The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) expressed in regions of the brain that control motor function, cognition, and motivation. As a result, D2R is involved in the pathophysiology of disorders such as schizophrenia and drug addiction. Understanding the signaling pathways activated by D2R is crucial to finding new therapeutic targets for these disorders. D2R stimulation by its agonist, dopamine, causes desensitization and internalization of the receptor. A previous study found that inhibitors of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK) blocked D2R desensitization in neurons in the ventral tegmental area of the brain. In the present study, using a cell-based system, we investigated whether ALK regulates D2R internalization. The ALK inhibitor alectinib completely inhibited dopamine-induced D2R internalization. Since GPCRs can transactivate receptor tyrosine kinases, we also examined if D2R stimulation activated ALK signaling. ALK phosphorylation increased by almost 2-fold after dopamine treatment and ALK coimmunoprecipitated with D2R. To identify the signaling pathways downstream of ALK that might regulate D2R internalization, we used pharmacological inhibitors of proteins activated by ALK signaling. Protein kinase Cγ was activated by dopamine in an ALK-dependent manner, and a protein kinase C inhibitor completely blocked dopamine-induced D2R internalization. Taken together, these results identify ALK as a receptor tyrosine kinase transactivated by D2R that promotes its internalization, possibly through activation of protein kinase C. ALK inhibitors could be useful in enhancing D2R signaling.

SIGNIFICANCE STATEMENT Receptor internalization is a mechanism by which receptors are desensitized. In this study we found that agonist-induced internalization of the dopamine D2 receptor is regulated by the receptor tyrosine kinase ALK. ALK was also transactivated by and associated with dopamine D2 receptor. Dopamine activated protein kinase C in an ALK-dependent manner and a PKC inhibitor blocked dopamine D2 receptor internalization. These results indicate that ALK regulates dopamine D2 receptor trafficking, which has implications for psychiatric disorders involving dysregulated dopamine signaling.

Phosphodiesterase type 4 (PDE4) inhibitors prevent hydrolysis of cyclic adenosine monophosphate and increase protein kinase A (PKA)-mediated phosphorylation. PDE4 inhibitors also regulate responses to ethanol and GABAergic drugs. We investigated mechanisms by which the PDE4 inhibitor, apremilast, regulates acute effects of ethanol and GABAergic drugs in male and female mice. Apremilast prolonged the sedative-hypnotic effects of gaboxadol, zolpidem, and propofol but did not alter etomidate effects, and unexpectedly shortened the sedative-hypnotic effects of diazepam. Apremilast prolonged rotarod ataxia induced by zolpidem, propofol, and loreclezole, shortened recovery from diazepam, but had no effect on ataxia induced by gaboxadol or etomidate. The PKA inhibitor H-89 blocked apremilast's ability to prolong the sedative-hypnotic effects of ethanol, gaboxadol, and propofol and to prolong ethanol- and propofol-induced ataxia. H-89 also blocked apremilast's ability to shorten the sedative-hypnotic and ataxic effects of diazepam. The β1-specific antagonist, salicylidene salicylhydrazide (SCS), produced faster recovery from ethanol- and diazepam-induced ataxia, but did not alter propofol- or etomidate-induced ataxia. SCS shortened the sedative-hypnotic effects of ethanol and diazepam but not of propofol. In Xenopus oocytes, a phosphomimetic (aspartate) mutation at the PKA phosphorylation site in β1 subunits decreased the maximal GABA current in receptors containing α1 or α3, but not α2 subunits. In contrast, phosphomimetic mutations at PKA sites in β3 subunits increased the maximal GABA current in receptors containing α1 or α2, but not α3 subunits. The GABA potency and allosteric modulation by ethanol, propofol, etomidate, zolpidem, flunitrazepam, or diazepam were not altered by these mutations. We propose a model whereby apremilast increases PKA-mediated phosphorylation of β1-and β3-containing GABA_A receptors and selectively alters acute tolerance to ethanol and GABAergic drugs.

