Increased reactivity of the paraventricular nucleus of the hypothalamus and decreased threat responding in male rats following psilocin administration.

Psychedelics have experienced renewed interest following positive clinical effects, however the neurobiological mechanisms underlying effects remain unclear. The paraventricular nucleus of the hypothalamus (PVN) plays an integral role in stress response, autonomic function, social behavior, and other affective processes. We investigated the effect of psilocin, the psychoactive metabolite of psilocybin, on PVN reactivity in Sprague Dawley rats. Psilocin increased stimulus-independent PVN activity as measured by c-Fos expression in male and female rats. Psilocin increased PVN reactivity to an aversive air-puff stimulus in males but not females. Reactivity was restored at 2- and 7-days post-injection with no group differences. Additionally, prior psilocin injection did not affect PVN reactivity following acute restraint stress. Experimental groups sub-classified by baseline threat responding indicate that increased male PVN reactivity is driven by active threat responders. These findings identify the PVN as a significant site of psychedelic drug action with implications for threat responding behavior.

Negative modulation of AMPA receptors bound to transmembrane AMPA receptor regulatory protein γ-8 blunts the positive reinforcing properties of alcohol and sucrose in a brain region-dependent manner in male mice.

RATIONALE: 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. OBJECTIVE: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Sex-specific plasticity in CRF regulation of inhibitory control in central amygdala CRF1 neurons after chronic voluntary alcohol drinking.

Despite strong preclinical evidence for the ability of corticotropin releasing factor 1 (CRF1) antagonists to regulate alcohol consumption, clinical trials have not yet demonstrated therapeutic effects of these compounds in alcohol use disorder (AUD) patients. Several confounding factors may limit the translation of preclinical CRF1 research to patients, including reliance on experimenter-administered alcohol instead of voluntary consumption, a preponderance of evidence collected in male subjects only and an inability to assess the effects of alcohol on specific brain circuits. A population of particular interest is the CRF1-containing neurons of the central amygdala (CeA). CRF1 CeA neurons are sensitive to ethanol, but the effects of alcohol drinking on CRF signalling within this population are unknown. In the present study, we assessed the effects of voluntary alcohol drinking on inhibitory control of CRF1+ CeA neurons from male and female CRF1:GFP mice using ex vivo electrophysiology and determined the contributions of CRF1 signalling to inhibitory control and voluntary alcohol drinking. Chronic alcohol drinking produced neuroadaptations in CRF1+ neurons that increased the sensitivity of GABAA receptor-mediated sIPSCs to the acute effects of alcohol, CRF and the CRF1 antagonist R121919, but these adaptations were more pronounced in male versus female mice. The CRF1 antagonist CP-154,526 reduced voluntary alcohol drinking in both sexes and abolished sex differences in alcohol drinking. The lack of alcohol-induced adaptation in the female CRF1 system may be related to the elevated alcohol intake exhibited by female mice and could contribute to the ineffectiveness of CRF1 antagonists in female AUD patients.

Inhibition of AMPA receptors (AMPARs) containing transmembrane AMPAR regulatory protein γ-8 with JNJ-55511118 shows preclinical efficacy in reducing chronic repetitive alcohol self-administration.

