Craving is a complex construct that is not fully understood. Craving for alcohol is an important motivator of alcohol use, a known risk factor for return-to-drinking, and one of the Diagnostic and Statistical Manual of Mental Disorders, fifth edition (DSM-5) diagnostic criteria for alcohol use disorder (AUD). Craving can be assessed retrospectively over a defined period (tonic) or in response to alcohol cues in real-time, e.g., through alcohol cue exposure (CE) paradigms (phasic). We conducted exploratory analyses of a clinical study including three matched groups: currently drinking (CD, N=9) and abstinent (AB, N=10) people with AUD and healthy controls (HC, N=12). During two alcohol CE sessions (CE1, CE2), tonic craving was assessed using the Penn Alcohol Craving Scale (PACS) and the Obsessive-Compulsive Drinking Scale (OCDS). Phasic craving was assessed using the Alcohol Urge Questionnaire (AUQ) alongside the Alcohol Attention Scale (AAS). The CD group showed higher AUQ and AAS than AB and HC groups (main effect of group, CE1: P=.048; CE2: P=.047). Group differences were also observed for tonic craving: the CD group reported higher PACS scores than HC (P=.001), while AB and CD groups showed higher OCDS scores than HC (HC vs. CD P<.0001, HC vs. AB P=.015). PACS scores correlated positively with OCDS (r=0.565, P=.001) and AUQ (r=0.595, P=.0004) scores in the full sample; within-group analyses showed a positive correlation between PACS and AUQ only in the AB group. Given the small sample size, these exploratory results should be considered preliminary and require confirmation in larger studies.

Traumatic stress is linked to problematic alcohol use, yet its persistent effects on alcohol sensitivity remain unclear. This study examined the long-term effects of traumatic stress on alcohol sensitivity in male and female Long-Evans rats. Rats were trained to discriminate alcohol (2 g/kg, i.g.) from water, and two weeks after a single, inescapable exposure to the predator odor stressor TMT, substitution for an alcohol dose curve and GABAA agonism were assessed both systemically (pentobarbital, i.p.) and site-specifically [muscimol in the prelimbic cortex (PrL) or anterior insular cortex (aIC)]. Additional experiments in alcohol-naive rats examined the effects of TMT on alcohol-induced behaviors, c-Fos expression, and GABAA receptor gene expression in the PrL and aIC. TMT exposure produced a leftward shift in the alcohol dose-response curve, indicating increased interoceptive sensitivity to alcohol in males, but not females. The alcohol-like effects of systemic pentobarbital were unaltered by TMT exposure. TMT reduced Gabra1 expression and increased c-Fos in the PrL of males, whereas TMT increased Gabra1 expression without altering c-Fos in the PrL of females. Alcohol-induced effects on locomotion and startle response were not observed in the TMT-exposed group when analyzed in both sexes. These findings highlight sex-specific effects of traumatic stress on alcohol sensitivity, with evidence that stress-induced PrL GABAA adaptations may contribute to increased interoceptive sensitivity to alcohol in males. These results may help to better understand the association between traumatic stress and alcohol use disorder (AUD).

Rationale: The subjective effects of alcohol are associated with alcohol use disorder (AUD) vulnerability and treatment outcomes. The interoceptive effects of alcohol are part of these subjective effects and can be measured in animal models using drug discrimination procedures. The newly developed mGlu2 and mGlu3 negative allosteric modulators (NAMs) are potential therapeutics for AUD and may alter interoceptive sensitivity to alcohol. Objectives: To determine the effects of mGlu2 and mGlu3 NAMs on the interoceptive effects of alcohol in rats. Methods: Long-Evans rats were trained to discriminate the interoceptive stimulus effects of alcohol (2.0 g/kg, i.g.) from water using both operant (males only) and Pavlovian (male and female) drug discrimination techniques. Following acquisition training, an alcohol dose-response (0, 0.5, 1.0, 2.0 g/kg) experiment was conducted to confirm stimulus control over behavior. Next, to test the involvement of mGlu2 and mGlu3, rats were pretreated with the mGlu2-NAM (VU6001966; 0, 3, 6, 12 mg/kg, i.p.) or the mGlu3-NAM (VU6010572; 0, 3, 6, 12 mg/kg, i. p.) before alcohol administration (2.0 g/kg, i.g.). Results: In Pavlovian discrimination, male rats showed greater interoceptive sensitivity to 1.0 and 2.0 g/kg alcohol compared to female rats. Both mGlu2-NAM and mGlu3-NAM attenuated the interoceptive effects of alcohol in male and female rats using Pavlovian and operant discrimination. There may be a potential sex difference in response to the mGlu2-NAM at the highest dose tested. Conclusions: Male rats may be more sensitive to the interoceptive effects of the 2.0 g/kg alcohol training dose compared to female rats. Both mGlu2-and mGlu3-NAM attenuate the interoceptive effects of alcohol in male and female rats. These drugs may have potential for treatment of AUD in part by blunting the subjective effects of alcohol.