Atrial Naturietic Peptide (ANP) is a neuropeptide that regulates function of the hypothalamic-pituitary-adrenal (HPA) axis, immune and neuroimmune system, and epigenetic factors. Research has indicated that ANP may mediate alcohol intake, withdrawal, and craving like behaviors. ANP receptors are present in the mesocorticolimbic (MCL) reward pathway of the brain, which includes the nucleus accumbens (Acb) and the ventral tegmental area (VTA). The objectives of the present study were to examine the effects of ANP microinjected into Acb subregions (Shell (Sh), Core (Co), ventral to AcbSh) on operant ethanol (EtOH) self-administration and into posterior VTA (pVTA) on EtOH-seeking behavior of female alcohol-preferring (P) rats. In the first experiment, ANP (0, 10 μg, or 100 μg) was microinjected into subregions of the Acb to determine its effects on EtOH self-administration. In the second experiment, ANP was microinjected into pVTA to determine its effects on Pavlovian Spontaneous Recovery (PSR) of responding, a measure of context-induced EtOH-seeking behavior. Administration of ANP directly into the AcbSh significantly reduced EtOH self-administration compared to vehicle, whereas ANP into the AcbCo or areas directly ventral to the AcbSh did not alter responding for EtOH. Microinjection of ANP into the pVTA significantly reduced responding on the EtOH-associated lever during the PSR test. The data indicate that activation of ANP systems in the (a) AcbSh can inhibit EtOH intake, and (b) in the pVTA can inhibit EtOH-seeking behavior. The results suggest that manipulations of the ANP system could be a potential target for pharmacotherapeutic intervention to treat alcohol use disorder. Supported in part by AA07462, AA07611, AA10717, AA10721, AA013522, AA019366, AA020908, AA022287, and AA024612.

Keywords: Alcohol; Alcohol preferring P rats; Atrial natriuretic peptide; Nucleus accumbens; Ventral tegmental Area.

Administration of selective serotonin reuptake inhibitors (SSRIs), typically used as antidepressants, induces long-lasting behavioral changes associated with alcohol use disorder (AUD). However, the contribution of SSRI (fluoxetine)-induced alterations in neurobiological processes underlying alcohol relapse such as endocannabinoid and glutamate signaling in the central amygdala (CeA) remains largely unknown. We utilized an integrative approach to study the effects of repeated fluoxetine administration during abstinence on ethanol drinking. Gene expression and biochemical and electrophysiological studies explored the hypothesis that dysregulation in glutamatergic and endocannabinoid mechanisms in the CeA underlie the susceptibility to alcohol relapse. Cessation of daily treatment with fluoxetine (10 mg/kg) during abstinence resulted in a marked increase in ethanol seeking during re-exposure periods. The increase in ethanol self-administration was associated with (a) reductions in levels of the endocannabinoids N-arachidonoylethanolomine and 2-arachidonoylglycerol in the CeA, (b) increased amygdalar gene expression of cannabinoid type-1 receptor (CB1), N-acyl phosphatidylethanolamine phospholipase D (Nape-pld), fatty acid amid hydrolase (Faah), (c) decreased amygdalar gene expression of ionotropic AMPA (GluA2 and GluA4) and metabotropic (mGlu3) glutamate receptors, and (d) increased glutamatergic receptor function. Overall, our data suggest that the administration of the antidepressant fluoxetine during abstinence dysregulates endocannabinoid signaling and glutamatergic receptor function in the amygdala, facts that likely facilitate alcohol drinking behavior during relapse.

Binge drinking is a dangerous pattern of behavior. We tested whether chronically manipulating nucleus accumbens (NAc) activity (via clozapine-N-oxide (CNO) and Designer Receptors Exclusively Activated by Designer Drugs (DREADD)) could produce lasting effects on ethanol binge-like drinking in mice selectively bred to drink to intoxication. We found chronically increasing NAc activity (4 weeks, via CNO and the excitatory DREADD, hM3Dq) decreased binge-like drinking, but did not observe CNO-induced changes in drinking with the inhibitory DREADD, hM4Di. The CNO/hM3Dq-induced reduction in ethanol drinking persisted for at least one week, suggesting adaptive neuroplasticity via transcriptional and epigenetic mechanisms. Therefore, we defined this plasticity at the morphological and transcriptomic levels. We found that chronic binge drinking (6 weeks) altered neuronal morphology in the NAc, an effect that was ameliorated with CNO/hM3Dq. Moreover, we detected significant changes in expression of several plasticity-related genes with binge drinking that were ameliorated with CNO treatment (e.g., Hdac4). Lastly, we found that LMK235, an HDAC4/5 inhibitor, reduced binge-like drinking. Thus, we were able to target specific molecular pathways using pharmacology to mimic the behavioral effects of DREADDs.

