Chronic ethanol exposure induces impairments in CNS excitatory and inhibitory activity. These impairments are associated with glutamatergic dysfunction, including altered neuroplasticity. This study examined the effects of 6-week ethanol (15% and 30% v/v) consumption, by male alcohol-preferring P rats, on protein expression associated with neuroplasticity and glutamate transporter-1 (GLT-1) function. The latter regulates intra- and extra-synaptic glutamate levels. We focused on the shell and core subregions of the nucleus accumbens (Acb); i.e., shell (AcbSh) and core (AcbCo), for these measures. Chronic ethanol exposure increased the expression of BDNF, Arc and phosphorylated (p)-post-synaptic density protein-95 (p-PSD-95) in the AcbSh of P rats. Moreover, the ratio of phospho-neuronal nitric oxide synthase (p-nNOS) to total nNOS was also increased in the AcbSh. These changes in BDNF, Arc and p-nNOS/nNOS ratio were not observed in the AcbCo. Furthermore, chronic ethanol consumption reduced GLT-1 expression in the AcbSh. Alternatively, treatment with ceftriaxone (CEF), a known GLT-1 upregulator, abolished the effect of chronic ethanol consumption on BDNF expression in the AcbSh. Overall, the present findings confirm that chronic ethanol consumption modulates activity-associated synaptic proteins, including BDNF, Arc and nNOS in a subregion-specific (i.e., in the AcbSh but not AcbCo) manner. Thus, alterations in mesocorticolimbic glutamatergic homeostasis and neuroplasticity are possible functional targets for the treatment of alcohol use disorders.

Keywords: Arc; BDNF; Ethanol dependence; GLT-1; glutamate; nNOS; nucleus accumbens.

 

Abstract

Background

Rodent models of high alcohol drinking offer opportunities to better understand factors for alcohol use disorders (AUD) and test potential treatments. Selective breeding was carried out to create 2 unique High Drinking in the Dark (HDID‐1, HDID‐2) mouse lines that represent models of genetic risk for binge‐like drinking. A number of studies have indicated that neuroimmune genes are important for regulation of alcohol drinking. We tested whether compounds shown to reduce drinking in other models also reduce alcohol intake in these unique genetic lines.

Methods

We report tests of gabapentin, tesaglitazar, fenofibrate, caffeic acid phenethyl ester (CAPE), ibrutinib, and rolipram. Although these compounds have different mechanisms of action, they have all been shown to reduce inflammatory responses. We evaluated effects of these compounds on alcohol intake. In order to facilitate comparison with previously published findings for some compounds, we employed similar schedules that were previously used for that compound.

Results

Gabapentin increased ethanol (EtOH) binge‐like alcohol drinking in female HDID‐1 and HS/NPT mice. Tesaglitazar and fenofibrate did not alter 2‐bottle choice (2BC) drinking in male HDID‐1 or HS/NPT mice. However, tesaglitazar had no effect on DID EtOH intake but reduced blood alcohol levels (BAL), and fenofibrate increased DID intake with no effects on BAL. CAPE had no effect on EtOH intake. Ibrutinib reduced intake in female HDID‐1 in initial testing, but did not reduce intake in a second week of testing. Rolipram reduced DID intake and BALs in male and female HDID‐1, HDID‐2, and HS/NPT mice.

Conclusions

A number of compounds shown to reduce EtOH drinking in other models, and genotypes are not effective in HDID mice or their genetically heterogeneous founders, HS/NPT. The most promising compound was the PDE4 inhibitor, rolipram. These results highlight the importance of assessing generalizability when rigorously testing compounds for therapeutic development.

High Drinking in the Dark (HDID-1) mice represent a unique genetic risk model of binge-like drinking and a novel means of screening potential pharmacotherapies to treat alcohol use disorders (AUDs). We tested the effects of tacrolimus (0, 0.5, 1, and 2 mg/kg), sirolimus (0, 5, 10, and 20 mg/kg), palmitoylethanolamide (PEA; 0, 75, 150, and 225 mg/kg), and secukinumab (0, 5, 20, and 60 mg/kg) on binge-like ethanol intake (2-day, "Drinking in the Dark" [DID]) and blood alcohol levels (BALs) in HDID-1 mice. Tacrolimus reduced ethanol intake and BALs. Tacrolimus had no effect on water intake, but reduced saccharin intake. There was no effect of sirolimus, PEA, or secukinumab on ethanol intake or BALs. These results compare and contrast with previous work addressing these compounds or their targeted mechanisms of action on ethanol drinking, highlighting the importance of screening a wide range of models and genotypes to inform the role of neuroimmune signaling in AUDs.