Existing literature indicates that communication between the central nervous system and the peripheral nervous system is disrupted by substance use disorders (SUDs), including alcohol use disorder (AUD). Fibroblast growth factor 21 (FGF21), a liver-brain axis hormone governing energy homeostasis, has been shown to modulate alcohol intake/preference and other substances. To further elucidate the relationship between FGF21, alcohol use, and other substance use, we conducted a scoping review to explore the association between FGF21 and SUDs. Increases in FGF21 reduce alcohol consumption while suppressing FGF21 increases alcohol consumption, demonstrating an inverse relationship. Alcohol elevates FGF21 levels primarily via the liver, subsequently promoting neuronal signals to curb alcohol intake. FGF21 activation engages molecular pathways that defend against alcohol-induced fat accumulation, oxidative stress, and inflammation. Considering the bidirectional association between FGF21 and alcohol, further studies on the FGF21 system as a potential pharmacotherapy for AUD and alcohol-associated liver disease are warranted.

Background:Traumatic stress is associated with high rates of problematic alcohol use, but how the persistent effects of trauma impact sensitivity to alcohol remain unknown. This study examined the persistent effects of traumatic stress exposure on sensitivity to alcohol and underlying neurobiological mechanisms in rats. Methods:Male (N=98) and female (N=98) Long-Evans rats were exposed to the predator odor TMT, and two weeks later, molecular, neuronal, and behavioral sensitivity to alcohol were assessed. Next, rats were trained to discriminate alcohol from water (male N=70; female N=56), and the impact of TMT on interoceptive sensitivity to alcohol and the alcohol-like effects of systemic GABAA receptor activation were evaluated. Lastly, functional involvement of GABAA and NMDA receptors in the prelimbic cortex (PrL) and the anterior insular cortex (aIC) was investigated. Results:TMT exposure sex-dependently altered PrL Gabra1, and elevated aIC Grin2b and Grin2c in males. TMT increased PrL c-Fos in males, which was attenuated by alcohol administration. Alcohol-induced locomotor and startle response effects were attenuated in the TMT group in both sexes. TMT exposure potentiated interoceptive sensitivity to alcohol in males but not in females, and this effect was driven by GABAA receptors in the PrL. Greater stress reactivity during TMT exposure was associated with higher interoceptive sensitivity to alcohol, and alcohol exposure history was linked to a heightened stress response to TMT in males. Conclusions:Traumatic stress increased interoceptive sensitivity to alcohol in males, but not females, through PrL GABAA receptor adaptations, potentially enhancing the stimulatory, and by extension the rewarding, effects of alcohol.