BACKGROUND: A prominent therapeutic indication for alcohol use disorder (AUD) is reduction in chronic repetitive alcohol use. Glutamate α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (AMPARs) regulate chronic alcohol self-administration in preclinical models. Recent evidence indicates that the expression and function of AMPARs require the transmembrane AMPAR regulatory protein γ-8 (TARP γ-8). This study evaluated the preclinical efficacy of JNJ-55511118, a novel, selective, high-affinity inhibitor of TARP γ-8-bound AMPARs, in reducing chronic operant alcohol self-administration. METHODS: Separate groups of male and female C57BL/6J mice (n = 8/sex/group) were trained to lever press for sweetened alcohol (9% v/v + sucrose 2% w/v) or sucrose only (2% w/v) in operant conditioning chambers using an FR-4 schedule of reinforcement. After a 40-day baseline, JNJ-55511118 (0, 1, and 10 mg/kg, p.o.) was administered in randomized order 1 h before self-administration sessions. Parameters of operant behavior including response rate, total reinforcers, and head entries in the drinking troughs were computer recorded. RESULTS: During baseline, responding to alcohol, but not sucrose, was greater in female than male mice. In male mice, both doses of JNJ-55511118 decreased multiple parameters of alcohol self-administration but did not reduce behavior-matched sucrose-only self-administration. JNJ-55511118 had no effect on sweetened alcohol or sucrose self-administration in female mice. Subsequent tests of motor function showed that the lowest effective dose of JNJ-55511118 (1 mg/kg) had no effect on open-field activity in male mice. CONCLUSIONS: This study shows for the first time that TARP γ-8-bound AMPARs regulate a behavioral pathology associated with addiction. The preclinical efficacy of JNJ-55511118 in reducing alcohol self-administration in male mice suggests that inhibition of TARP γ-8-bound AMPARs is a novel and highly significant neural target for developing medications to treat AUD and other forms of addiction.

Calcium-permeable AMPA receptor activity and GluA1 trafficking in the basolateral amygdala regulate operant alcohol self-administration.

Addiction is viewed as maladaptive glutamate-mediated neuroplasticity that is regulated, in part, by calcium-permeable AMPA receptor (CP-AMPAR) activity. However, the contribution of CP-AMPARs to alcohol-seeking behavior remains to be elucidated. We evaluated CP-AMPAR activity in the basolateral amygdala (BLA) as a potential target of alcohol that also regulates alcohol self-administration in C57BL/6J mice. Operant self-administration of sweetened alcohol increased spontaneous EPSC frequency in BLA neurons that project to the nucleus accumbens as compared with behavior-matched sucrose controls indicating an alcohol-specific upregulation of synaptic activity. Bath application of the CP-AMPAR antagonist NASPM decreased evoked EPSC amplitude only in alcohol self-administering mice indicating alcohol-induced synaptic insertion of CP-AMPARs in BLA projection neurons. Moreover, NASPM infusion in the BLA dose-dependently decreased the rate of operant alcohol self-administration providing direct evidence for CP-AMPAR regulation of alcohol reinforcement. As most CP-AMPARs are GluA1-containing, we asked if alcohol alters the activation state of GluA1-containing AMPARs. Immunocytochemistry results showed elevated GluA1-S831 phosphorylation in the BLA of alcohol as compared with sucrose mice. To investigate mechanistic regulation of alcohol self-administration by GluA1-containing AMPARs, we evaluated the necessity of GluA1 trafficking using a TET-ON AAV encoding a dominant-negative GluA1 c-terminus (GluA1ct) that blocks activity-dependent synaptic delivery of native GluA1-containing AMPARs. GluA1ct expression in the BLA reduced alcohol self-administration with no effect on sucrose controls. These results show that CP-AMPAR activity and GluA1 trafficking in the BLA mechanistically regulate the reinforcing effects of sweetened alcohol. Pharmacotherapeutic targeting these mechanisms of maladaptive neuroplasticity may aid medical management of alcohol use disorder.

Pharmacological inhibition of glycogen synthase kinase 3 increases operant alcohol self-administration in a manner associated with altered pGSK-3ß, protein interacting with C kinase and GluA2 protein expression in the reward pathway of male C57BL/6J mice.

