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Cue-induced reinstatement of alcohol-seeking is a hallmark behavioral pathology of addiction. Evidence suggests that reinstatement (e.g., relapse), may be regulated by cell signaling systems that underlie neuroplasticity. A variety of plasticity events require activation of calcium calmodulin-dependent protein kinase II (CaMKII) in components of the reward pathway, such as the nucleus accumbens and amygdala. We sought to determine if cue-induced reinstatement of alcohol-seeking behavior is associated with changes in the activation state (e.g., phosphorylation) of CaMKII-T286. Male C57BL/6J mice (n = 14) were trained to lever press on a fixed-ratio-4 schedule of sweetened alcohol (2% sucrose + 9% EtOH) reinforcement. After 14-d of extinction (no cues or reinforcers), mice underwent a response-contingent reinstatement (n = 7) vs. an additional day of extinction (n = 7). Brains were removed immediately after the test and processed for evaluation of pCaMKII-T286 immunoreactivity (IR). Number of pCaMKII-T286 positive cells/mm2 was quantified from coronal brain sections using Bioquant Image Analysis software. Mice emitted significantly more responses on the alcohol vs. the inactive lever throughout the baseline phase with average alcohol intake of 1.1 ± 0.03 g/kg/1-h. During extinction, responses on the alcohol lever decreased to inactive lever levels by day 7. Significant cue-induced reinstatement of alcohol-seeking was observed during a single test with no effects on the inactive lever. Reinstatement was associated with increased pCaMKII-T286 IR specifically in amygdala (LA and BLA), nucleus accumbens (AcbSh), lateral septum, mediodorsal thalamus, and piriform cortex as compared to extinction control. Brain regions showing no change included the dorsal striatum, medial septum, cingulate cortex, habenula, paraventricular thalamus, and ventral hypothalamus. These results show response contingent cue-induced reinstatement of alcohol-seeking behavior is associated with selective increases in pCaMKII-T286 in specific reward- and memory-related brain regions of male C57BL/6J mice. Primary molecular mechanisms of associative learning and memory may regulate relapse in alcohol addiction.

Extracellular signal-regulated protein kinase (ERK1/2) is a member of the mitogen-activated protein kinase (MAPK) signaling pathway and a key molecular target for ethanol (EtOH) and other drugs of abuse.

Despite worldwide consumption of moderate amounts of alcohol, the neural mechanisms that mediate the transition from use to abuse are not fully understood.

Repeated and/or heightened elevations in glucocorticoids (e.g., repeated stress) can promote escalated drug-taking behaviors and induce compromised HPA axis function. Given that interoceptive/subjective drug cues are a fundamental factor in drug-taking behavior, we sought to determine the effects of exposure to repeated elevations in the glucocorticoid corticosterone (CORT) on the interoceptive effects of alcohol in rats using drug discrimination techniques.

Background:  Neuroactive steroids modulate ethanol intake in several self‐administration models with variable effects. The purpose of this work was to examine the effects of the long‐acting synthetic GABAergic neurosteroid ganaxolone and the endogenous neurosteroid pregnenolone, a precursor of all GABAergic neuroactive steroids, on the maintenance of ethanol self‐administration in an animal model of elevated drinking—the alcohol‐preferring (P) rats.Methods:  P rats were trained to self‐administer ethanol (15% v/v) versus water on a concurrent schedule of reinforcement, and the effects of ganaxolone (0 to 30 mg/kg, subcutaneous [SC]) and pregnenolone (0 to 75 mg/kg, intraperitoneal [IP]) were evaluated on the maintenance of ethanol self‐administration. After completion of self‐administration testing, doses of the neuroactive steroids that altered ethanol self‐administration were assessed on spontaneous locomotor activity. Finally, the effect of pregnenolone administration on cerebral cortical levels of the GABAergic neuroactive steroid (3α,5α)‐3‐hydroxypregnan‐20‐one (allopregnanolone, 3α,5α‐THP) was determined in both ethanol‐experienced and ethanol‐inexperienced P rats because pregnenolone is a precursor of these steroids.Results:  Ganaxolone produced a dose‐dependent biphasic effect on ethanol reinforcement, as the lowest dose (1 mg/kg) increased and the highest dose (30 mg/kg) decreased ethanol‐reinforced responding. However, the highest ganaxolone dose also produced a nonspecific reduction in locomotor activity. Pregnenolone treatment significantly reduced ethanol self‐administration (50 and 75 mg/kg), without altering locomotor activity. Pregnenolone (50 mg/kg) produced a significant increase in cerebral cortical allopregnanolone levels. This increase was observed in the self‐administration trained animals, but not in ethanol‐naïve P rats.Conclusions:  These results indicate that pregnenolone dose‐dependently reduces operant ethanol self‐administration in P rats without locomotor impairment, suggesting that it may have potential as a novel therapeutic for reducing chronic alcohol drinking in individuals that abuse alcohol.

