Alcohol Self-administration and Relapse-like Behavior: A Functional Role for Enhanced Activity at AMPA Receptors

Mechanisms underlying excessive alcohol drinking behavior and relapse are not fully understood and are critical for mapping the pathological course of alcohol use disorders (AUD). Long-term ethanol consumption results in strengthened excitatory neurotransmission and increased AMPA receptor signaling in animal models. However, the mechanistic role of enhanced AMPA receptor activity in ethanol-reinforcement and alcohol-seeking behavior remains unclear. Thus, the experiments in this dissertation sought to elucidate the behavioral and molecular mechanisms that underlie AMPA receptor-mediated ethanol reinforcement processes and relapse to ethanol-seeking behavior using a preclinical model of high alcohol preference, the alcohol-preferring (P-) rat. Enhancement of AMPA receptor signaling by systemically administered aniracetam (AMPA receptor positive allosteric modulator) significantly increased ethanol self-administration in a reinforcer-specific manner. Moreover, aniracetam potentiated cue-induced reinstatement in P-rats, which suggest that enhanced activity at AMPA receptors promotes reinforcement and ethanol-seeking behavior. Experiments further characterized enhanced AMPA receptor signaling in modulating operant self-administration and relapse-like behavior by examining neuroanatomical contributions to AMPA receptor-mediated alterations in ethanol reinforcement. Since AMPA receptor activity is potentiated by post-translational modification (e.g. phosphorylation of GluA1subunits), immunohistochemistry was used to examine neuroadaptive changes in pGluA1 in limbic brain regions after a history of ethanol self-administration. Increased pGluA1 immunoreactivity was observed in sub-nuclei of the amygdala and nucleus accumbens of ethanol self-administering P-rats relative to the sucrose controls. Guided by immunohistochemistry results, the effects of aniracetam on ethanol self-administration were examined via site-specific microinjections in the amygdala and nucleus accumbens. Intra-amygdala, but not intra-accumbens, aniracetam increased ethanol self-administration in a reinforcer-specific manner. Furthermore, coadministration of intra-amygdala aniracetam and myristolated AIP (CaMKII peptide inhibitor) blocked aniracetam-induced increased ethanol self-administration; which demonstrates a critical role for amygdala CaMKII activity in AMPA receptor-mediated potentiation of ethanol reinforcement. These data suggest that enhanced amygdala AMPA receptor activity promotes drinking and ultimately could contribute to alcohol use disorders. In contrast, intra-amygdala aniracetam did not significantly alter cue-induced reinstatement; which suggest that enhanced AMPA receptor activity in this region may not significantly contribute to promoting cue-induced ethanol-seeking. Collectively, key experimental results provide novel insight into AMPA receptor-related mechanisms in excessive alcohol drinking behavior and vulnerability to relapse.Doctor of Philosoph

The nicotine + alcohol interoceptive drug state: contribution of the components and effects of varenicline in rats

Nicotine and alcohol co-use is highly prevalent, and as such, individuals experience the interoceptive effects of both substances together. Therefore, examining sensitivity to a compound nicotine and alcohol (N+A) interoceptive cue is critical to broaden our understanding of mechanisms that may contribute to nicotine and alcohol co-use

Transient increase in alcohol self-administration following a period of chronic exposure to corticosterone

Stressful life events and chronic stressors have been associated with escalations in alcohol drinking. Stress exposure leads to the secretion of glucocorticoids (cortisol in the human; corticosterone (CORT) in the rodent). To model a period of heightened elevations in CORT, the present work assessed the effects of chronic exposure to the stress hormone CORT on alcohol self-administration. Male Long Evans rats were trained to self-administer a sweetened alcohol solution (2% sucrose/15% alcohol) resulting in moderate levels of daily alcohol intake (0.5–0.7 g/kg). Following stable baseline operant self-administration, rats received CORT in the drinking water for 7 days. A transient increase in alcohol self-administration was observed on the first self-administration session following CORT exposure, and behavior returned to control levels by the second session. Control experiments determined that this increase in alcohol self-administration was specific to alcohol, unrelated to general motor activation, and functionally dissociated from decreased CORT levels at the time of testing. These results indicate that repeated exposure to heightened levels of stress hormone (e.g., as may be experienced during stressful episodes) has the potential to lead to exacerbated alcohol intake in low to moderate drinkers. Given that maladaptive drinking patterns, such as escalated alcohol drinking following stressful episodes, have the potential to put an individual at risk for future drinking disorders, utilization of this model will be important for examination of neuroadaptations that occur as a consequence of CORT exposure in order to better understand escalated drinking following stressful episodes in nondependent individuals

Enhanced AMPA receptor activity increases operant alcohol self-administration and cue-induced reinstatement: AMPA receptors and alcohol