After decades of uncertainties and drawbacks, the study on the role and significance of acetaldehyde in the effects of ethanol seemed to have found its main paths. Accordingly, the effects of acetaldehyde, after its systemic or central administration and as obtained following ethanol metabolism, looked as they were extensively characterized. However, almost 5 years after this research appeared at its highest momentum, the investigations on this topic have been revitalized on at least three main directions: (1) the role and the behavioral significance of acetaldehyde in different phases of ethanol self-administration and in voluntary ethanol consumption; (2) the distinction, in the central effects of ethanol, between those arising from its non-metabolized fraction and those attributable to ethanol-derived acetaldehyde; and (3) the role of the acetaldehyde-dopamine condensation product, salsolinol. The present review article aims at presenting and discussing prospectively the most recent data accumulated following these three research pathways on this never-ending story in order to offer the most up-to-date synoptic critical view on such still unresolved and exciting topic.

Keywords: ethanol, acetaldehyde, salsolinol, ethanol metabolism, epigenetics, neuroinflammation, mesolimbic system, dopamine

Elevations in estrogen (17β-estradiol, E2) are associated with increased alcohol drinking by women and experimentally in rodents. E2 alters the activity of the dopamine system, including the VTA and its projection targets, which plays an important role in binge drinking. A previous study demonstrated that, during high E2 states, VTA neurons in female mice are more sensitive to ethanol excitation. However, the mechanisms responsible for the ability of E2 to enhance ethanol sensitivity of VTA neurons have not been investigated. In this study, we used selective agonists and antagonists to examine the role of ER subtypes (ERα and ERβ) in regulating the ethanol sensitivity of VTA neurons in female mice and found that ERα promotes the enhanced ethanol response of VTA neurons. We also demonstrated that enhancement of ethanol excitation requires the activity of the metabotropic glutamate receptor, mGluR1, which is known to couple with ERα at the plasma membrane. To investigate the behavioral relevance of these findings, we administered lentivirus-expressing short hairpin RNAs targeting either ERα or ERβ into the VTA and found that knockdown of each receptor in the VTA reduced binge-like ethanol drinking in female, but not male, mice. Reducing ERα in the VTA had a more dramatic effect on binge-like drinking than reducing ERβ, consistent with the ability of ERα to alter ethanol sensitivity of VTA neurons. These results provide important insight into sex-specific mechanisms that drive excessive alcohol drinking.

The level of response (LR) to alcohol as measured with the Self-Report of the Effects of Alcohol Retrospective Questionnaire (SRE) evaluates the number of standard drinks usually required for up to four effects. The need for a higher number of drinks for effects is genetically influenced and predicts higher risks for heavy drinking and alcohol problems. We conducted genome-wide association study (GWAS) in the African-American (COGA-AA, N = 1527 from 309 families) and European-American (COGA-EA, N = 4723 from 956 families) subsamples of the Collaborative Studies on the Genetics of Alcoholism (COGA) for two SRE scores: SRE-T (average of first five times of drinking, the period of heaviest drinking, and the most recent 3 months of consumption) and SRE-5 (the first five times of drinking). We then meta-analyzed the two COGA subsamples (COGA-AA + EA). Both SRE-T and SRE-5 were modestly heritable (h2 : 21%-31%) and genetically correlated with alcohol dependence (AD) and DSM-IV AD criterion count (rg : 0.35-0.76). Genome-wide significant associations were observed (SRE-T: chromosomes 6, rs140154945, COGA-EA P = 3.30E-08 and 11, rs10647170, COGA-AA+EA P = 3.53E-09; SRE-5: chromosome13, rs4770359, COGA-AA P = 2.92E-08). Chromosome 11 was replicated in an EA dataset from the National Institute on Alcohol Abuse and Alcoholism intramural program. In silico functional analyses and RNA expression analyses suggest that the chromosome 6 locus is an eQTL for KIF25. Polygenic risk scores derived using the COGA SRE-T and SRE-5 GWAS predicted 0.47% to 2.48% of variances in AD and DSM-IV AD criterion count in independent datasets. This study highlights the genetic contribution of alcohol response phenotypes to the etiology of alcohol use disorders.

