The orphan G-protein-coupled receptor GPR88 is highly expressed in the striatum. Studies using GPR88 knockout mice have suggested that the receptor is implicated in alcohol seeking and drinking behaviors. To date, the biological effects of GPR88 activation are still unknown due to the lack of a potent and selective agonist appropriate for in vivo investigation. In this study, we report the discovery of the first potent, selective, and brain-penetrant GPR88 agonist RTI-13951-33 (6). RTI-13951-33 exhibited an EC50 of 25 nM in an in vitro cAMP functional assay and had no significant off-target activity at 38 GPCRs, ion channels, and neurotransmitter transporters that were tested. RTI-13951-33 displayed enhanced aqueous solubility compared to (1R,2R)-2-PCCA (2) and had favorable pharmacokinetic properties for behavioral assessment. Finally, RTI-13951-33 significantly reduced alcohol self-administration and alcohol intake in a dose-dependent manner without effects on locomotion and sucrose self-administration in rats when administered intraperitoneally.

The insular cortex (IC) is a region proposed to modulate, in part, interoceptive states and motivated behavior. Interestingly, IC dysfunction and deficits in interoceptive processing are often found among individuals with substance-use disorders. Furthermore, the IC projects to the nucleus accumbens core (AcbC), a region known to modulate the discriminative stimulus/interoceptive effects of alcohol and other drug-related behaviors. Therefore, the goal of the present work was to investigate the possible role of the IC ➔ AcbC circuit in modulating the interoceptive effects of alcohol. Thus, we utilized a chemogenetic technique (hM4D designer receptor activation by designer drugs) to silence neuronal activity in the IC of rats trained to discriminate alcohol (1 g/kg, IG) versus water using an operant or Pavlovian alcohol discrimination procedure. Chemogenetic silencing of the IC or IC ➔ AcbC neuronal projections resulted in potentiated sensitivity to the interoceptive effects of alcohol in both the operant and Pavlovian tasks. Together, these data provide critical evidence for the nature of the complex IC circuitry and, specifically, suppression of the insular-striatal circuit in modulating behavior under a drug stimulus control.

Background: Growing evidence suggests that neuroimmune signaling via Toll-like receptors (TLRs) alters brain circuitry related to alcohol use disorders. Both ethanol (EtOH) exposure and the TLR3 agonist, poly(I:C), increase brain TLR3 expression in neurons and glia. Furthermore, previous studies have shown that cortical TLR3 expression is correlated with lifetime EtOH intake in humans.Methods: The current experiments investigated the consequences of poly(I:C) treatment on gene expression in 2 brain regions contributing to alcohol reinforcement, the insular cortex (IC) and nucleus accumbens (Acb) and on operant EtOH self-administration, in Long Evans rats.Results: TLR3 activation increased mRNA levels of neuroimmune genes (TLR3, COX2), glutamatergic genes (mGluR2, mGluR3, GLT1), and the trophic factor BDNF in Acb and IC. Furthermore, increases in each of these genes were correlated with increases in TLR3 mRNA, suggesting that TLR3 induction of these genes may impact excitatory transmission in IC and Acb. TLR3 activation also increased EtOH self-administration 18 days postinjection and enhanced the effects of the mGluR2/3 agonist LY379268 to reduce EtOH self-administration following poly(I:C).Conclusions: Together, these findings suggest lasting consequences of TLR3 activation on gene expression including increases in Group II mGluRs in the Acb. Furthermore, we show an important role for TLR3 signaling in EtOH intake, and a functional involvement of Group II mGluRs.

Stress can drive adaptive changes to maintain survival during threatening stimuli. Chronic stress exposure, however, may result in pathological adaptations. A key neurotransmitter involved in stress signaling is norepinephrine. Previous studies show that stress elevates norepinephrine levels in the bed nucleus of the stria terminalis (BNST), a critical node regulating anxiety and upstream of stress responses. Here, we use mice expressing channelrhodopsin in norepinephrine neurons to selectively activate terminals in the BNST, and measure norepinephrine release with fast-scan cyclic voltammetry. Mice exposed to a single restraint session show an identical norepinephrine release profile compared to that of unexposed mice. Mice experiencing five days of restraint stress, however, show elevated norepinephrine release across multiple stimulation parameters, and reduced sensitivity to the α 2 -adrenergic receptor antagonist idazoxan. These data are the first to examine norepinephrine release in the BNST to tonic and phasic stimulation frequencies, and confirm that repeated stress alters autoreceptor sensitivity.

