified into "Sensitive" and "Resilient" subgroups, based on the change in ethanol drinking from BL after PS2-4. Then, mice received injections of vehicle or drug (NBI or TC-G) in a within-subjects design. Control studies examined the effects of NBI or TC-G on binge drinking, locomotor activity, and saccharin intake. RESULTS:NBI and TC-G significantly suppressed binge drinking in male and female mice in the control studies. However, sensitivity to the ability of the compounds to decrease PS-enhanced drinking did not differ between animals in the "PS-sensitive" versus "PS-resilient" subgroups, and female mice were insensitive to TC-G in the traumatic stress drinking model. Specifically, NBI doses of 5 and 10 mg/kg (males) and 12.5 mg/kg (females) significantly decreased PS-associated drinking in both subgroups. TC-G (7.5 mg/kg) significantly decreased PS-associated drinking in both subgroups of male mice but not in female mice. CONCLUSIONS:The present findings suggest that stress sensitivity and subsequent enhanced ethanol drinking in the "Sensitive" subgroup may not increase sensitivity to CRF-R1 antagonism or GPR39 agonism.

Clinical and preclinical evidence indicate that both peripheral and central elements of the hypothalamic-pituitary-thyroid (HPT) axis are dysregulated in alcohol use disorder, and that thyroid hormone system dysregulation is associated alcohol craving and co-morbid mood and depression-related disorders. Yet, no study has investigated if central nervous system (CNS) thyroid hormone receptors, primary targets of thyroid hormone and major regulators of the HPT axis are involved in alcohol consumption. We utilized a 24-h access two-bottle choice (2BC) voluntary ethanol (EtOH) drinking paradigm to assess if the expression of CNS thyroid hormone receptors is sensitive to voluntary alcohol consumption in C57BL/6 J mice. We found that thyroid hormone receptor-beta (Thrb/THRβ) mRNA expression was significantly reduced in the paraventricular nucleus of the hypothalamus of EtOH drinking mice compared to water controls. In addition, EtOH drinking mice exhibited peripheral elevation of serum triiodothyronine. Next, we utilized the CNS selective THRβ agonist, Sob-AM2, to determine if central activation of THRβ would influence voluntary alcohol drinking in mice in the same EtOH 2BC drinking paradigm. We found that repeated treatment with Sob-AM2 significantly reduced daily EtOH intake and preference, while in conjunction increasing water intake. In summary, we found that hypothalamic Thrb expression is sensitive to voluntary alcohol drinking, and that CNS THRβ activity regulates alcohol consumption in mice. Taken together, our results highlight an important role of central thyroid hormone receptor signalling in alcohol drinking and indicate therapeutic potential of CNS selective thyromimetics in treatment of alcohol use disorder.

AIMS:Excessive alcohol use is often triggered by traumatic experiences, when subjects consume alcohol-containing beverages as a passive coping mechanism to relieve negative affect. There are no FDA-approved medications that are specifically recommended for patients with alcohol use disorder who use alcohol to decrease the consequences of trauma. The current study used a mouse model of predator stress-enhanced drinking to test whether administration of oxytocin (OXT) could selectively target subjects with increased sensitivity to psychological trauma. METHODS:Male and female C57BL/6J mice established consumption of 10% ethanol in a 2-bottle choice procedure and then were exposed to predator odor (soiled rat bedding) during four intermittent 30-minute sessions. Mice were designated as Sensitive, increasing ethanol intake, or Resilient, showing no increases in intake, following the predator odor exposures. Effects of OXT (1 mg/kg) on ethanol intake were examined at two and at four hours following treatment using an automated lickometer system. RESULTS:OXT non-selectively decreased ethanol and water intake in male and female mice during the first two hours after administration, suggesting sedative effects. Importantly, when analyzed at four hours post-injection, OXT selectively decreased ethanol, but not water intake, in male mice and in the Sensitive subgroup of female mice and had no significant effects on ethanol intake in the Resilient female mice. CONCLUSIONS:These results indicate that the predator odor model can help screen for pharmacotherapies to treat patients consuming alcohol to passively cope with trauma-induced negative affect. Further studies need to test whether OXT is preferentially effective in such subjects.