Keywords: RNA expression; genetic correlation; genome-wide association study (GWAS); heritability; polygenic risk score; self-rating of the effects of ethanol (SRE).

Background

The inbred mouse strain C57BL/6 is widely used in both models of addiction and immunological disease. However, there are pronounced phenotypic differences in ethanol (EtOH) consumption and innate immune response between C57BL/6 substrains. The focus of this study was to examine the effects of substrain on innate immune response and neuroimmune‐induced escalation of voluntary EtOH consumption. The main goal was to identify whether substrain differences in immune response can account for differences in EtOH behavior.

Methods

We compared acute innate immune response with a viral dsRNA mimic, polyinosinic:polycytidylic acid (poly(I:C)), in brain using qRT‐PCR in both C57BL/6N and C57BL/6J mice. Next, we used a neuroimmune model of escalation using poly(I:C) to compare drinking behavior between substrains. Finally, we compared brain neuroimmune response with both EtOH and repeated poly(I:C) in both substrains as a way to account for differences in EtOH behavior.

Results

We found that C57BL/6 substrains have differing immune response and drinking behaviors. C57BL/6N mice have a shorter but more robust inflammatory response to acute poly(I:C). In contrast, C57BL/6J mice have a smaller but longer‐lasting acute immune response to poly(I:C). In our neuroimmune‐induced escalation model, C57BL/6J mice but not C57BL/6N mice escalate EtOH intake after poly(I:C). Finally, only C57BL/6J mice show enhanced proinflammatory transcript abundance after poly(I:C) and EtOH, suggesting that longer‐lasting immune responses are critical to neuroimmune drinking phenotypes.

Conclusions

Altogether, this work has elucidated additional influences that substrain has on both innate immune response and drinking phenotypes. Our observations highlight the importance of considering and reporting the source and background used for production of transgenic and knockout mice. These data provide further evidence that genetic background must be carefully considered when investigating the role of neuroimmune signaling in EtOH abuse.

Post-mortem neuropathological and in vivo neuroimaging methods have demonstrated the vulnerability of the inferior colliculus to the sequelae of thiamine deficiency as occurs in Wernicke-Korsakoff Syndrome (WKS). A rich literature in animal models ranging from mice to monkeys—including our neuroimaging studies in rats—has shown involvement of the inferior colliculi in the neural response to thiamine depletion, frequently accomplished with pyrithiamine, an inhibitor of thiamine metabolism. In uncomplicated alcoholism (i.e., absent diagnosable neurological concomitants), the literature citing involvement of the inferior colliculus is scarce, has nearly all been accomplished in preclinical models, and is predominately discussed in the context of ethanol withdrawal. Our recent work using novel, voxel-based analysis of structural Magnetic Resonance Imaging (MRI) has demonstrated significant, persistent shrinkage of the inferior colliculus using acute and chronic ethanol exposure paradigms in two strains of rats. We speculate that these consistent findings should be considered from the perspective of the inferior colliculi having a relatively high CNS metabolic rate. As such, they are especially vulnerable to hypoxic injury and may be provide a common anatomical link among a variety of disparate insults. An argument will be made that the inferior colliculi have functions, possibly related to auditory gating, necessary for awareness of the external environment. Multimodal imaging including diffusion methods to provide more accurate in vivo visualization and quantification of the inferior colliculi may clarify the roles of brain stem nuclei such as the inferior colliculi in alcoholism and other neuropathologies marked by altered metabolism.

Background

N‐methyl‐D‐aspartate receptors (NMDARs) are glutamate‐activated, heterotetrameric ligand‐gated ion channels critically important in virtually all aspects of glutamatergic signaling. Ethanol (EtOH) inhibition of NMDARs is thought to mediate specific actions of EtOH during acute and chronic exposure. Studies from our laboratory, and others, identified EtOH‐sensitive sites within specific transmembrane (TM) domains involved in channel gating as well as those in subdomains of extracellular and intracellular regions of GluN1 and GluN2 subunits that affect channel function. In this study, we characterize for the first time the physiological and behavioral effects of EtOH on knock‐in mice expressing a GluN2A subunit that shows reduced sensitivity to EtOH.