Glycogen synthase kinase 3 (GSK-3) is a constitutively active serine-threonine kinase that regulates numerous signaling pathways and has been implicated in neurodegenerative and neuropsychiatric diseases. Alcohol exposure increases GSK-3ß (ser9) phosphorylation (pGSK-3ß); however, few studies have investigated whether GSK-3 regulates the positive reinforcing effects of alcohol, which drive repetitive drug use. To address this goal, male C57BL/6J mice were trained to lever press on a fixed-ratio 4 schedule of sweetened alcohol or sucrose-only reinforcement in operant conditioning chambers. The GSK-3 inhibitor CHIR 99021 (0-10 mg/kg, i.p.) was injected 45 minutes prior to self-administration sessions. After completion of the self-administration dose-effect curve, potential locomotor effects of the GSK-3 inhibitor were assessed. To determine molecular efficacy, CHIR 99021 (10 mg/kg, i.p.) was evaluated on pGSK-3ß, GSK-3ß, protein interacting with C kinase (PICK1), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluA2 subunit protein expression in amygdala, nucleus accumbens (NAcb), and frontal cortex. Results showed that CHIR 99021 (10 mg/kg) dose-dependently increased alcohol reinforced responding with no effect on sucrose self-administration or locomotor activity. CHIR 99021 (10 mg/kg) significantly decreased pGSK-3ß expression in all brain regions tested, reduced PICK1 and increased GluA2 total expression only in the NAcb. We conclude that GSK-3 inhibition increased the reinforcing effects of alcohol in mice. This was associated with reduced pGSK-3ß and PICK1, and increased GluA2 expression. Given prior results showing that AMPA receptor activity regulates alcohol self-administration, we propose that signaling through the GSK-3/PICK1/GluA2 molecular pathway drives the positive reinforcing effects of the drug, which are required for abuse liability.

Manipulations of Central Amygdala Neurotensin Neurons Alter the Consumption of Ethanol and Sweet Fluids in Mice.

The central nucleus of the amygdala plays a significant role in alcohol use and other affective disorders; however, the genetically-defined neuronal subtypes and projections that govern these behaviors are not well known. Here we show that neurotensin neurons in the central nucleus of the amygdala of male mice are activated by in vivo ethanol consumption and that genetic ablation of these neurons decreases ethanol consumption and preference in non-ethanol-dependent animals. This ablation did not impact preference for sucrose, saccharin, or quinine. We found that the most robust projection of the central amygdala neurotensin neurons was to the parabrachial nucleus, a brain region known to be important in feeding behaviors, conditioned taste aversion, and alarm. Optogenetic stimulation of projections from these neurons to the parabrachial nucleus is reinforcing, and increases ethanol drinking as well as consumption of sucrose and saccharin solutions. These data suggest that this central amygdala to parabrachial nucleus projection influences the expression of reward-related phenotypes and is a novel circuit promoting consumption of ethanol and palatable fluids.SIGNIFICANCE STATEMENT Alcohol use disorder (AUD) is a major health burden worldwide. Although ethanol consumption is required for the development of AUD, much remains unknown regarding the underlying neural circuits that govern initial ethanol intake. Here we show that ablation of a population of neurotensin-expressing neurons in the central amygdala decreases intake of and preference for ethanol in non-dependent animals, whereas the projection of these neurons to the parabrachial nucleus promotes consumption of ethanol as well as other palatable fluids.