Women tend to progress from initial alcohol use to Alcohol Use Disorder (AUD) more quickly than men, highlighting the need to study sex differences in models of early-stage alcohol use. In humans and rodents, the nucleus accumbens (NAc) regulates alcohol binge drinking, a risk factor for developing AUD. However, it is unknown whether similar brain regions and NAc inputs are engaged in males and females during binge-like drinking. We labeled NAc inputs with a viral retrograde tracer (GFP) and quantified whole-brain c-Fos to determine the regions and NAc inputs differentially engaged in male and female mice during binge-like drinking. Mice underwent a 4-day Drinking-in-the-Dark task for either ethanol or water. Immediately following drinking on day 4, periorbital blood samples were collected for determination of blood ethanol concentration, and brains were collected. c-Fos expression and c-Fos + GFP colocalization was quantified for 426 brain areas using SmartAnalytics. We employed several data and network analysis approaches to identify NAc circuits and regions engaged, and network dynamics altered by binge-like drinking. We found that ethanol engaged significantly more NAc inputs in binge-like ethanol drinking females, as compared to males, including various GABAergic and glutamatergic regions. Moreover, we found that binge-like drinking females had 129 brain areas with greater c-Fos than males. Relative to water controls, ethanol increased network modularity and decreased connectivity in both sexes and did so more dramatically in males. Our results support that early-stage binge-like ethanol drinking engages brain regions and NAc inputs and alters network dynamics in a sex-specific manner, which may help to explain the faster transition from initial alcohol use to AUD in women.

Social hierarchies are a prevalent feature of all animal groups, and an individual's rank within the group can significantly affect their overall health, typically at the greatest expense of the lowest-ranked individuals, or omegas. These subjects have been shown to exhibit various stress-related phenotypes, such as increased hypothalamic-pituitary axis activity and increased amygdalar corticotropin-releasing factor levels compared to higher-ranked subjects. However, these findings have been primarily characterized in males and in models requiring exhibition of severe aggression. The goals of the current study, therefore, were to characterize the formation and maintenance of social hierarchies using the tube test and palatable liquid competition in same-sex groups of male and female C57BL/6 J mice. We also aimed to examine the effects of tube test-determined social rank on plasma and hypothalamic oxytocin and vasopressin levels, peptides with established roles in social behaviors and the stress response. Lastly, we assessed the effects of environmental enrichment and length of testing on the measures outlined above. Overall, we demonstrated that males and females develop social hierarchies and that these hierarchies can be determined using the tube test. While we were unable to establish a consistent connection between peptide levels and social rank, we observed transient changes in these peptides reflecting complex interactions between social rank, sex, environment, and length of testing. We also found that many male and female omegas began to exhibit passive coping behavior after repeated tube test losses, demonstrating the potential of this assay to serve as a model of chronic, mild psychosocial stress.

Growing parity in Alcohol Use Disorder (AUD) diagnoses in men and women necessitates consideration of sex as a biological variable. In humans and rodents, the nucleus accumbens core (NAcc) regulates alcohol binge drinking, a risk factor for developing AUD. We labeled NAcc inputs with a viral retrograde tracer and quantified whole-brain c-Fos to determine the regions and NAcc inputs differentially engaged in male and female mice during binge-like ethanol drinking. We found that binge-like ethanol drinking females had 129 brain areas with greater c-Fos than males. Moreover, ethanol engaged more NAcc inputs in binge-like ethanol drinking females (as compared with males), including GABAergic and glutamatergic inputs. Relative to water controls, ethanol increased network modularity and decreased connectivity in both sexes and did so more dramatically in males. These results demonstrate that early-stage binge-like ethanol drinking engages brain regions and NAcc-inputs and alters network dynamics in a sex-specific manner.

Alcohol use disorder (AUD) is a highly prevalent public health problem. The ghrelin system has been identified as a potential target for therapeutic intervention for AUD. Previous work showed that systemic administration of the growth hormone secretagogue receptor (GHSR) antagonist DLys reduced alcohol intake and preference in male mice. Yet, it is unclear whether central or peripheral GHSRs mediated these effects. We hypothesized that alcohol consumption is driven by central GHSRs and addressed this hypothesis by testing the effects of central administration of DLys. Male C57BL/6J mice consumed alcohol in a two-bottle choice procedure (10% ethanol versus water). DLys (2 nmol) was administered intracerebroventricularly for 7 days to examine alcohol intake and preference. DLys decreased alcohol intake and preference but had no effect on food intake. The effects on alcohol intake and preference persisted after several administrations, indicating lack of tolerance to DLys' effects. These results suggest that central administration of DLys is sufficient to reduce alcohol drinking and that DLys remains effective after several administrations when given intracerebroventricularly. Moreover, this work suggests that the effects of intracerebroventricularly administered DLys are specific to alcohol and do not generalize to other calorie-driven behaviors.