Alcohol use disorders (AUDs) impose an enormous societal and financial burden, and world-wide, alcohol misuse is the 7th leading cause of premature death [1]. Despite this, there are currently only 3 FDA approved pharmacological approaches for the treatment of AUDs in the United States. The neurotensin (Nts) system has long been implicated in modulating behaviors associated with alcohol misuse. Recently, a novel compound, SBI-553, that biases the action of Nts receptor 1 (NTSR1) activation, has shown promise in preclinical models of psychostimulant use. Here we investigate the efficacy of this compound to alter ethanol-mediated behaviors in a comprehensive battery of experiments assessing ethanol consumption, behavioral responses to ethanol, physiological sensitivity to ethanol, and ethanol metabolism. Additionally, we investigated behavior in avoidance and cognitive assays to monitor potential side effects of SBI-553. We find that SBI-553 reduces ethanol consumption in mice without altering avoidance behavior or novel object recognition. We also observe sex-dependent differences in physiological responses to sequential ethanol injections in mice. In rats, we show that SBI-553 attenuates sensitivity to the interoceptive effects of ethanol (using a Pavlovian drug discrimination task). Our data suggest that targeting NTSR1 signaling may be promising to attenuate alcohol misuse, and adds to a body of literature that suggests NTSR1 may be a common downstream target involved in the psychoactive effects of multiple reinforcing substances.

The neurosteroid 3α,5α-THP is a potent GABAA receptor-positive modulator and its regulatory action on the HPA axis stress response has been reported in numerous preclinical and clinical studies. We previously demonstrated that 3α,5α-THP down-regulation of HPA axis activity during stress is sex-, brain region- and stressor-dependent. In this study, we observed a deleterious submersion behavior in response to 3α,5α-THP (15 mg/kg) during forced swim stress (FSS) that led us to investigate how 3α,5α-THP might affect behavioral coping strategies engaged in by the animal. Given the well-established involvement of the opioid system in HPA axis activation and its interaction with GABAergic neurosteroids, we explored the synergic effects of 3α,5α-THP/opiate system activation in this behavior. Serum β-endorphin (β-EP) was elevated by FSS and enhanced by 3α,5α-THP + FSS. Hypothalamic Mu-opiate receptors (MOP) were increased in female rats by 3α,5α-THP + FSS. Pretreatment with the MOP antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP; 2 mg/kg, IP) reversed submersion behavior in males. Moreover, in both males and females, CTAP pretreatment decreased immobility episodes while increasing immobility duration but did not alter swimming duration. This interaction between 3α,5α-THP and the opioid system in the context of FSS might be important in the development of treatment for neuropsychiatric disorders involving HPA axis activation.

Metabotropic glutamate (mGlu) receptors are promising targets for the treatment of affective disorders and alcohol use disorder (AUD). Nonspecific ligands for Group II (mGlu2 and mGlu3) mGlu receptors have demonstrated consistent therapeutic potential for affective disorders in preclinical models. Disentangling the specific roles of mGlu2 versus mGlu3 receptors in these effects has persisted as a major challenge, in part due to pharmacological limitations. However, the recent development of highly specific allosteric modulators for both mGlu2 and mGlu3 receptors have enabled straightforward and rigorous investigations into the specific function of each receptor. Here, we review recent experiments using these compounds that have demonstrated both similar and distinct receptor functions in behavioral, molecular, and electrophysiological measures associated with basal function and preclinical models of affective disorders. Studies using these selective drugs have demonstrated that mGlu2 is the predominant receptor subclass involved in presynaptic neurotransmitter release in prefrontal cortex. By contrast, the activation of postsynaptic mGlu3 receptors induces a cascade of cellular changes that results in AMPA receptor internalization, producing long-term depression and diminishing excitatory drive. Acute stress decreases the mGlu3 receptor function and dynamically alters transcript expression for both mGlu2 (Grm2) and mGlu3 (Grm3) receptors in brain areas involved in reward and stress. Accordingly, both mGlu2 and mGlu3 negative allosteric modulators show acute antidepressant-like effects and potential prophylactic effects against acute and traumatic stressors. The wide array of effects displayed by these new allosteric modulators of mGlu2 and mGlu3 receptors suggest that these drugs may act through improving endophenotypes of symptoms observed across several neuropsychiatric disorders. Therefore, recently developed allosteric modulators selective for mGlu2 or mGlu3 receptors show promise as potential therapeutics for affective disorders and AUD.