Alcohol drinking exacerbates neural and behavioral pathology in the 3xTg-AD mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that represents the most common cause of dementia in the United States. Although the link between alcohol use and AD has been studied, preclinical research has potential to elucidate neurobiological mechanisms that underlie this interaction. This study was designed to test the hypothesis that nondependent alcohol drinking exacerbates the onset and magnitude of AD-like neural and behavioral pathology. We first evaluated the impact of voluntary 24-h, two-bottle choice home-cage alcohol drinking on the prefrontal cortex and amygdala neuroproteome in C57BL/6J mice and found a striking association between alcohol drinking and AD-like pathology. Bioinformatics identified the AD-associated proteins MAPT (Tau), amyloid beta precursor protein (APP), and presenilin-1 (PSEN-1) as the main modulators of alcohol-sensitive protein networks that included AD-related proteins that regulate energy metabolism (ATP5D, HK1, AK1, PGAM1, CKB), cytoskeletal development (BASP1, CAP1, DPYSL2 [CRMP2], ALDOA, TUBA1A, CFL2, ACTG1), cellular/oxidative stress (HSPA5, HSPA8, ENO1, ENO2), and DNA regulation (PURA, YWHAZ). To address the impact of alcohol drinking on AD, studies were conducted using 3xTg-AD mice that express human MAPT, APP, and PSEN-1 transgenes and develop AD-like brain and behavioral pathology. 3xTg-AD and wild-type mice consumed alcohol or saccharin for 4 months. Behavioral tests were administered during a 1-month alcohol-free period. Alcohol intake induced AD-like behavioral pathologies in 3xTg-AD mice including impaired spatial memory in the Morris Water Maze, diminished sensorimotor gating as measured by prepulse inhibition, and exacerbated conditioned fear. Multiplex immunoassay conducted on brain lysates showed that alcohol drinking upregulated primary markers of AD pathology in 3xTg-AD mice: Aß 42/40 ratio in the lateral entorhinal and prefrontal cortex and total Tau expression in the lateral entorhinal cortex, medial prefrontal cortex, and amygdala at 1-month post alcohol exposure. Immunocytochemistry showed that alcohol use upregulated expression of pTau (Ser199/Ser202) in the hippocampus, which is consistent with late-stage AD. According to the NIA-AA Research Framework, these results suggest that alcohol use is associated with Alzheimer's pathology. Results also showed that alcohol use was associated with a general reduction in Akt/mTOR signaling via several phosphoproteins (IR, IRS1, IGF1R, PTEN, ERK, mTOR, p70S6K, RPS6) in multiple brain regions including hippocampus and entorhinal cortex. Dysregulation of Akt/mTOR phosphoproteins suggests alcohol may target this pathway in AD progression. These results suggest that nondependent alcohol drinking increases the onset and magnitude of AD-like neural and behavioral pathology in 3xTg-AD mice.

MGluR5 activity is required for the induction of ethanol behavioral sensitization and associated changes in ERK MAP kinase phosphorylation in the nucleus accumbens shell and lateral habenula.

Metabotropic glutamate receptor subtype-5 (mGluR5) activity regulates a variety of behavioral pathologies associated with alcohol addiction. The main goal of this study was to determine if mGluR5 regulates the induction of ethanol-induced locomotor sensitization, which is a model of experience-dependent plasticity following initial exposure to drugs of abuse. The extracellular signal-regulated kinase (ERK1/2) pathway is downstream of mGluR5 and implicated in alcohol addiction; however, its role in sensitization remains unexplored. We sought to determine if mGluR5-mediated changes in ethanol-induced sensitization are associated with changes in ERK1/2 phosphorylation (pERK1/2) in specific brain regions. Adult male DBA/2 J mice were tested for acute locomotor response to ethanol (0 or 2 g/kg, IP) followed by a 9-day induction period in which the mGluR5 antagonist MPEP (0 or 30 mg/kg, IP) was administered prior to ethanol (0 or 2.5 g/kg, IP). One day later, ethanol (2 g/kg) produced a robust within- and between-group increase in locomotor activity, indicating sensitization in mice that received MPEP (0 mg/kg) during induction. MPEP (30 mg/kg) treatment during induction resulted in locomotor response to ethanol (2 g/kg) challenge that was equivalent to an acute response, indicating full blockade of sensitization. Sensitization was associated with increased pERK1/2 immunoreactivity (IR) in nucleus accumbens shell (AcbSh) and a reduction in lateral habenula (LHb), both of which were blocked by MPEP treatment during induction. Sensitization was also associated with mGluR5-independent increases in pERK1/2 IR in the nucleus accumbens core and decreases in the dentate gyrus and lateral septum. These data indicate that mGluR5 activity is required for the induction of ethanol locomotor sensitization and associated changes in ERK1/2 phosphorylation in the AcbSh and LHb, which raises the hypothesis that mGluR5-mediated cell signaling in these brain regions may mediate the induction of sensitization. Elucidating mechanisms of sensitization may increase understanding of how ethanol hijacks behavioral functions during the development of addiction.

