Publications

In Press
Kircher DM, Aziz H, Mangieri R, R M. Ethanol experience enhances glutamatergic ventral hippocampal inputs to D1 receptor-expressing medium spiny neurons in the nucleus accumbens shell. The Journal of neuroscience [Internet]. In Press;39 (13) :2459-2469. Publisher's VersionAbstract

Nucleus accumbens dopamine D1 receptor-expressing medium spiny neurons (D1-MSNs) have been implicated in the formation of dependence to many drugs of abuse including alcohol. Previous studies have revealed that acute alcohol exposure suppresses glutamatergic signaling within the accumbens and repeated alcohol exposure enhances glutamatergic signaling. D1-MSNs receive glutamatergic input from several brain regions and it is not currently known how individual inputs onto D1-MSNs are altered by alcohol experience. To Address this, we used virally mediated expression of Channelrhodopsin (ChR2) in ventral hippocampal (vHipp) glutamate neurons to selectively activate vHipp to D1-MSN synapses and compared synaptic adaptations in response to low and high alcohol experience in vitro and in vivo. Alcohol experience enhanced glutamatergic activity and abolished long-term depression (LTD) at ventral hippocampal (vHipp) to D1-MSN synapses. Following chronic alcohol experience GluA2-lacking AMPA receptors, which are Ca-permeable, were inserted into vHipp to D1-MSN synapses. These alcohol-induced adaptations of glutamatergic signaling occurred at lower levels of exposure than previously reported. The loss of LTD expression and enhancement in glutamatergic signaling from the vHipp to D1-MSNs in the nucleus accumbens may play a critical role in the formation of alcohol dependence and enhancements in ethanol consumption. Reversal of alcohol-induced insertion of Ca-permeable AMPA receptors and enhancement of glutamatergic activity at vHipp to D1-MSNs presents potential targets for intervention during early exposure to alcohol.

SIGNIFICANCE STATEMENT The work presented here is the first to elucidate how an individual glutamatergic input onto D1-MSNs of the accumbens shell (shNAc) are altered by repeated ethanol exposure. Our findings suggest that glutamatergic input from the ventral hippocampus (vHipp) onto D1-MSNs is enhanced following drinking in a two-bottle choice (2BC) paradigm and is further enhanced by chronic intermittent ethanol (CIE) vapor exposure which escalated volitional ethanol intake. A critical finding was the insertion of Ca-permeable AMPA receptors into vHipp-shNAc D1-MSN synapses following CIE exposure, and more importantly following ethanol consumption in the absence of vapor exposure. These findings suggest that enhancements of glutamatergic input from the vHipp and insertion of Ca-permeable AMPARs play a role in the formation of ethanol dependence.

Varodayan FP, Minnig MA, Steinman MS, Oleata CS, Riley MW, Sabino V, Roberto M. PACAP regulation of central amygdala GABAergic synapses is altered by restraint stress. Neuropharmacology [Internet]. In Press;168 :107752. Publisher's VersionAbstract
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.
Suárez J, Khom S, Alén F, Natividad LA, Varodayan FP, Patel RR, Kirson D, Arco R, Ballesta A, Bajo M, et al. Cessation of fluoxetine treatment increases alcohol seeking during relapse and dysregulates endocannabinoid and glutamatergic signaling in the central amygdala. Addict Biol. In Press.Abstract
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.
Lo CL, Lumeng L, Bell RL, Liang T, Lossie AC, Muir WM, Zhou FC. Cis-acting allele specific expression (ASE) differences induced by alcohol and impacted by sex as well as parental genotype of origin. Alcohol Clin Exp. In Press.Abstract

BACKGROUND:

Alcohol use disorders (AUDs) are influenced by complex interactions between the genetics of the individual and their environment. We have previously identified hundreds of polygenic genetic variants between the selectively bred high- and low-alcohol drinking (HAD and LAD) rat lines. Here, we report allele-specific expression (ASE) differences, between the HAD2 and LAD2 rat lines.

METHODS:

The HAD2 and LAD2 rats, which have been sequenced, were reciprocally crossed to generate 10 litters of F1 progeny. For 5 of these litters, the sire was HAD2, and for the other 5 litters, the sire was a LAD2. From these 10 litters, 2 males and 2 females were picked from each F1 litter (N = 40 total). The F1 pups were divided, balancing for sex and direction of cross, into an alcohol (15%) versus a water control group. Alcohol drinking started in the middle of adolescence (~postnatal day 35) and lasted 9 weeks. At the end of these treatments, rats were euthanized, the nucleus accumbens was dissected, and RNA was processed for RNA-sequencing and ASE analyses.