All drugs with the potential for addiction liability, including alcohol, produce distinct interoceptive stimulus effects that influence drug intake. Our lab has shown involvement of Group II metabotropic receptors (mGlu2/3) in the expression of alcohol interoceptive effects using a nonspecific mGlu2/3 agonist and antagonist. Furthermore, the anterior insular cortex (aIC) is a hub for interoceptive processing, including the interoceptive effects of alcohol. Therefore, the goal of the present work was to assess the independent functional contribution of mGlu2 and mGlu3, and the role of mGlu3 in the aIC, in the interoceptive effects of alcohol. As such, male, Long-Evans rats were trained to discriminate the stimulus effects of alcohol (2 g/kg, i.g.) from water (i.g.) using an operant, 2-lever drug discrimination procedure. To assess involvement of mGlu2 and mGlu3 in the discriminative stimulus effects of alcohol, rats were administered the mGlu2 negative allosteric modulator (NAM, VU6001966; 0, 3, 6 mg/kg, i.p.) and the mGlu3-NAM (VU6010572; 0, 3, 6, 12 mg/kg, i.p.) before alcohol (2 g/kg, i.g.). mGlu2-NAM (6 mg/kg) and mGlu3-NAM (12 mg/kg) decreased alcohol-appropriate lever responses without affecting response rate, reflecting diminished interoceptive alcohol sensitivity. mGlu3-NAM microinjection (3 μg) into the aIC before alcohol decreased response rate to such an extent that alcohol-appropriate lever responding could not be determined. As such, mGlu3-NAM was tested in the aIC before water administration to determine if VU6010572 substitutes for alcohol. mGlu3-NAM VU6010572 (3 μg) in the aIC produced partial alcohol-like stimulus effects without affecting response rate. These data indicate that the newly developed mGlu2 and mGlu3 NAMs can modulate interoceptive effects of alcohol. The opposing effects of systemic vs. microinjected (aIC) mGlu3-NAM on alcohol interoceptive effects may reflect differences in receptor binding, a functionally relevant metabolite, and/or brain region differences in receptor function.

A stressor can trigger lasting adaptations that contribute to neuropsychiatric disorders. Predator odor (TMT) exposure is an innate stressor that may activate the metabotropic glutamate receptor 3 (mGlu3) to produce stress adaptations. To evaluate functional involvement, the mGlu3 negative allosteric modulator (NAM, VU6010572; 3 mg/kg, i.p.) was administered before TMT exposure in male, Long Evans rats. Two weeks after, rats underwent context re-exposure, elevated zero maze (ZM), and acoustic startle (ASR) behavioral tests, followed by RT-PCR gene expression in the insular cortex and bed nucleus of the stria terminalis (BNST) to evaluate lasting behavioral and molecular adaptations from the stressor. Rats displayed stress-reactive behaviors in response to TMT exposure that were not affected by VU6010572. Freezing and hyperactivity were observed during the context re-exposure, and mGlu3-NAM pretreatment during stressor prevented the context freezing response. TMT exposure did not affect ZM or ASR measures, but VU6010572 increased time spent in the open arms of the ZM and ASR habituation regardless of stressor treatment. In the insular cortex, TMT exposure increased expression of mGlu (Grm3, Grm5) and NMDA (GriN2A, GriN2B, GriN2C, GriN3A, GriN3B) receptor transcripts, and mGlu3-NAM pretreatment blocked GriN3B upregulation. In the BNST, TMT exposure increased expression of GriN2B and GriN3B in vehicle-treated rats, but decreased expression in the mGlu3-NAM group. Similar to the insular cortex, mGlu3-NAM reversed the stressor-induced upregulation of GriN3B in the BNST. mGlu3-NAM also upregulated GriN2A, GriN2B, GriN3B and Grm2 in the control group, but not the TMT group. Together, these data implicate mGlu3 receptor signaling in some lasting adaptations of predator odor stressor and anxiolytic-like effects.

