Kristen R. Fisher

Enhanced AMPA receptor activity increases operant alcohol self‐administration and cue‐induced reinstatement

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 α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate receptors (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 pre‐treated 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 pre‐treatment significantly increased alcohol‐reinforced responses relative to vehicle treatment. This increase was not attributed to aniracetam‐induced hyperactivity as aniracetam pre‐treatment did not alter locomotor activity. AMPA receptor involvement was confirmed because 6,7‐dinitroquinoxaline‐2,3‐dione (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 pre‐treatment 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.

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.

Overexpression of the Steroidogenic Enzyme Cytochrome P450 Side Chain Cleavage in the Ventral Tegmental Area Increases 3α,5α-THP and Reduces Long-Term Operant Ethanol Self-Administration

Neuroactive steroids are endogenous neuromodulators capable of altering neuronal activity and behavior. In rodents, systemic administration of endogenous or synthetic neuroactive steroids reduces ethanol self-administration. We hypothesized this effect arises from actions within mesolimbic brain regions that we targeted by viral gene delivery. Cytochrome P450 side chain cleavage (P450scc) converts cholesterol to pregnenolone, the rate-limiting enzymatic reaction in neurosteroidogenesis. Therefore, we constructed a recombinant adeno-associated serotype 2 viral vector (rAAV2), which drives P450scc expression and neuroactive steroid synthesis. The P450scc-expressing vector (rAAV2-P450scc) or control GFP-expressing vector (rAAV2-GFP) were injected bilaterally into the ventral tegmental area (VTA) or nucleus accumbens (NAc) of alcohol preferring (P) rats trained to self-administer ethanol. P450scc overexpression in the VTA significantly reduced ethanol self-administration by 20% over the 3 week test period. P450scc overexpression in the NAc, however, did not alter ethanol self-administration. Locomotor activity was unaltered by vector administration to either region. P450scc overexpression produced a 36% increase in (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP, allopregnanolone)-positive cells in the VTA, but did not increase 3α,5α-THP immunoreactivity in NAc. These results suggest that P450scc overexpression and the resultant increase of 3α,5α-THP-positive cells in the VTA reduces ethanol reinforcement. 3α,5α-THP is localized to neurons in the VTA, including tyrosine hydroxylase neurons, but not astrocytes. Overall, the results demonstrate that using gene delivery to modulate neuroactive steroids shows promise for examining the neuronal mechanisms of moderate ethanol drinking, which could be extended to other behavioral paradigms and neuropsychiatric pathology.

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.

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.

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

Extracellular signal-regulated kinase (ERK(1/2)) 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 ERK(1/2) in modulating the discriminative stimulus effects of alcohol. Using drug discrimination procedures, rats were trained to discriminate a moderate intragastric (IG) alcohol dose (1g/kg) versus water (IG). Following an alcohol (1g/kg) discrimination session phosphorylated ERK(1/2) (pERK(1/2)) immunoreactivity (IR) was significantly elevated in the amygdala, but not the nucleus accumbens. Therefore, we hypothesized that intra-amygdala inhibition of ERK(1/2) would disrupt expression of the discriminative stimulus effects of alcohol. However, intra-amygdala or accumbens administration of the MEK/ERK(1/2) inhibitor U0126 (1 and 3μg) had no effect on the discriminative stimulus effects of the training dose of alcohol (1g/kg). Contrary to our hypothesis, intra-amygdala infusion of U0126 (3μg) potentiated the discriminative stimulus effects of a low alcohol dose (0.5g/kg) and had no effect following nucleus accumbens infusion. Importantly, site-specific inhibition of pERK(1/2) in each brain region was confirmed. Therefore, the increase in pERK(1/2) 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 ERK(1/2). These findings show that activity of the ERK signaling pathway in the amygdala can influence the discriminative stimulus effects of alcohol.

Potentiation of amygdala AMPA receptor activity selectively promotes escalated alcohol self-administration in a CaMKII-dependent manner

Growing evidence indicates that drugs of abuse gain control over the individual by usurping glutamate‐linked mechanisms of neuroplasticity in reward‐related brain regions. Accordingly, we have shown that glutamate α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPAR) activity in the amygdala is required for the positive reinforcing effects of alcohol, which underlie the initial stages of addiction. It is unknown, however, if enhanced AMPAR activity in the amygdala facilitates alcohol self‐administration, which is a kernel premise of glutamate hypotheses of addiction. Here, we show that low‐dose alcohol (0.6 g/kg/30 minutes) self‐administration increases phosphorylation (activation) of AMPAR subtype GluA1 S831 (pGluA1 S831) in the central amygdala (CeA), basolateral amygdala and nucleus accumbens core (AcbC) of selectively bred alcohol‐preferring P‐rats as compared with behavior‐matched (non‐drug) sucrose controls. The functional role of enhanced AMPAR activity was assessed via site‐specific infusion of the AMPAR positive modulator, aniracetam, in the CeA and AcbC prior to alcohol self‐administration. Intra‐CeA aniracetam increased alcohol‐reinforced but not sucrose‐reinforced responding and was ineffective following intra‐AcbC infusion. Because GluA1 S831 is a Ca2+/calmodulin‐dependent protein kinase II (CaMKII) substrate, we sought to determine if AMPAR regulation of enhanced alcohol self‐administration is dependent on CaMKII activity. Intra‐CeA infusion of the cell‐permeable CaMKII peptide inhibitor myristolated autocamtide‐2‐related inhibitory peptide (m‐AIP) dose‐dependently reduced alcohol self‐administration. A subthreshold dose of m‐AIP also blocked the aniracetam‐induced escalation of alcohol self‐administration, demonstrating that AMPAR‐mediated potentiation of alcohol reinforcement requires CaMKII activity in the amygdala. Enhanced activity of plasticity‐linked AMPAR‐CaMKII signaling in the amygdala may promote escalated alcohol use via increased positive reinforcement during the initial stages of addiction.