Methods

A battery of tests evaluating locomotion, anxiety, sedation, motor coordination, and voluntary alcohol intake were performed in wild‐type mice and those expressing the GluN2A A825W knock‐in mutation. Whole‐cell patch‐clamp electrophysiological recordings were used to confirm reduced EtOH sensitivity of NMDAR‐mediated currents in 2 separate brain regions (mPFC and the cerebellum) where the GluN2A subunit is known to contribute to NMDAR‐mediated responses.

Results

Male and female mice homozygous for the GluN2A(A825W) knock‐in mutation showed reduced EtOH inhibition of NMDAR‐mediated synaptic currents in mPFC and cerebellar neurons as compared to their wild‐type counterparts. GluN2A(A825W) male but not female mice were less sensitive to the sedative and motor‐incoordinating effects of EtOH and showed a rightward shift in locomotor‐stimulating effects of EtOH. There was no effect of the mutation on EtOH‐induced anxiolysis or voluntary EtOH consumption in either male or female mice.

Conclusions

These findings show that expression of EtOH‐resistant GluN2A NMDARs results in selective and sex‐specific changes in the behavioral sensitivity to EtOH.

Background

Microglia, the primary immune cells of the brain, are implicated in alcohol use disorder. However, it is not known if microglial activation contributes to the transition from alcohol use to alcohol use disorder or is a consequence of alcohol intake.

Methods

We investigated the role of microglia in a mouse model of alcohol dependence using a colony stimulating factor 1 receptor inhibitor (PLX5622) to deplete microglia and a chronic intermittent ethanol vapor two-bottle choice drinking procedure. Additionally, we examined anxiety-like behavior during withdrawal. We then analyzed synaptic neuroadaptations in the central nucleus of the amygdala (CeA) and gene expression changes in the medial prefrontal cortex and CeA from the same animals used for behavioral studies.

Results

PLX5622 prevented escalations in voluntary alcohol intake and decreased anxiety-like behavior associated with alcohol dependence. PLX5622 also reversed expression changes in inflammatory-related genes and glutamatergic and GABAergic (gamma-aminobutyric acidergic) genes in the medial prefrontal cortex and CeA. At the cellular level in these animals, microglia depletion reduced inhibitory GABA _A and excitatory glutamate receptor-mediated synaptic transmission in the CeA, supporting the hypothesis that microglia regulate dependence-induced changes in neuronal function.

Conclusions

Our multifaceted approach is the first to link microglia to the molecular, cellular, and behavioral changes associated with the development of alcohol dependence, suggesting that microglia may also be critical for the development and progression of alcohol use disorder.

The medial habenula (MHb) is considered a brain center regulating aversive states. The mu opioid receptor (MOR) has been traditionally studied at the level of nociceptive and mesolimbic circuits, for key roles in pain relief and reward processing. MOR is also densely expressed in MHb, however, MOR function at this brain site is virtually unknown. Here we tested the hypothesis that MOR in the MHb (MHb-MOR) also regulates aversion processing. We used chnrb4-Cre driver mice to delete the Oprm1 gene in chnrb4-neurons, predominantly expressed in the MHb. Conditional mutant (B4MOR) mice showed habenula-specific reduction of MOR expression, restricted to chnrb4-neurons (50% MHb-MORs). We tested B4MOR mice in behavioral assays to evaluate effects of MOR activation by morphine, and MOR blockade by naloxone. Locomotor, analgesic, rewarding, and motivational effects of morphine were preserved in conditional mutants. In contrast, conditioned place aversion (CPA) elicited by naloxone was reduced in both naïve (high dose) and morphine-dependent (low dose) B4MOR mice. Further, physical signs of withdrawal precipitated by either MOR (naloxone) or nicotinic receptor (mecamylamine) blockade were attenuated. These data suggest that MORs expressed in MHb B4-neurons contribute to aversive effects of naloxone, including negative effect and aversive effects of opioid withdrawal. MORs are inhibitory receptors, therefore we propose that endogenous MOR signaling normally inhibits chnrb4-neurons of the MHb and moderates their known aversive activity, which is unmasked upon receptor blockade. Thus, in addition to facilitating reward at several brain sites, tonic MOR activity may also limit aversion within the MHb circuitry.