Sex differences in chronic pain and alcohol abuse are not well understood. The development of rodent models is imperative for investigating the underlying changes behind these pathological states. In the present study, we investigated whether hind paw treatment with the inflammatory agent Complete Freund’s Adjuvant (CFA) could generate hyperalgesia and alter alcohol consumption in male and female C57BL/6J mice. CFA treatment led to greater nociceptive sensitivity for both sexes in the Hargreaves test, and increased alcohol drinking for males in a continuous access two-bottle choice (CA2BC) paradigm. Regardless of treatment, female mice exhibited greater alcohol drinking than males. Following a 2-hour terminal drinking session, CFA treatment failed to produce changes in alcohol drinking, blood ethanol concentration (BEC), and plasma corticosterone (CORT) for both sexes. 2-hr alcohol consumption and CORT was higher in females than males, irrespective of CFA treatment. Taken together, these findings have established that male mice are more susceptible to escalations in alcohol drinking when undergoing pain, despite higher levels of total alcohol drinking and CORT in females. Furthermore, the exposure of CFA-treated C57BL/6J mice to the CA2BC drinking paradigm has proven to be a useful model for studying the relationship between chronic pain and alcohol abuse. Future applications of the CFA/CA2BC model should incorporate manipulations of stress signaling and other related biological systems to improve our mechanistic understanding of pain and alcohol interactions.

Cortisol/corticosterone and the hypothalamic-pituitary-adrenal (HPA) axis serve an important role in modulating alcohol drinking behaviors. To date most alcohol research has focused on the functional involvement of corticosterone and the glucocorticoid receptor (GR), the primary receptor for corticosterone. Recent studies have indicated that the related mineralocorticoid receptor (MR), which binds both corticosterone and aldosterone, may also play a role in alcohol drinking. Therefore, the purpose of the present study was to test the functional role of MR signaling in alcohol self-administration via pharmacological antagonism of the MR with spironolactone. Male and female Long-Evans rats were trained to self-administer a sweetened alcohol solution (15% (v/v) alcohol +2% (w/v) sucrose). The effects of spironolactone (0, 10, 25, 50 mg/kg; IP) were tested on alcohol self-administration and under "probe extinction" conditions to measure the persistence of responding in the absence of the alcohol reinforcer. Parallel experiments in sucrose self-administration trained rats were used to confirm the specificity of spironolactone effects to an alcohol reinforcer. In female rats spironolactone (50 mg/kg) reduced alcohol self-administration and persistence of alcohol responding. In male rats spironolactone (25 and 50 mg/kg) reduced alcohol self-administration, but not persistence of alcohol responding. Spironolactone reduced sucrose intake in female rats only, and locomotion in male and female rats during sucrose self-administration. There was no effect of spironolactone on persistence of sucrose responding. These studies add to growing evidence that the MR is involved in alcohol drinking, while underscoring the importance of studying both male and female animals.

Post-traumatic stress disorder (PTSD) is a psychiatric illness that can increase the risk for developing an alcohol use disorder (AUD). While clinical data has been useful in identifying similarities in the neurobiological bases of these disorders, preclinical models are essential for understanding the mechanism(s) by which PTSD increases the risk of developing AUD. The purpose of these studies was to examine if exposure of male Long-Evans rats to the synthetically produced predator odor 2,5-dihydro-2,4,5trimethylthiazoline (TMT) would increase alcohol self-administration, potentially by facilitating transfer of salience towards cues, and alter neuronal response to alcohol as measured by the immediate early gene c-Fos. In Experiment 1 rats exposed to repeated (4x) TMT showed reductions in goal-tracking behavior in Pavlovian conditioned approach, and increases in alcohol self-administration. In Experiment 2 rats exposed to repeated TMT showed blunted basolateral amygdala c-Fos response to alcohol, and increased correlation between medial prefrontal cortex and amygdala subregions. In Experiment 3 rats exposed to single, but not repeated TMT showed increases in alcohol self-administration, and no change in anxiety-like behavior or hyperarousal. In Experiment 4, rats showed no habituation of corticosterone response after 4 TMT exposures. In summary, exposure of male rats to TMT can cause escalations in alcohol self-administration, reductions in goal-tracking behavior, and reduction in BLA response to alcohol. These studies outline and utilize a novel preclinical model that can be used to further neurobiological understanding of the relationship between PTSD and AUD.

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