Corticotropin-releasing factor (CRF) regulates the stress response in the hypothalamus and modulates neurotransmission across the brain through CRF receptors. Acute stress increases hypothalamic CRF and the GABAergic neurosteroid (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP). We previously showed that 3α,5α-THP regulation of CRF is sex and brain region dependent. In this study, we investigated 3α,5α-THP regulation of stress-induced hypothalamic CRF, CRF receptor type 1 (CRFR1), CRF binding protein (CRFBP), pro-opiomelanocortin (POMC), and glucocorticoid receptor (GR) by western blot and circulating corticosterone (CORT) by enzyme-linked immunosorbent assay (ELISA) in male and female Sprague Dawley rats. Tissue was collected after rats were injected with 3α,5α-THP (15 mg/kg, IP) or vehicle 15 min prior to 30 min of restraint stress (RS), or 10 min of forced swim stress (FSS) and 20 min recovery. The initial exposure to a stress stimulus increased circulating CORT levels in both males and females, but 3α,5α-THP attenuated the CORT response only in females after RS. 3α,5α-THP reduced GR levels in male and females, but differently between stressors. 3α,5α-THP decreased the CRF stress response after FSS in males and females, but after RS, only in female rats. 3α,5α-THP reduced the CRFR1, CRFBP, and POMC increases after RS and FSS in males, but in females only after FSS. Our results showed different stress responses following different types of stressors: 3α,5α-THP regulated the HPA axis at different levels, depending on sex.

Post-traumatic stress disorder (PTSD) confers enhanced vulnerability to develop comorbid alcohol use disorder (AUD). Exposure to the scent of a predator, such as the fox odor TMT, has been used to model a traumatic stressor with relevance to PTSD symptomatology. Alcohol produces distinct interoceptive (subjective) effects that may influence vulnerability to problem drinking and AUD. As such, understanding the lasting impact of stressor on sensitivity to the interoceptive effects of alcohol is clinically relevant. The present study used a 2-lever, operant drug discrimination procedure to train male, Long-Evans rats to discriminate the interoceptive effects of alcohol (2 g/kg, i.g.) from water. Upon stable performance, rats underwent a 15-min exposure to TMT. Two weeks later, an alcohol dose-response curve was conducted to evaluate the lasting effects of the TMT stressor on the interoceptive effects of alcohol. The TMT group showed a leftward shift in ED 50 of the dose response curve compared to controls, reflecting potentiated interoceptive sensitivity to alcohol. TMT exposure did not affect response rate. GABAergic signaling in both the anterior insular cortex (aIC) and the nucleus accumbens (Acb) is involved in the interoceptive effects of alcohol and stressor-induced adaptations. As such, follow-up experiments in alcohol-naïve rats examined neuronal activation (as measured by c-Fos immunoreactivity) following TMT and showed that TMT exposure increased c-Fos expression in the aIC and the nucleus accumbens core (AcbC). 2 weeks after TMT exposure, Gad-1 gene expression was elevated in the aIC and Gat-1 was increased in the Acb compared to controls. Lastly, the alcohol discrimination and alcohol-naïve groups displayed dramatic differences in stress reactive behaviors during the TMT exposure, suggesting that alcohol exposure may alter the behavioral response to predator odor. Together, these data suggest that predator odor stressor results in potentiated sensitivity to alcohol possibly through GABAergic adaptations in the aIC and Acb, which may be relevant to understanding PTSD-AUD comorbidity.