Mining the nucleus accumbens proteome for novel targets of alcohol self-administration in male C57BL/6J mice.

RATIONALE: There is a clear need for discovery of effective medications to treat behavioral pathologies associated with alcohol addiction, such as chronic drinking. OBJECTIVE: The goal of this preclinical study was to assess effects of chronic alcohol drinking on the nucleus accumbens (NAcb) proteome to identify and validate novel targets for medications development. MATERIALS AND METHODS: Two-dimensional difference in-gel electrophoresis (2D-DIGE) with matrix-assisted laser desorption ionization tandem time-of-flight (MALDI-TOF/TOF) was used to assess effects of chronic voluntary home-cage (24-h access) alcohol drinking on the NAcb proteome of C57BL/6J mice. To extend these findings to a model of alcohol self-administration and reinforcement, we investigated potential regulation of the positive reinforcing effects of alcohol by the target protein glutathione S-transferase Pi 1 (GSTP1) using a pharmacological inhibition strategy in mice trained to self-administer alcohol or sucrose. RESULTS: Expression of 52 unique proteins in the NAcb was changed by chronic alcohol drinking relative to water control (23 upregulated, 29 downregulated). Ingenuity Pathway Analysis showed that alcohol drinking altered an array of protein networks associated with neurological and psychological disorders, molecular and cellular functions, and physiological systems and development. DAVID functional annotation analysis identified 9 proteins (SNCA, GSTP1, PRDX3, PPP3R1, EIF5A, PHB, PEBP1/RKIP, GAPDH, AND SOD1) that were significantly overrepresented in a functional cluster that included the Gene Ontology categories "response to alcohol" and "aging." Immunoblots confirmed changes in Pebp1 (RKIP) and GSTP1 in NAcb with no change in amygdala or frontal cortex, suggesting anatomical specificity. Systemic inhibition of GSTP1 with Ezatiostat (0-30 mg/kg, i.p.) dose-dependently reduced the reinforcing effects of alcohol as measured by operant self-administration, in the absence of motor effects. Sucrose self-administration was also reduced but in a manner associated with nonspecific motor inhibition. CONCLUSIONS: Protein expression profiling identified an array of proteins and networks in the NAcb, including GSTP1, that are novel molecular targets of chronic alcohol drinking. Pharmacological inhibition of GSTP1 significantly reduced the positive reinforcing effects of alcohol, which regulate repetitive use and abuse liability. The observation that this protein was both upregulated after chronic drinking and that its inhibition could modulate the reinforcing properties of alcohol suggests that it is a key target for alcohol-related pathologies. Proteomic strategies combined with specific preclinical models has potential to identify and validate novel targets of alcohol that may be useful in the medical management of alcohol addiction.

Cue-induced reinstatement of alcohol-seeking behavior is associated with increased CaMKII T286 phosphorylation in the reward pathway of mice.

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±0.03g/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.

Comparison of the adolescent and adult mouse prefrontal cortex proteome.

Adolescence is a developmental period characterized by unique behavioral phenotypes (increased novelty seeking, risk taking, sociability and impulsivity) and increased risk for destructive behaviors, impaired decision making and psychiatric illness. Adaptive and maladaptive adolescent traits have been associated with development of the medial prefrontal cortex (mPFC), a brain region that mediates regulatory control of behavior. However, the molecular changes that underlie brain development and behavioral vulnerability have not been fully characterized. Using high-throughput 2D DIGE spot profiling with identification by MALDI-TOF mass spectrometry, we identified 62 spots in the PFC that exhibited age-dependent differences in expression. Identified proteins were associated with diverse cellular functions, including intracellular signaling, synaptic plasticity, cellular organization and metabolism. Separate Western blot analyses confirmed age-related changes in DPYSL2, DNM1, STXBP1 and CFL1 in the mPFC and expanded these findings to the dorsal striatum, nucleus accumbens, motor cortex, amygdala and ventral tegmental area. Ingenuity Pathway Analysis (IPA) identified functional interaction networks enriched with proteins identified in the proteomics screen, linking age-related alterations in protein expression to cellular assembly and development, cell signaling and behavior, and psychiatric illness. These results provide insight into potential molecular components of adolescent cortical development, implicating structural processes that begin during embryonic development as well as plastic adaptations in signaling that may work in concert to bring the cortex, and other brain regions, into maturity.