Evidence suggests the hypothalamic–pituitary–adrenal (HPA) axis is involved in Alcohol Use Disorders (AUDs), which might be mediated by an imbalance of glucocorticoid receptor (GR), GRα and GRβ, activity. GRβ antagonizes the GRα isoform to cause glucocorticoid (GC) resistance. In the present study, we aimed to investigate the effects of chronic continuous free-choice access to ethanol on GR isoform expression in subregions of the mesocorticolimbic reward circuit. Adult male alcohol-preferring (P) rats had concurrent access to 15% and 30% ethanol solutions, with ad lib access to lab chow and water, for six weeks. Quantitative Real-time PCR (RT-PCR) analysis showed that chronic ethanol consumption reduced GRα expression in the nucleus accumbens shell (NAcsh) and hippocampus, whereas ethanol drinking reduced GRβ in the nucleus accumbens core (NAcc), prefrontal cortex (PFC), and hippocampus. An inhibitor of GRα, microRNA-124-3p (miR124-3p) was significantly higher in the NAcsh, and GC-induced gene, GILZ, as a measure of GC-responsiveness, was significantly lower. These were not changed in the NAcc. Likewise, genes associated with HPA axis activity were not significantly changed by ethanol drinking [i.e., corticotrophin-releasing hormone (Crh), adrenocorticotrophic hormone (Acth), and proopiomelanocortin (Pomc)] in these brain regions. Serum corticosterone levels were not changed by ethanol drinking. These data indicate that the expression of GRα and GRβ isoforms are differentially affected by ethanol drinking despite HPA-associated peptides remaining unchanged, at least at the time of tissue harvesting. Moreover, the results suggest that GR changes may stem from ethanol-induced GC-resistance in the NAcsh. These findings confirm a role for stress in high ethanol drinking, with GRα and GRβ implicated as targets for the treatment of AUDs.

Background

Genome‐wide profiling to examine brain transcriptional features associated with excessive ethanol (EtOH) consumption has been applied to a variety of species including rodents, nonhuman primates (NHPs), and humans. However, these data were obtained from cross‐sectional samples which are particularly vulnerable to individual variation when obtained from small outbred populations typical of human and NHP studies. In the current study, a novel within‐subject design was used to examine the effects of voluntary EtOH consumption on prefrontal cortex (PFC) gene expression in a NHP model.

Methods

Two cohorts of cynomolgus macaques (n  = 23) underwent a schedule‐induced polydipsia procedure to establish EtOH self‐administration followed by 6 months of daily open access to EtOH (4% w/v) and water. Individual daily EtOH intakes ranged from an average of 0.7 to 3.7 g/kg/d. Dorsal lateral PFC area 46 (A46) brain biopsies were collected in EtOH‐naïve and control monkeys; contralateral A46 biopsies were collected from the same monkeys following the 6 months of fluid consumption. Gene expression changes were assessed using RNA‐Seq paired analysis, which allowed for correction of individual baseline differences in gene expression.

Results

A total of 675 genes were significantly down‐regulated following EtOH consumption; these were functionally enriched for immune response, cell adhesion, plasma membrane, and extracellular matrix. A total of 567 genes that were up‐regulated following EtOH consumption were enriched in microRNA target sites and included target sites associated with Toll‐like receptor pathways. The differentially expressed genes were also significantly enriched in transcription factor binding sites.

Conclusions

The data presented here are the first to use a longitudinal biopsy strategy to examine how chronic EtOH consumption affects gene expression in the primate PFC. Prominent effects were seen in both cell adhesion and neuroimmune pathways; the latter contained both pro‐ and antiinflammatory genes. The data also indicate that changes in miRNAs and transcription factors may be important epigenetic regulators of EtOH consumption.

Background: Genome-wide profiling to examine brain transcriptional features associated with excessive ethanol (EtOH) consumption has been applied to a variety of species including rodents, nonhuman primates (NHPs), and humans. However, these data were obtained from cross-sectional samples which are particularly vulnerable to individual variation when obtained from small outbred populations typical of human and NHP studies. In the current study, a novel within-subject design was used to examine the effects of voluntary EtOH consumption on prefrontal cortex (PFC) gene expression in a NHP model.