Long-term alcohol exposure produces neuroadaptations that contribute to the progression of alcohol abuse disorders. Chronic alcohol consumption results in strengthened excitatory neurotransmission and increased AMPA receptor signaling in animal models. However, the mechanistic role of enhanced AMPA receptor activity in alcohol reinforcement and alcohol-seeking behavior remains unclear. This study examined the role of enhanced AMPA receptor function using the selective positive allosteric modulator, aniracetam, in modulating operant alcohol self-administration and cue-induced reinstatement. Male alcohol-preferring (P-) rats, trained to self-administer alcohol (15%, v/v) versus water were pretreated with aniracetam to assess effects on maintenance of alcohol self-administration. To determine reinforcer specificity, P-rats were trained to self-administer sucrose (0.8%, w/v) versus water, and effects of aniracetam were tested. The role of aniracetam in modulating relapse of alcohol-seeking was assessed using a response-contingent cue-induced reinstatement procedure in P-rats trained to self-administer 15% alcohol. Aniracetam pretreatment significantly increased alcohol-reinforced responses relative to vehicle treatment. This increase was not attributed to aniracetam-induced hyperactivity as aniracetam pretreatment did not alter locomotor activity. AMPA receptor involvement was confirmed because DNQX (AMPA receptor antagonist) blocked the aniracetam-induced increase in alcohol self-administration. Aniracetam did not alter sucrose-reinforced responses in sucrose-trained P-rats, suggesting that enhanced AMPA receptor activity is selective in modulating the reinforcing function of alcohol. Finally, aniracetam pretreatment potentiated cue-induced reinstatement of alcohol-seeking behavior versus vehicle treated-P-rats. These data suggest that enhanced glutamate activity at AMPA receptors may be key in facilitating alcohol consumption and seeking behavior which could ultimately contribute to the development of alcohol abuse disorders

Stress Hormone Exposure Reduces mGluR5 Expression in the Nucleus Accumbens: Functional Implications for Interoceptive Sensitivity to Alcohol

Escalations in alcohol drinking associated with experiencing stressful life events and chronic life stressors may be related to altered sensitivity to the interoceptive/subjective effects of alcohol. Indeed, through the use of drug discrimination methods, rats show decreased sensitivity to the discriminative stimulus (interoceptive) effects of alcohol following exposure to the stress hormone corticosterone (CORT). This exposure produces heightened elevations in plasma CORT levels (eg, as may be experienced by an individual during stressful episodes). We hypothesized that decreased sensitivity to alcohol may be related, in part, to changes in metabotropic glutamate receptors-subtype 5 (mGluR5) in the nucleus accumbens, as these receptors in this brain region are known to regulate the discriminative stimulus effects of alcohol. In the accumbens, we found reduced mGluR5 expression (immunohistochemistry and Western blot) and decreased neural activation (as measured by c-Fos immunohistochemistry) in response to a moderate alcohol dose (1 g/kg) following CORT exposure (7 days). The functional role of these CORT-induced adaptations in relation to the discriminative stimulus effects of alcohol was confirmed, as both the systemic administration of 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) an mGluR5 positive allosteric modulator and the intra-accumbens administration of (R,S)-2-Amino-2-(2-chloro-5-hydroxyphenyl)acetic acid sodium salt (CHPG) an mGluR5 agonist restored sensitivity to alcohol in discrimination-trained rats. These results suggest that activation of mGluR5 may alleviate the functional impact of the CORT-induced downregulation of mGluR5 in relation to sensitivity to alcohol. Understanding the contribution of such neuroadaptations to the interoceptive effects of alcohol may enrich our understanding of potential changes in subjective sensitivity to alcohol during stressful episodes

Intra-amygdala inhibition of ERK1/2 potentiates the discriminative stimulus effects of alcohol

Extracellular signal-regulated kinase (ERK1/2) has been implicated in modulating drug seeking behavior and is a target of alcohol and other drugs of abuse. Given that the discriminative stimulus (subjective/interoceptive) effects of drugs are determinants of abuse liability and can influence drug seeking behavior, we examined the role of ERK1/2 in modulating the discriminative stimulus effects of alcohol. Using drug discrimination procedures, rats were trained to discriminate a moderate intragastric (IG) alcohol dose (1 g/kg) versus water (IG). Following an alcohol (1 g/kg) discrimination session phosphorylated ERK1/2 (pERK1/2) immunoreactivity (IR) was significantly elevated in the amygdala, but not the nucleus accumbens. Therefore, we hypothesized that intra-amygdala inhibition of ERK1/2 would disrupt expression of the discriminative stimulus effects of alcohol. However, intra-amygdala or accumbens administration of the MEK/ERK1/2 inhibitor U0126 (1 and 3 μg) had no effect on the discriminative stimulus effects of the training dose of alcohol (1 g/kg). Contrary to our hypothesis, intra-amygdala infusion of U0126 (3 μg) potentiated the discriminative stimulus effects of a low alcohol dose (0.5 g/kg) and had no effect following nucleus accumbens infusion. Importantly, site-specific inhibition of pERK1/2 in each brain region was confirmed. Therefore, the increase in pERK1/2 IR in the amygdala following systemic alcohol administration may be reflective of the widespread effects of alcohol on the brain (activation/inhibition of brain circuits), whereas the site specific microinjection studies confirmed functional involvement of intra-amygdala ERK1/2. These findings show that activity of the ERK signaling pathway in the amygdala can influence the discriminative stimulus effects of alcohol