CRF is the main activator of the hypothalamic-pituitary-adrenal (HPA) axis in response to stress. CRF neurons are found mainly in the hypothalamus, but CRF positive cells and CRF1 receptors are also found in extra-hypothalamic structures, including amygdala (CeA), hippocampus, NAc and VTA. CRF release in the hypo-thalamus is regulated by inhibitory GABAergic interneurons and extrahypothalamic glutamatergic inputs, and disruption of this balance is found in stress-related disorders and addiction. (3 alpha,5 alpha)3-hydroxypregnan-20-one (3 alpha,5 alpha-THP), the most potent positive modulator of GABAA receptors, attenuates the stress response reducing hypothalamic CRF mRNA expression and ACTH and corticosterone serum levels. In this study, we explored 3 alpha,5 alpha-THP regulation of hypothalamic and extrahypothalamic CRF mRNA and peptide expression, in male and female Sprague Dawley rats, following vehicle or 3 alpha,5 alpha-THP administration (15 mg/kg). In the hypothalamus, we found sex differences in CRF mRNA expression (females +74%, p < 0.01) and CRF peptide levels (females-71%, p < 0.001). 3 alpha,5 alpha-THP administration reduced hypothalamic CRF mRNA expression only in males (-50%, p < 0.05) and did not alter CRF peptide expression in either sex. In hippocampus and CeA, 3 alpha,5 alpha-THP administration reduced CRF peptide concentrations only in the male (hippocampus-29%, p < 0.05; CeA-62%, p < 0.01). In contrast, 3 alpha,5 alpha-THP injection increased CRF peptide concentration in the VTA of both males (+32%, p < 0.01) and females (+26%, p < 0.01). The results show sex and region-specific regulation of CRF signals and the response to 3 alpha,5 alpha-THP administration. This data may be key to successful development of therapeutic approaches for stress-related disorders and addiction.

Interoception refers to the perception of the internal state of the body and is increasingly being recognized as an important factor in mental health disorders. Drugs of abuse produce powerful interoceptive states that are upstream of behaviors that drive and influence drug intake, and addiction pathology is impacted by interoceptive processes. The goal of the present review is to discuss interoceptive processes related to alcohol. We will cover physiological responses to alcohol, how interoceptive states can impact drinking, and the recruitment of brain networks as informed by clinical research. We also review the molecular and brain circuitry mechanisms of alcohol interoceptive effects as informed by preclinical studies. Finally, we will discuss emerging treatments with consideration of interoception processes. As our understanding of the role of interoception in drug and alcohol use grows, we suggest that the convergence of information provided by clinical and preclinical studies will be increasingly important. Given the complexity of interoceptive processing and the multitude of brain regions involved, an overarching network-based framework can provide context for how focused manipulations modulate interoceptive processing as a whole. In turn, preclinical studies can systematically determine the roles of individual nodes and their molecular underpinnings in a given network, potentially suggesting new therapeutic targets and directions. As interoceptive processing drives and influences motivation, emotion, and subsequent behavior, consideration of interoception is important for our understanding of processes that drive ongoing drinking and relapse.

A stressor can trigger adaptations that contribute to neuropsychiatric disorders. Predator odor (TMT) exposure is an innate stressor that produces lasting adaptations. TMT exposure may activate metabotropic glutamate receptor 3 (mGlu 3 ), triggering excitatory corticolimbic adaptations that underlie behavioral changes. To evaluate functional involvement, the mGlu 3 negative allosteric modulator (NAM, VU6010572; 3 mg/kg, i.p.) was administered before TMT exposure in male, Long Evans rats. Two weeks after stressor, rats underwent behavioral testing (context re-exposure, zero maze and acoustic startle response) followed by RT-PCR gene expression in the insular cortex and BNST. During the TMT exposure, rats displayed stress-reactive behaviors that were not affected by the VU6010572. During the context re-exposure, prior TMT exposure and VU6010572 pretreatment both produced a hyperactive response. TMT exposure did not affect zero maze or ASR measures, but VU6010572 increased time spent in the open arms and habituation to ASR, indicating anxiolytic-like effects. In the insular cortex, TMT exposure resulted in excitatory adaptations as shown by increased expression of mGlu ( Grm3, Grm5 ), NMDA ( GriN2A, GriN2B, GriN2C, GriN3A, GriN3B ) and AMPA ( GriA3 ) receptor transcripts. Interestingly, mGlu 3 signaling during stressor mediated GriN3B upregulation. Stress reactivity during TMT exposure was associated with Grm5, GriN2A, GriN2C , and GriA3 upregulation in the insular cortex and context re-exposure reactivity in the TMT/vehicle, but not the TMT/mGlu 3 NAM group. In the BNST, GriN2A, GriN2B and GriN3B were increased by VU6010572, but TMT prevented these effects. These data demonstrate that mGlu 3 signaling contributes to the lasting behavioral and molecular adaptations of predator odor stressor.