Research confirms that stress is associated with alcohol drinking and relapse in males and females and that there are sex differences in the alcohol-related adaptations of stress pathways. The predator stress (PS) model of traumatic stress produces an increase in alcohol drinking or self-administration in a subpopulation of rodents, so it is utilized as an animal model of comorbid alcohol use disorder (AUD) and post-traumatic stress disorder (PTSD). Previous work determined that sensitivity to PS-enhanced drinking produced sex differences in proteins related to stress-regulating systems in the medial prefrontal cortex and hippocampus. The present studies examined whether male and female C57BL/6J mice differ in sensitivity to the ability of the corticotropin releasing factor receptor 1 antagonist CP-376395 to decrease PS-enhanced drinking. In control studies, CP-376395 doses of 5, 10, and 20 mg/kg dose-dependently decreased 4-h ethanol drinking. Next, CP-376395 doses of 5 and 10 mg/kg were tested for effects on ethanol drinking in mice with differential sensitivity to PS-enhanced drinking. Subgroups of "Sensitive" and "Resilient" male and female mice were identified based on changes in ethanol intake in an unrestricted-access ethanol-drinking procedure following four exposures to PS (dirty rat bedding). During the first 2 h post-injection of CP-376395, both doses significantly decreased ethanol licks versus vehicle in the females, with no significant interaction between subgroups, whereas the 10 mg/kg dose significantly decreased ethanol licks versus vehicle in the "Resilient" males. Thus, sensitivity to the suppressive effect of CP-376395 on stress-induced ethanol intake was greater in females versus males, whereas sensitivity and resilience to PS-enhanced drinking produced differential sensitivity to the ability of CP-376395 to decrease ethanol drinking only in male mice. Our results argue against greater efficacy of CRF-R1's ability to decrease ethanol intake in subjects with traumatic stress-enhanced ethanol drinking.

Purpose of ReviewStress is associated with alcohol drinking, and epidemiological studies document the comorbidity of alcohol use disorder (AUD) and post-traumatic stress disorder (PTSD), with higher comorbid prevalence in females than in males. The aim of this paper is to highlight information related to sex differences in stress-enhanced alcohol drinking from clinical studies and from preclinical studies utilizing an animal model of traumatic stress.Recent FindingsStress is associated with alcohol drinking and relapse in males and females, but there are sex differences in the alcohol-related adaptation of stress pathways and in the association of different prefrontal regions with stress-induced anxiety. The predator stress model of traumatic stress produced enhanced alcohol drinking in a subgroup of stress-sensitive male and female animals, which could be associated with sex and subgroup differences in stress axis responsivity, behavioral responses to predator odors, and epigenetic mechanisms engaged by traumatic experiences.SummaryWhile additional studies in females are necessary, existing clinical and preclinical evidence suggests that biological mechanisms underlying stress-enhanced drinking likely differ between males and females. Thus, effective treatment strategies may differ between the sexes.

BackgroundExcessive alcohol consumption leads to serious health problems. Mechanisms regulating the consumption of alcohol are insufficiently understood. Previous preclinical studies suggested that non-social environmental and social environmental complexities can regulate alcohol consumption in opposite directions. However, previous studies did not include all conditions and/or did not include female rodents. Therefore, in this study, we examined the effects of social versus single housing in standard versus non-standard housing conditions in male and female mice.MethodsAdult C57BL/6 J mice were housed in either standard shoebox cages or in automated Herdsman 2 (HM2) cages and exposed to a two-bottle choice procedure with 3% or 6% ethanol versus water for 5 days. The HM2 cages use radiotracking devices to measure the fluid consumption of individual mice in an undisturbed and automated manner. In both housing conditions, mice were housed either at one or at four per cage.ResultsIn standard cages, group housing of animals decreased alcohol consumption and water consumption. In HM2 cages, group housing significantly increased ethanol preference and decreased water intake. There were no significant differences in these effects between male and female animals. These observations were similar for 3 and 6% ethanol solutions but were more pronounced for the latter. The effects of social environment on ethanol preference in HM2 cages were accompanied by an increase in the number of approaches to the ethanol solution and a decrease in the number of approaches to water. The differences in ethanol intake could not be explained by differences in locomotor or exploratory activity as socially housed mice showed fewer non-consummatory visits to the ethanol solutions than single-housed animals. In addition, we observed that significant changes in behaviors measuring the approach to the fluid were not always accompanied by significant changes in fluid consumption, and vice versa, suggesting that it is important to assess both measures of motivation to consume alcohol.ConclusionOur results indicate that the direction of the effects of social environment on alcohol intake in mice depends on the non-social housing environment. Understanding mechanisms by which social and non-social housing conditions modulate alcohol intake could suggest approaches to counteract environmental factors enhancing hazardous alcohol consumption.