Background:  Emerging evidence indicates that Group I metabotropic glutamate receptors (mGluR1 and mGluR5) differentially regulates ethanol self‐administration in several rodent behavioral models. The purpose of this work was to further characterize involvement of Group I mGluRs in the reinforcing effects of ethanol using a progressive ratio schedule of reinforcement.Methods:  Alcohol‐preferring (P) rats were trained to self‐administer ethanol (15% v/v) versus water on a concurrent schedule of reinforcement, and the effects of the Group I mGluR antagonists were evaluated on progressive ratio performance. The rats were then trained to self‐administer sucrose (0.4% w/v) versus water, and the effects of the antagonists were tested on progressive ratio performance.Results:  The mGluR1 antagonist, 3,4‐dihydro‐2H‐pyrano[2,3]b quinolin‐7‐yl (cis‐4‐methoxycyclohexyl) methanone (JNJ 16259685; 0 to 1 mg/kg) and the mGluR5 antagonist, 6‐methyl‐2‐(phenylethynyl) pyridine (MPEP; 0 to 10 mg/kg) dose‐dependently reduced ethanol break point. In separate locomotor activity assessments, the lowest effective dose of JNJ 16259685 (0.3 mg/kg) produced a motor impairment, whereas the lowest effective dose of MPEP (3 mg/kg) did not. Thus, the reduction in ethanol break point by mGluR1 antagonism was probably a result of a motor impairment. JNJ 16259685 (0.3 mg/kg) and MPEP (10 mg/kg) reduced sucrose break point and produced motor impairments. Thus, the reductions in sucrose break point produced by both Group I antagonists were probably because of nonspecific effects on motor activity.Conclusions:  Together, these results suggest that glutamate activity at mGluR5 regulates motivation to self‐administer ethanol.

The discriminative stimulus properties of ethanol are mediated in part by positive modulation of GABAA receptors. Recent evidence indicates that metabotropic glutamate receptor subtype 5 (mGluR5) activity can influence GABAA receptor function. Therefore, the purpose of this work was to examine the potential involvement of mGluR5 in the discriminative stimulus effects of ethanol. In rats trained to discriminate ethanol (1 g/kg, intragastric gavage (i.g.)) from water, 2-methyl-6-(phenylethyl)-pyridine (MPEP) (1–50 mg/kg, i.p.) a selective noncompetitive antagonist of the mGlu5 receptor did not produce ethanol-like stimulus properties. However, pretreatment with MPEP (30 mg/kg) reduced the stimulus properties of ethanol as indicated by significant reductions in ethanol-appropriate responding, specifically at 0.5 and 1 g/kg ethanol, and a failure of ethanol test doses (1 and 2 g/kg) to fully substitute for the ethanol training dose. To test whether mGluR5 antagonism altered the GABAA receptor component of the ethanol stimulus, the ability of MPEP to modulate pentobarbital and diazepam substitution for ethanol was assessed. Pentobarbital substitution (1–10 mg/kg, i.p.) for ethanol was not altered by MPEP pretreatment. However, MPEP pretreatment inhibited the ethanol-like stimulus properties of diazepam (5 mg/kg, i.p.). To examine a potential anatomical basis for these pharmacological findings, expression patterns of mGluR5- and benzodiazepine-sensitive GABAA α1-containing receptors were examined by dual-label fluorescent immunohistochemistry with visualization by confocal microscopy. Results indicated that mGluR5- and GABAA α1-containing receptors were both coexpressed in limbic brain regions and colocalized on the same cells in specific brain regions including the amygdala, hippocampus, globus pallidus, and ventral pallidum. Together, these findings suggest an interaction between mGluR5- and benzodiazepine-sensitive GABAA receptors in mediating ethanol discrimination.

The development and progression of alcohol use disorder (AUD) are widely viewed as maladaptive neuroplasticity. The transmembrane alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) regulatory protein γ8 (TARP γ-8) is a molecular mechanism of neuroplasticity that has not been evaluated in AUD or other addictions.To address this gap in knowledge, we evaluated the mechanistic role of TARP γ-8 bound AMPAR activity in the basolateral amygdala (BLA) and ventral hippocampus (vHPC) in the positive reinforcing effects of alcohol, which drive repetitive alcohol use throughout the course of AUD, in male C57BL/6 J mice. These brain regions were selected because they exhibit high levels of TARP γ-8 expression and send glutamate projections to the nucleus accumbens (NAc), which is a key nucleus in the brain reward pathway.Site-specific pharmacological inhibition of AMPARs bound to TARP γ-8 in the BLA via bilateral infusion of the selective negative modulator JNJ-55511118 (0-2 µg/µl/side) significantly decreased operant alcohol self-administration with no effect on sucrose self-administration in behavior-matched controls. Temporal analysis showed that reductions in alcohol-reinforced response rate occurred > 25 min after the onset of responding, consistent with a blunting of the positive reinforcing effects of alcohol in the absence of nonspecific behavioral effects. In contrast, inhibition of TARP γ-8 bound AMPARs in the vHPC selectively decreased sucrose self-administration with no effect on alcohol.This study reveals a novel brain region-specific role of TARP γ-8 bound AMPARs as a molecular mechanism of the positive reinforcing effects of alcohol and non-drug rewards.