Methods: Two cohorts of cynomolgus macaques (n = 23) underwent a schedule-induced polydipsia procedure to establish EtOH self-administration followed by 6 months of daily open access to EtOH (4% w/v) and water. Individual daily EtOH intakes ranged from an average of 0.7 to 3.7 g/kg/d. Dorsal lateral PFC area 46 (A46) brain biopsies were collected in EtOH-naïve and control monkeys; contralateral A46 biopsies were collected from the same monkeys following the 6 months of fluid consumption. Gene expression changes were assessed using RNA-Seq paired analysis, which allowed for correction of individual baseline differences in gene expression.

Results: A total of 675 genes were significantly down-regulated following EtOH consumption; these were functionally enriched for immune response, cell adhesion, plasma membrane, and extracellular matrix. A total of 567 genes that were up-regulated following EtOH consumption were enriched in microRNA target sites and included target sites associated with Toll-like receptor pathways. The differentially expressed genes were also significantly enriched in transcription factor binding sites.

Conclusions: The data presented here are the first to use a longitudinal biopsy strategy to examine how chronic EtOH consumption affects gene expression in the primate PFC. Prominent effects were seen in both cell adhesion and neuroimmune pathways; the latter contained both pro- and antiinflammatory genes. The data also indicate that changes in miRNAs and transcription factors may be important epigenetic regulators of EtOH consumption.

Keywords: Brain Gene Expression; Cortex; Cynomolgus Macaque; EtOH.

We previously reported that paternal preconception chronic ethanol exposure in mice imparts adult male offspring with reduced ethanol drinking preference and consumption, increased ethanol sensitivity, and attenuated stress responsivity. That same chronic ethanol exposure paradigm was later revealed to affect the sperm epigenome by altering the abundance of several small noncoding RNAs, a mechanism that mediates the intergenerational effects of numerous paternal environmental exposures. Although recent studies have revealed that the unique RNA signature of sperm is shaped during maturation in the epididymis via extracellular vesicles (EVs), formal demonstration that EVs mediate the effects of paternal preconception perturbations is lacking. Therefore, in the current study we tested the hypothesis that epididymal EV preparations are sufficient to induce intergenerational effects of paternal preconception ethanol exposure on offspring. To test this hypothesis, sperm from ethanol-naïve donors were incubated with epididymal EV preparations from chronic ethanol (Ethanol EV-donor) or control-treated (Control EV-donor) mice prior to in vitro fertilization (IVF) and embryo transfer. Progeny were examined for ethanol- and stress-related behaviors in adulthood. Ethanol EV-donors imparted reduced body weight at weaning and imparted modestly increased limited access ethanol intake to male offspring. Ethanol-EV donors also imparted increased basal anxiety-like behavior and reduced sensitivity to ethanol-induced anxiolysis to female offspring. Although Ethanol EV-donor treatment did not recapitulate the ethanol- or stress-related intergenerational effects of paternal ethanol following natural mating, these results demonstrate that coincubation of sperm with epididymal EV preparations is sufficient to impart intergenerational effects of ethanol through the male germline. This mechanism may generalize to the intergenerational effects of a wide variety of paternal preconception perturbations.

Alcohol misuse and dependence is a widespread health problem. The central nucleus of the amygdala (CeA) plays important roles in both the anxiety associated with alcohol (ethanol) dependence and the increased alcohol intake that is observed during withdrawal in dependent animals. We and others have shown the essential involvement of the corticotropin releasing factor (CRF) system in alcohol's synaptic effects on the CeA and in the development of ethanol dependence. Another system that has been shown to be critically involved in the molecular underpinnings of alcohol dependence is the norepinephrine (NE) system originating in the locus coeruleus. Both the CRF and NE systems act in concert to facilitate a stress response: central amygdalar afferents release CRF in the locus coeruleus promoting widespread release of NE. In this study, we are the first to use fast-scan cyclic voltammetry to classify local electrically-evoked NE release in the CeA and to determine if acute alcohol and CRF modulate it. Evoked NE release is action potential dependent, is abolished after depletion of monoaminergic vesicles, differs pharmacologically from dopamine release, is insensitive to acute alcohol, and decreases in response to locally applied CRF. Taken together, these results indicate that NE release in the CeA is released canonically in a vesicular-dependent manner, and that while acute alcohol does not directly alter NE release, CRF decreases it. Our results suggest that CRF acts locally on NE terminals as negative feedback and potentially prevents hyperactivation of the CRF-norepinephrine stress pathway.