There is much interest in targeting the activity in the oxytocin system to regulate social bonding. However, studies with exogenous administration of oxytocin face the caveats of its low stability, poor brain permeability and insufficient receptor specificity. The use of a small-molecule oxytocin receptor-specific agonist could overcome these caveats. Prior to testing the potential effects of a brain-penetrant oxytocin receptor agonist in clinical settings, it is important to assess how such an agonist would affect social bonds in animal models. The facultatively monogamous prairie voles (Microtus ochrogaster), capable of forming long-term social attachments between adult individuals, are an ideal rodent model for such testing. Therefore, in a series of experiments we investigated the effects of the recently developed oxytocin receptor-specific agonist LIT-001 on the acquisition and expression of partner preference, a well-established model of pair bonding, in prairie voles. LIT-001 (10 mg/kg, intraperitoneal), as expected, facilitated the acquisition of partner preference when administered prior to a 4hr cohabitation. In contrast, while animals injected with vehicle after the 4hr cohabitation exhibited significant partner preference, animals that were injected with LIT-001 did not show such partner preference. This result suggests that OXTR activation during expression of pair bonding can inhibit partner preference. The difference in effects of LIT-001 on acquisition versus expression was not due to basal differences in partner preference between the experiments, as LIT-001 had no significant effects on expression of partner preference if administered following a shorter (2hr-long) cohabitation. Instead, this difference agrees with the hypothesis that the activation of oxytocin receptors acts as a signal of presence of a social partner. Our results indicate that the effects of pharmacological activation of oxytocin receptors crucially depend on the phase of social attachments.

BACKGROUND Social interactions with subjects experiencing pain can increase nociceptive sensitivity in observers, even without direct physical contact. In previous experiments, extended indirect exposure to soiled bedding from mice with alcohol withdrawal-related hyperalgesia enhanced nociception in their conspecifics. This finding suggested that olfactory cues could be sufficient for nociceptive hypersensitivity in otherwise untreated animals (also known as "bystanders"). AIM The current study addressed this possibility using an inflammation-based hyperalgesia model and long- and short-term exposure paradigms in C57BL/6J mice. MATERIALS & METHOD Adult male and female mice received intraplantar injection of complete Freund's adjuvant (CFA) and were used as stimulus animals to otherwise naïve same-sex bystander mice (BS). Another group of untreated mice (OLF) was simultaneously exposed to the bedding of the stimulus mice. RESULTS In the long-term, 15-day exposure paradigm, the presence of CFA mice or their bedding resulted in reduced von Frey threshold but not Hargreaves paw withdrawal latency in BS or OLF mice. In the short-term paradigm, 1-hr interaction with CFA conspecifics or 1-hr exposure to their bedding induced mechanical hypersensitivity in BS and OLF mice lasting for 3 hrs. Chemical ablation of the main olfactory epithelium prevented bedding-induced and stimulus mice-induced mechanical hypersensitivity. Gas chromatography-mass spectrometry (GC-MS) analysis of the volatile compounds in the bedding of experimental mice revealed that CFA-treated mice released an increased number of compounds indicative of disease states. DISCUSSION AND CONCLUSION These results demonstrate that CFA-induced inflammatory pain can modulate nociception in bystander mice via an olfactory mechanism involving dynamic changes in volatile compounds detectable in the rodent bedding. SIGNIFICANCE Social context can influence nociceptive sensitivity. Recent studies suggested involvement of olfaction in this influence. In agreement with this idea, the present study shows that the presence of mice with inflammatory pain produces nociceptive hypersensitivity in nearby conspecifics. This enhanced nociception occurs via olfactory cues present in the mouse bedding. Analysis of the bedding from mice with inflammatory pain identifies a number of compounds indicative of disease states. These findings demonstrate the importance of olfactory system in influencing pain states.