The medial habenula (MHb) is considered a brain center regulating aversive states. The mu opioid receptor (MOR) has been traditionally studied at the level of nociceptive and mesolimbic circuits, for key roles in pain relief and reward processing. MOR is also densely expressed in MHb, however, MOR function at this brain site is virtually unknown. Here we tested the hypothesis that MOR in the MHb (MHb-MOR) also regulates aversion processing. We used chnrb4-Cre driver mice to delete the Oprm1 gene in chnrb4-neurons, predominantly expressed in the MHb. Conditional mutant (B4MOR) mice showed habenula-specific reduction of MOR expression, restricted to chnrb4-neurons (50% MHb-MORs). We tested B4MOR mice in behavioral assays to evaluate effects of MOR activation by morphine, and MOR blockade by naloxone. Locomotor, analgesic, rewarding, and motivational effects of morphine were preserved in conditional mutants. In contrast, conditioned place aversion (CPA) elicited by naloxone was reduced in both naïve (high dose) and morphine-dependent (low dose) B4MOR mice. Further, physical signs of withdrawal precipitated by either MOR (naloxone) or nicotinic receptor (mecamylamine) blockade were attenuated. These data suggest that MORs expressed in MHb B4-neurons contribute to aversive effects of naloxone, including negative effect and aversive effects of opioid withdrawal. MORs are inhibitory receptors, therefore we propose that endogenous MOR signaling normally inhibits chnrb4-neurons of the MHb and moderates their known aversive activity, which is unmasked upon receptor blockade. Thus, in addition to facilitating reward at several brain sites, tonic MOR activity may also limit aversion within the MHb circuitry.

A generally accepted framework derived predominately from animal models asserts that repeated cycles of chronic intermittent ethanol (EtOH; CIE) exposure cause progressive brain adaptations associated with anxiety and stress that promote voluntary drinking, alcohol dependence, and further brain changes that contribute to the pathogenesis of alcoholism. The current study used CIE exposure via vapor chambers to test the hypothesis that repeated episodes of withdrawals from chronic EtOH would be associated with accrual of brain damage as quantified using in vivo magnetic resonance imaging (MRI), diffusion tensor imaging (DTI), and MR spectroscopy (MRS). The initial study group included 16 male (~325g) and 16 female (~215g) wild-type Wistar rats exposed to 3 cycles of 1-month in vapor chambers + 1 week of abstinence. Half of each group (n = 8) was given vaporized EtOH to blood alcohol levels approaching 250 mg/dL. Blood and behavior markers were also quantified. There was no evidence for dependence (i.e., increased voluntary EtOH consumption), increased anxiety, or an accumulation of pathology. Neuroimaging brain responses to exposure included increased cerebrospinal fluid (CSF) and decreased gray matter volumes, increased Choline/Creatine, and reduced fimbria-fornix fractional anisotropy (FA) with recovery seen after one or more cycles and effects in female more prominent than in male rats. These results show transient brain integrity changes in response to CIE sufficient to induce acute withdrawal but without evidence for cumulative or escalating damage. Together, the current study suggests that nutrition, age, and sex should be considered when modeling human alcoholism.

The pituitary adenylate cyclase-activating polypeptide (PACAP) system plays a central role in the brain's emotional response to psychological stress by activating cellular processes and circuits associated with threat exposure. The neuropeptide PACAP and its main receptor PAC1 are expressed in the rodent central amygdala (CeA), a brain region critical in negative emotional processing, and CeA PACAPergic signaling drives anxiogenic and stress coping behaviors. Despite this behavioral evidence, PACAP's effects on neuronal activity within the medial subdivision of the CeA (CeM, the major output nucleus for the entire amygdala complex) during basal conditions and after psychological stress remain unknown. Therefore, in the present study, male Wistar rats were subjected to either restraint stress or control conditions, and PACAPergic regulation of CeM cellular function was assessed using immunohistochemistry and whole-cell patch-clamp electrophysiology. Our results demonstrate that PACAP-38 potentiates GABA release in the CeM of naïve rats, via its actions at presynaptic PAC1. Basal PAC1 activity also enhances GABA release in an action potential-dependent manner. Notably, PACAP-38's facilitation of CeM GABA release was attenuated after a single restraint stress session, but after repeated sessions returned to the level observed in naïve animals. A single restraint session also significantly decreased PAC1 levels in the CeM, with repeated restraint sessions producing a slight recovery. Collectively our data reveal that PACAP/PAC1 signaling enhances inhibitory control of the CeM and that psychological stress can modulate this influence to potentially disinhibit downstream effector regions that mediate anxiety and stress-related behaviors. This article is part of the special issue on 'Neuropeptides'.