Activation of Group II Metabotropic Glutamate Receptors Inhibits the Discriminative Stimulus Effects of Alcohol via Selective Activity Within the Amygdala

Metabotropic glutamate receptor subtypes (mGlu2/3) regulate a variety of alcohol-associated behaviors, including alcohol reinforcement, and relapse-like behavior. To date, the role of mGlu2/3 receptors in modulating the discriminative stimulus effects of alcohol has not been examined. Given that the discriminative stimulus effects of drugs are determinants of abuse liability and can influence drug seeking, we examined the contributions of mGlu2/3 receptors in modulating the discriminative stimulus effects of alcohol. In male Long-Evans rats trained to discriminate between alcohol (1 g/kg, IG) and water, the mGlu2/3 agonist LY379268 (0.3–10 mg/kg) did not produce alcohol-like stimulus effects. However, pretreatment with LY379268 (1 and 3 mg/kg; in combination with alcohol) inhibited the stimulus effects of alcohol (1 g/kg). Systemic LY379268 (3 mg/kg, i.p.) was associated with increases in neuronal activity within the amygdala, but not the nucleus accumbens, as assessed by c-Fos immunoreactivity. Intra-amygdala activation of mGlu2/3 receptors by LY379268 (6 μg) inhibited the discriminative stimulus effects of alcohol, without altering response rate. In contrast, intra-accumbens LY379268 (3 μg) profoundly reduced response rate; however, at lower LY379268 doses (0.3, 1 μg), the discriminative stimulus effects of alcohol and response rate were not altered. These data suggest that amygdala mGlu2/3 receptors have a functional role in modulating the discriminative stimulus properties of alcohol and demonstrate differential motor sensitivity to activation of mGlu2/3 receptors in the amygdala and the accumbens. Understanding the neuronal mechanisms that underlie the discriminative stimulus effects of alcohol may prove to be important for future development of pharmacotherapies for treating alcoholism

The effects of repeated corticosterone exposure on the interoceptive effects of alcohol in rats

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

Metabotropic Glutamate Receptor 5 Activity in the Nucleus Accumbens Is Required for the Maintenance of Ethanol Self-Administration in a Rat Genetic Model of High Alcohol Intake

Systemic modulation of Group I and II metabotropic glutamate receptors (mGluRs) regulate ethanol self-administration in a variety of animal models. Although these receptors are expressed in reward-related brain regions, the anatomical specificity of their functional involvement in ethanol self-administration remains to be characterized. This study sought to evaluate the functional role of Group I (mGluR5) and Group II (mGluR2/3) in mesocorticolimbic brain regions in ethanol self-administration

KCNN Genes that Encode Small-Conductance Ca(2+)-Activated K(+) Channels Influence Alcohol and Drug Addiction.

Small-conductance Ca(2+)-activated K(+) (KCa2) channels control neuronal excitability and synaptic plasticity, and have been implicated in substance abuse. However, it is unknown if genes that encode KCa2 channels (KCNN1-3) influence alcohol and drug addiction. In the present study, an integrative functional genomics approach shows that genetic datasets for alcohol, nicotine, and illicit drugs contain the family of KCNN genes. Alcohol preference and dependence QTLs contain KCNN2 and KCNN3, and Kcnn3 transcript levels in the nucleus accumbens (NAc) of genetically diverse BXD strains of mice predicted voluntary alcohol consumption. Transcript levels of Kcnn3 in the NAc negatively correlated with alcohol intake levels in BXD strains, and alcohol dependence enhanced the strength of this association. Microinjections of the KCa2 channel inhibitor apamin into the NAc increased alcohol intake in control C57BL/6J mice, while spontaneous seizures developed in alcohol-dependent mice following apamin injection. Consistent with this finding, alcohol dependence enhanced the intrinsic excitability of medium spiny neurons in the NAc core and reduced the function and protein expression of KCa2 channels in the NAc. Altogether, these data implicate the family of KCNN genes in alcohol, nicotine, and drug addiction, and identify KCNN3 as a mediator of voluntary and excessive alcohol consumption. KCa2.3 channels represent a promising novel target in the pharmacogenetic treatment of alcohol and drug addiction. Neuropsychopharmacology 2015; 40:1928-39