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.

Cannabinoid CB1 receptor inhibition blunts adolescent-typical increased binge alcohol and sucrose consumption in male C57BL/6J mice.

Increased binge alcohol consumption has been reported among adolescents as compared to adults in both humans and rodent models, and has been associated with serious long-term health consequences. However, the neurochemical mechanism for age differences in binge drinking between adolescents and adults has not been established. The present study was designed to evaluate the mechanistic role of the cannabinoid CB1 receptor in adolescent and adult binge drinking. Binge consumption was established in adolescent and adult male C57BL/6J mice by providing access to 20% alcohol or 1% sucrose for 4h every other day. Pretreatment with the CB1 antagonist/inverse agonist AM-251 (0, 1, 3, and 10mg/kg) in a Latin square design dose-dependently reduced adolescent alcohol consumption to adult levels without altering adult intake. AM-251 (3mg/kg) also reduced adolescent but not adult sucrose consumption. Adolescent reductions in alcohol and sucrose were not associated with alterations in open-field locomotor activity or thigmotaxis. These findings point to age differences in CB1 receptor activity as a functional mediator of adolescent-typical increased binge drinking as compared to adults. Developmental alterations in endocannabinoid signaling in the adolescent brain may therefore be responsible for the drinking phenotype seen in this age group.

Moderate Alcohol Drinking and the Amygdala Proteome: Identification and Validation of Calcium/Calmodulin Dependent Kinase II and AMPA Receptor Activity as Novel Molecular Mechanisms of the Positive Reinforcing Effects of Alcohol.

BACKGROUND: Despite worldwide consumption of moderate amounts of alcohol, the neural mechanisms that mediate the transition from use to abuse are not fully understood. METHODS: Here, we conducted a high-throughput screen of the amygdala proteome in mice after moderate alcohol drinking (n = 12/group) followed by behavioral studies (n = 6-8/group) to uncover novel molecular mechanisms of the positive reinforcing properties of alcohol that strongly influence the development of addiction. RESULTS: Two-dimensional difference in-gel electrophoresis with matrix assisted laser desorption ionization tandem time-of-flight identified 29 differentially expressed proteins in the amygdala of nondependent C57BL/6J mice following 24 days of alcohol drinking. Alcohol-sensitive proteins included calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) and a network of functionally linked proteins that regulate neural plasticity and glutamate-mediated synaptic activity. Accordingly, alcohol drinking increased α-amino-3-hydroxy-5-methyl-4-isooxazole receptor (AMPAR) in central amygdala (CeA) and phosphorylation of AMPAR GluA1 subunit at a CaMKII locus (GluA1-Ser831) in CeA and lateral amygdala. Further, CaMKIIα-Thr286 and GluA1-Ser831 phosphorylation was increased in CeA and lateral amygdala of mice that lever-pressed for alcohol versus the nondrug reinforcer sucrose. Mechanistic studies showed that targeted pharmacologic inhibition of amygdala CaMKII or AMPAR activity specifically inhibited the positive reinforcing properties of alcohol but not sucrose. CONCLUSIONS: Moderate alcohol drinking increases the activity and function of plasticity-linked protein networks in the amygdala that regulate the positive reinforcing effects of the drug. Given the prominence of positive reinforcement in the etiology of addiction, we propose that alcohol-induced adaptations in CaMKIIα and AMPAR signaling in the amygdala may serve as a molecular gateway from use to abuse.