Keywords: Anxiety; GABA; PAC1; PACAP-38; Pituitary adenylate cyclase-activating polypeptide; Synaptic transmission; sIPSC.

The pituitary adenylate cyclase-activating polypeptide (PACAP) system plays a central role in the brain's emotional response to psychological stress by activating cellular processes and circuits associated with threat exposure. The neuropeptide PACAP and its main receptor PAC1 are expressed in the rodent central amygdala (CeA), a brain region critical in negative emotional processing, and CeA PACAPergic signaling drives anxiogenic and stress coping behaviors. Despite this behavioral evidence, PACAP's effects on neuronal activity within the medial subdivision of the CeA (CeM, the major output nucleus for the entire amygdala complex) during basal conditions and after psychological stress remain unknown. Therefore, in the present study, male Wistar rats were subjected to either restraint stress or control conditions, and PACAPergic regulation of CeM cellular function was assessed using immunohistochemistry and whole-cell patch-clamp electrophysiology. Our results demonstrate that PACAP-38 potentiates GABA release in the CeM of naïve rats, via its actions at presynaptic PAC1. Basal PAC1 activity also enhances GABA release in an action potential-dependent manner. Notably, PACAP-38's facilitation of CeM GABA release was attenuated after a single restraint stress session, but after repeated sessions returned to the level observed in naïve animals. A single restraint session also significantly decreased PAC1 levels in the CeM, with repeated restraint sessions producing a slight recovery. Collectively our data reveal that PACAP/PAC1 signaling enhances inhibitory control of the CeM and that psychological stress can modulate this influence to potentially disinhibit downstream effector regions that mediate anxiety and stress-related behaviors.

One of the most pernicious characteristics of alcohol use disorder is the compulsion to drink despite negative consequences. The insular cortex controls decision making under conditions of risk or conflict. Cortical activity is tightly controlled by inhibitory interneurons that are often enclosed by specialized extracellular matrix structures known as perineuronal nets (PNNs), which regulate neuronal excitability and plasticity. The density of PNNs in the insula increases after repeated bouts of binge drinking, suggesting that they may play a role in the transition from social to compulsive, or aversion-resistant, drinking. Here, we investigated whether insular PNNs play a role in aversion-resistant alcohol drinking using a mouse model in which ethanol was adulterated with the bitter tastant quinine. Disrupting PNNs in the insula rendered mice more sensitive to quinine-adulterated ethanol but not ethanol alone. Activation of the insula, as measured by c-fos expression, occurred during aversion-resistant drinking and was further enhanced by elimination of PNNs. These results demonstrate that PNNs control the activation of the insula during aversion-resistant drinking and suggest that proper excitatory/inhibitory balance is important for decision making under conditions of conflict. Disrupting PNNs in the insula or optimizing insula activation may be a novel strategy to reduce aversion-resistant drinking.

Of the more than 100 studies that have examined relationships between excessive ethanol consumption and the brain transcriptome, few rodent studies have examined chronic consumption. Heterogeneous stock collaborative cross mice freely consumed ethanol vs. water for 3 months. Transcriptional differences were examined for the central nucleus of the amygdala, a brain region known to impact ethanol preference. Early preference was modestly predictive of final preference and there was significant escalation of preference in females only. Genes significantly correlated with female preference were enriched in annotations for the primary cilium and extracellular matrix. A single module in the gene co-expression network was enriched in genes with an astrocyte annotation. The key hub node was the master regulator, orthodenticle homeobox 2 (Otx2). These data support an important role for the extracellular matrix, primary cilium and astrocytes in ethanol preference and consumption differences among individual female mice of a genetically diverse population.