Comorbidity of post-traumatic stress disorder (PTSD) and alcohol use disorder (AUD) worsens the prognosis for each of these individual disorders. The current study aimed to identify neurocircuits potentially involved in regulation of PTSD-AUD comorbidity by mapping expression of c-Fos in male and female C57BL/6J mice following repeated predator stress (PS), modeled by exposure to dirty rat bedding. In experiment 1, the levels of c-Fos in the paraventricular nucleus of the hypothalamus (PVH) and the nucleus accumbens shell were higher after the second PS vs the first PS, indicating a sensitized response to this stressor. Additional brain regions showed varied sex-dependent and independent regulation by the two consecutive PS exposures. In experiment 2, mice that increased voluntary alcohol consumption following four exposures to PS (Sensitive subgroup) showed higher c-Fos induction in the PVH, piriform cortex and ventromedial hypothalamus than mice that decreased consumption following these exposures (Resilient subgroup). In contrast to these brain regions, c-Fos was higher in the anterior olfactory nucleus of Resilient vs Sensitive mice. Taken together, these data demonstrate that repeated PS exposure and voluntary alcohol consumption increase neuronal activity across neurocircuits in which specific components depend on the vulnerability of individual mice to these stressors. Increased PVH activity observed across both experiments suggests this brain area as a potential mediator of PS-induced increases in alcohol consumption. Future investigations of specific neuronal populations within the PVH activated by PS, and manipulation of these specific neuronal populations, could improve our understanding of the mechanisms leading to PTSD-AUD comorbidity.

The centrally-projecting Edinger-Westphal nucleus (EWcp) has been shown to contribute to regulation of multiple functions, including responses to stress and fear, attention, food consumption, addiction, body temperature and maternal behaviors. However, receptors involved in regulation of these behaviors through EWcp remain poorly characterized. On the other hand, the oxytocin peptide (OXT) is also known to regulate a substantial number of physiological responses and behaviors. Here we show that mRNA encoding OXT receptors (Oxtr) is expressed in EWcp of male and female C57BL/6J mice. These receptors are present on urocortin 1 (Ucn) mRNA-containing neurons and, to a lesser extent, on neurons in EWcp expressing the vesicular glutamate transporter 2 (Vglut2) mRNA of EWcp. Using RNAscope in situ hybridization, we show that neurons containing Ucn and Vglut2 mRNAs are two intermingled, but independent subpopulations in EWcp and characterize their relationship with other populations of neurons in the vicinity of this nucleus. Using immunohistochemistry, we show that intraperitoneal (IP) administration of OXT can induce FOS in Oxtr-containing neurons, suggesting that these receptors on EWcp neurons are functional. A follow up study showed that injection of OXT (2.3 or 7.7 mg/kg, IP) is accompanied by a decrease in body temperature. Since EWcp is known to be involved in regulation of body temperature, we hypothesize that OXT's effects on body temperature could be mediated through the EWcp. The contribution of OXTR in EWcp to regulation of various functions of EWcp and OXT needs to be deciphered.

Targeting the oxytocin (OXT) peptide system has emerged as a promising new approach for the treatment of alcohol use disorder (AUD). However, further advancements in this development depend on properly modeling various complex social aspects of AUD and its treatment. Here we examined behavioral and molecular underpinnings of OXT receptor (OXTR) agonism in prairie voles, a rodent species with demonstrated translational validity for neurobiological mechanisms regulating social affiliations. To further improve translational validity of these studies, we examined effects of intranasal (IN) OXT administration in male and female prairie voles socially housed in the presence of untreated cagemates. IN OXT selectively inhibited alcohol drinking in male, but not female, animals. Further, we confirmed that exogenously administered OXT penetrates the prairie vole brain and showed that Receptor for Advanced Glycation End-products assists this penetration after IN, but not intraperitoneal (IP), OXT administration. Finally, we demonstrated that IP administration of LIT-001, a small-molecule OXTR agonist, inhibits alcohol intake in male, but not female, prairie voles socially housed in the presence of untreated cagemates. Taken together, results of this study support the promise of selectively targeting OXTR for individualized treatment of AUD.