In a short period, many research publications that report sets of experimentally validated drugs as potential COVID-19 therapies have emerged. To organize this accumulating knowledge, we developed the COVID-19 Drug and Gene Set Library (https://amp.pharm.mssm.edu/covid19/), a collection of drug and gene sets related to COVID-19 research from multiple sources. The platform enables users to view, download, analyze, visualize, and contribute drug and gene sets related to COVID-19 research. To evaluate the content of the library, we compared the results from six in vitro drug screens for COVID-19 repurposing candidates. Surprisingly, we observe low overlap across screens while highlighting overlapping candidates that should receive more attention as potential therapeutics for COVID-19. Overall, the COVID-19 Drug and Gene Set Library can be used to identify community consensus, make researchers and clinicians aware of new potential therapies, enable machine-learning applications, and facilitate the research community to work together toward a cure.

The development of CRISPR/Cas9 technology has vastly sped up the process of genome editing by introducing a bacterial system that can be exploited for reverse genetics-based research. However, generating homozygous knockout (KO) animals using traditional CRISPR/Cas9-mediated techniques requires three generations of animals. A founder animal with a desired mutation is crossed to produce heterozygous F1 offspring which are subsequently interbred to generate homozygous F2 KO animals. This study describes a novel adaptation of the CRISPR/Cas9-mediated method to develop a homozygous gene-targeted KO animal cohort in one generation. A well-characterized ethanol-responsive gene, MyD88, was chosen as a candidate gene for generation of MyD88^-/- mice as proof of concept. Previous studies have reported changes in ethanol-related behavioral outcomes in MyD88 KO mice. Therefore, it was hypothesized that a successful one-generation KO of MyD88 should reproduce decreased responses to ethanols sedative effects, as well as increased ethanol consumption in males that were observed in previous studies. One-cell mouse embryos were simultaneously electroporated with four gRNAs targeting a critical Exon of MyD88 along with Cas9. DNA and RNA analysis of founder mice revealed a complex mix of genetic alterations, all of which were predicted to ablate MyD88 gene function. This study additionally compared responses of Mock treatment control mice generated through electroporation to controls purchased from a vendor. No substantial behavioral changes were noted between control cohorts. Overall, the CRISPR/Cas9 KO protocol reported here, which we call CRISPR Turbo Accelerated KnockOut (CRISPy TAKO), will be useful for reverse genetic in vivo screens of gene function in whole animals.

Binge drinking is a widespread public health concern with limited effective treatment options. To better select pharmaceutical targets, it is imperative to expand our knowledge of the underlying neural mechanisms involved in binge drinking. Our previous experiments in C57BL/6J female mice found that increasing activity in the nucleus accumbens (NAc) core using excitatory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) reduced binge-like drinking. These results differed from what has been found in males; however, it is unclear whether differences in experimental procedures or sex underlie these discrepancies. We matched the conditions used in our female study and asked whether bidirectional manipulation of NAc core activity has different effects on binge-like drinking in males. Male C57BL/6J mice were stereotaxically injected with AAV2 hSyn-HA hM3Dq (excitatory), -hM4Di (inhibitory), or -eGFP bilaterally into the NAc core. We tested the effects of altering NAc activity on binge-like ethanol intake using Drinking in the Dark (DID). During the first week, mice were pre-treated with vehicle to establish baseline ethanol intake. In week 2, mice were treated with 1 mg/kg CNO prior to DID to determine the effects of DREADD-induced changes in NAc core activity on ethanol intake. Decreasing activity via CNO/hM4Di significantly decreased binge-like drinking in male mice relative to eGFP and hM4Di groups. We also measured intake of sucrose, quinine, and water after CNO treatment and found that increasing NAc core activity via CNO/hM3Dq increased quinine intake, and increased water intake over time. We did not observe significant differences in the GFP or hM4Di groups. This work suggests there exist apparent sex-related differences in NAc core contributions to binge-like alcohol drinking, thus demonstrating the need for inclusion of both sexes in future work.

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.