CaMKII inhibition in the prefrontal cortex specifically increases the positive reinforcing effects of sweetened alcohol in C57BL/6J mice.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional enzyme that is required for synaptic plasticity and has been proposed to be a primary molecular component of the etiology of alcohol addiction. Chronic alcohol intake upregulates CaMKIIα protein expression in reward-related brain regions including the amygdala and nucleus accumbens, and CaMKIIα activity in the amygdala is required for the positive reinforcing effects of alcohol, suggesting this system promotes consumption in the early stages of alcohol addiction. Alternatively, the medial prefrontal cortex (mPFC) is known to inhibit limbic activity via CaMKII-dependent excitatory projections and may, therefore, enable top-down regulation of motivation. Here we sought to remove that regulatory control by site-specifically inhibiting CaMKII activity in the mPFC, and measured effects on the positive reinforcing effects of sweetened alcohol in C57BL/6J mice. Infusion of the CAMKII inhibitor KN-93 (0-10.0 µg) in the mPFC primarily increased alcohol+sucrose reinforced response rate in a dose- and time-dependent manner. KN-93 infusion reduced response rate in behavior-matched sucrose-only controls. Importantly, potentiation of operant responding for sweetened alcohol occurred immediately after infusion, at a time during which effects on sucrose responding were not observed, and persisted through the session. These results suggest that endogenous CaMKII activity in the mPFC exerts inhibitory control over the positive reinforcing effects of alcohol. Downregulation of CaMKII signaling in the mPFC might contribute to escalated alcohol use.

CaMKIIα-GluA1 Activity Underlies Vulnerability to Adolescent Binge Alcohol Drinking.

BACKGROUND: Binge drinking during adolescence is associated with increased risk for developing alcohol use disorders; however, the neural mechanisms underlying this liability are unclear. In this study, we sought to determine whether binge drinking alters expression or phosphorylation of 2 molecular mechanisms of neuroplasticity, calcium/calmodulin-dependent kinase II alpha (CaMKIIα) and the GluA1 subunit of AMPA receptors (AMPARs) in addiction-associated brain regions. We also asked whether activation of CaMKIIα-dependent AMPAR activity escalates binge-like drinking. METHODS: To address these questions, CaMKIIαT286 and GluA1S831 protein phosphorylation and expression were assessed in the amygdala and striatum of adolescent and adult male C57BL/6J mice immediately after voluntary binge-like alcohol drinking (blood alcohol >80 mg/dl). In separate mice, effects of the CaMKIIα-dependent GluA1S831 phosphorylation (pGluA1S831 )-enhancing drug tianeptine were tested on binge-like alcohol consumption in both age groups. RESULTS: Binge-like drinking decreased CaMKIIαT286 phosphorylation (pCaMKIIαT286 ) selectively in adolescent amygdala with no effect in adults. Alcohol also produced a trend for reduced pGluA1S831 expression in adolescent amygdala but differentially increased pGluA1S831 in adult amygdala. No effects were observed in the nucleus accumbens or dorsal striatum. Tianeptine increased binge-like alcohol consumption in adolescents but decreased alcohol consumption in adults. Sucrose consumption was similarly decreased by tianeptine pretreatment in both ages. CONCLUSIONS: These data show that the adolescent and adult amygdalae are differentially sensitive to effects of binge-like alcohol drinking on plasticity-linked glutamate signaling molecules. Tianeptine-induced increases in binge-like drinking only in adolescents suggest that differential CaMKIIα-dependent AMPAR activation may underlie age-related escalation of binge drinking.

Operant ethanol self-administration increases extracellular-signal regulated protein kinase (ERK) phosphorylation in reward-related brain regions: selective regulation of positive reinforcement in the prefrontal cortex of C57BL/6J mice.