The receptor tyrosine kinases (RTKs) are a large family of proteins that transduce extracellular signals to the inside of the cell to ultimately affect important cellular functions such as cell proliferation, survival, apoptosis, differentiation, and migration. They are expressed in the nervous system and can regulate behavior through modulation of neuronal and glial function. As a result, RTKs are implicated in neurodegenerative and psychiatric disorders such as depression and addiction. Evidence has emerged that 5 RTKs (tropomyosin-related kinase B (TrkB), RET proto-oncogene (RET), anaplastic lymphoma kinase (ALK), fibroblast growth factor receptor (FGFR), and epidermal growth factor receptor (EGFR)) modulate alcohol drinking and other behaviors related to alcohol addiction. RTKs are considered highly "druggable" targets and small-molecule inhibitors of RTKs have been developed for the treatment of various conditions, particularly cancer. These kinases are therefore attractive targets for the development of new pharmacotherapies to treat alcohol use disorder (AUD). This review will examine the preclinical evidence describing TrkB, RET, ALK, FGFR, and EGFR modulation of alcohol drinking and other behaviors relevant to alcohol abuse.

Rationale and objectives: Binge-like alcohol consumption during adolescence associates with several deleterious consequences during adulthood including an increased risk for developing alcohol use disorder (AUD) and other addictions. Replicated preclinical data has indicated that adolescent exposure to binge-like levels of alcohol results in a reduction of choline acetyltransferase (ChAT) and an upregulation in the α7 nicotinic receptor (α7). From this information, we hypothesized that the α7 plays a critical role in mediating the effects of adolescent alcohol exposure.

Methods: Male and female P rats were injected with the α7 agonist AR-R17779 (AR) once during 6 time points between post-natal days (PND) 29-37. Separate groups were injected with the α7 negative allosteric modulator (NAM) dehydronorketamine (DHNK) 2 h before administration of 4 g/kg EtOH (14 total exposures) during PND 28-48. On PND 75, all rats were given access to water and ethanol (15 and 30%) for 6 consecutive weeks (acquisition). All rats were then deprived of EtOH for 2 weeks and then, alcohol was returned (relapse).

Results: Administration of AR during adolescence significantly increased acquisition of alcohol consumption during adulthood and prolonged relapse drinking in P rats. In contrast, administration of DHNK prior to binge-like EtOH exposure during adolescence prevented the increase in alcohol consumption observed during acquisition of alcohol consumption and the enhancement of relapse drinking observed during adulthood.

Discussion: The data indicate that α7 mediates the effects of alcohol during adolescence. The data also indicate that α7 NAMs are potential prophylactic agents to reduce the deleterious effects of adolescent alcohol abuse.

Keywords: Acquisition; Addiction; Adolescence; Alcohol; Alcohol-preferring (P) rats; Ethanol; Relapse.

In humans, alcohol is consumed for its rewarding and anxiolytic effects. The Central Nucleus of the Amygdala (CeA) is considered a neuronal nexus that regulates fear, anxiety and drug self-administration. Manipulations of the CeA alter ethanol (EtOH) consumption under numerous EtOH self-administration models. The experiments determined if EtOH is reinforcing/anxiolytic within the CeA, if selective breeding for high alcohol consumption alters the rewarding properties of EtOH in the CeA, and if the reinforcing/anxiolytic effects of EtOH in the CeA are mediated by the neuropeptides corticotropin-releasing factor (CRF) and nociceptin. The reinforcing properties of EtOH were determined by having male Wistar and Taconic Alcohol-Preferring (tP) rats self-administer EtOH directly into the CeA. The expression of anxiety-like behaviors was assessed through multiple behavioral models (social interaction, acoustic startle, open field). Co-administration of EtOH and a CRF1 antagonist (NBI 35965) or nociceptin on self-administration into the CeA and anxiety-like behaviors was determined. EtOH was self-administered directly into the lateral CeA and tP rats self-administered a lower concentration of EtOH than Wistar rats. EtOH microinjected into the lateral CeA reduced the expression of anxiety-like behaviors, indicating an anxiolytic effect. The co-administration of NBI 35965 failed to alter the rewarding/anxiolytic properties of EtOH in the CeA. In contrast, co-administration of the nociceptin enhanced both EtOH reward and anxiolysis in the CeA. The data indicate that the lateral CeA is a key anatomical location that mediates the rewarding and anxiolytic effects of EtOH and activation of nociceptin receptors augment the effects of EtOH in this region.

SIGNIFICANCE STATEMENT Alcohol is consumed for the stimulatory, rewarding, and anxiolytic properties of the drug of abuse. The current data are the first to establish that alcohol is reinforcing and anxiolytic within the lateral central nucleus of the amygdala (CeA), and that the Nociceptin system regulates these effects of alcohol within the CeA.