RATIONALE: Extracellular-signal regulated protein kinase (ERK1/2) is activated by ethanol in reward-related brain regions. Accordingly, systemic inhibition of ERK1/2 potentiates ethanol reinforcement. However, the brain region(s) that mediate this effect are unknown. OBJECTIVE: This study aims to pharmacologically inhibit ERK1/2 in the medial prefrontal cortex (PFC), nucleus accumbens (NAC), and amygdala (AMY) prior to ethanol or sucrose self-administration, and evaluate effects of operant ethanol self-administration on ERK1/2 phosphorylation (pERK1/2). METHODS: Male C57BL/6J mice were trained to lever press on a fixed-ratio-4 schedule of 9% ethanol + 2% sucrose (ethanol) or 2% sucrose (sucrose) reinforcement. Mice were sacrificed immediately after the 30th self-administration session and pERK1/2 immunoreactivity was quantified in targeted brain regions. Additional groups of mice were injected with SL 327 (0-1.7 µg/side) in PFC, NAC, or AMY prior to self-administration. RESULTS: pERK1/2 immunoreactivity was significantly increased by operant ethanol (g/kg = 1.21 g/kg; BAC = 54.9 mg/dl) in the PFC, NAC (core and shell), and AMY (central nucleus) as compared to sucrose. Microinjection of SL 327 (1.7 µg) into the PFC selectively increased ethanol self-administration. Intra-NAC injection of SL 327 had no effect on ethanol- but suppressed sucrose-reinforced responding. Intra-AMY microinjection of SL 327 had no effect on either ethanol- or sucrose-reinforced responding. Locomotor activity was unaffected under all conditions. CONCLUSIONS: Operant ethanol self-administration increases pERK1/2 activation in the PFC, NAC, and AMY. However, ERK1/2 activity only in the PFC mechanistically regulates ethanol self-administration. These data suggest that ethanol-induced activation of ERK1/2 in the PFC is a critical pharmacological effect that mediates the reinforcing properties of the drug.

Alcohol alters the activation of ERK1/2, a functional regulator of binge alcohol drinking in adult C57BL/6J mice.

BACKGROUND: Binge alcohol drinking is a particularly risky pattern of alcohol consumption that often precedes alcohol dependence and addiction. The transition from binge alcohol drinking to alcohol addiction likely involves mechanisms of synaptic plasticity and learning in the brain. The mitogen-activated protein kinase (MAPK) signaling cascades have been shown to be involved in learning and memory, as well as the response to drugs of abuse, but their role in binge alcohol drinking remains unclear. The present experiments were designed to determine the effects of acute alcohol on extracellular signaling-related kinases (ERK1/2) expression and activity and to determine whether ERK1/2 activity functionally regulates binge-like alcohol drinking. METHODS: Adult male C57BL/6J mice were injected with ethanol (EtOH) (3.0 mg/kg, intraperitoneally) 10, 30, or 90 minutes prior to brain tissue collection. Next, mice that were brought to freely consume unsweetened EtOH in a binge-like access procedure were pretreated with the MEK1/2 inhibitor SL327 or the p38 MAPK inhibitor SB239063. RESULTS: Acute EtOH increased pERK1/2 immunoreactivity relative to vehicle in brain regions known to be involved in drug reward and addiction, including the central amygdala and prefrontal cortex. However, EtOH decreased pERK1/2 immunoreactivity relative to vehicle in the nucleus accumbens core. SB239063 pretreatment significantly decreased EtOH consumption only at doses that also produced nonspecific locomotor effects. SL327 pretreatment significantly increased EtOH, but not sucrose, consumption without inducing generalized locomotor effects. CONCLUSIONS: These findings indicate that ERK1/2 MAPK signaling regulates binge-like alcohol drinking. As alcohol increased pERK1/2 immunoreactivity relative to vehicle in brain regions known to regulate drug self-administration, SL327 may have blocked this direct pharmacological effect of alcohol and thereby inhibited the termination of binge-like drinking.

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.