RESULTS:

Analyses revealed that adolescent ethanol (EtOH) drinking, individual EtOH drinking levels, parentage, and sex-of-animal affected ASEs of about 300 genes. The identified genes included those associated with EtOH metabolism (e.g., Aldh2); neuromodulatory function (e.g., Cckbr, Slc6a7, and Slc1a1); ion channel activity (e.g., Kcnc3); and other synaptic and epigenetic functions.

CONCLUSIONS:

These data indicate that EtOH drinking differentially amplified paternal versus maternal allelic contribution to the transcriptome. We hypothesize that this was due, at least in part, to EtOH-induced changes in cis-regulation of polymorphisms previously identified between the HAD2 and LAD2 rat lines. This report highlights the complexity of gene-by-environment interactions mediating a genetic predisposition for, and/or the active development of, AUDs.

Iancu OD, Colville A, Wilmot B, Searles R, Darakjian P, Zheng C, McWeeney S, Kawane S, Crabbe JC, Metten P, et al. Gender specific effects of selection for drinking in the dark on the network roles of coding and non-coding RNAs. Alcohol Clin Exp. In Press.Abstract

BACKGROUND:

Transcriptional differences between heterogeneous stock mice and high drinking-in-the-dark selected mouse lines have previously been described based on microarray technology coupled with network-based analysis. The network changes were reproducible in 2 independent selections and largely confined to 2 distinct network modules; in contrast, differential expression appeared more specific to each selected line. This study extends these results by utilizing RNA-Seq technology, allowing evaluation of the relationship between genetic risk and transcription of noncoding RNA (ncRNA); we additionally evaluate sex-specific transcriptional effects of selection.

METHODS:

Naïve mice (N = 24/group and sex) were utilized for gene expression analysis in the ventral striatum; the transcriptome was sequenced with the Illumina HiSeq platform. Differential gene expression and the weighted gene co-expression network analysis were implemented largely as described elsewhere, resulting in the identification of genes that change expression level or (co)variance structure.

RESULTS:

Across both sexes, we detect selection effects on the extracellular matrix and synaptic signaling, although the identity of individual genes varies. A majority of nc RNAs cluster in a single module of relatively low density in both the male and female network. The most strongly differentially expressed transcript in both sexes was Gm22513, a small nuclear RNA with unknown function. Associated with selection, we also found a number of network hubs that change edge strength and connectivity. At the individual gene level, there are many sex-specific effects; however, at the annotation level, results are more concordant.

CONCLUSIONS:

In addition to demonstrating sex-specific effects of selection on the transcriptome, the data point to the involvement of extracellular matrix genes as being associated with the binge drinking phenotype.

Fritz M, Klawonn AM, Zahr NM. Neuroimaging in alcohol use disorder: From mouse to man. J Neurosci. In Press.Abstract
This article provides an overview of recent advances in understanding the effects of alcohol use disorders (AUD) on the brain from the perspective of magnetic resonance imaging (MRI) research in preclinical models and clinical studies. As a noninvasive investigational tool permitting assessment of morphological, metabolic, and hemodynamic changes over time, MRI offers insight into the dynamic course of alcoholism beginning with initial exposure through periods of binge drinking and escalation, sobriety, and relapse and has been useful in differential diagnosis of neurological diseases associated with AUD. Structural MRI has revealed acute and chronic effects of alcohol on both white and gray matter volumes. MR Spectroscopy, able to quantify brain metabolites in vivo, has shed light on biochemical alterations associated with alcoholism. Diffusion tensor imaging permits microstructural characterization of white matter fiber tracts. Functional MRI has allowed for elucidation of hemodynamic responses at rest and during task engagement. Positron emission tomography, a non-MRI imaging tool, has led to a deeper understanding of alcohol-induced receptor and neurotransmitter changes during various stages of drinking and abstinence. Together, such in vivo imaging tools have expanded our understanding of the dynamic course of alcoholism including evidence for regional specificity of the effects of AUD, hints at mechanisms underlying the shift from casual to compulsive use of alcohol, and profound recovery with sustained abstinence.
Chen H, Lasek AW. Perineuronal nets in the insula regulate aversion-resistant alcohol drinking. Addict Biol. In Press.Abstract
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.
2020
Zamudio PA, Smothers TC, Homanics GE, JJ W. Knock‐in Mice Expressing an Ethanol‐Resistant GluN2A NMDA Receptor Subunit Show Altered Responses to Ethanol. Alcoholism: Clinical and Experimental Research [Internet]. 2020;44 (2) :479-491. Publisher's VersionAbstract

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.

NM Z, Lenart AM, Karpf JA, Casey KM, KM P, EV S, Pfefferbaum A. Multi-modal imaging reveals differential brain volumetric, biochemical, and white matter fiber responsivity to repeated intermittent ethanol vapor exposure in male and female rats. Neuropharmacology [Internet]. 2020;170 :108066. Publisher's VersionAbstract
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.
Walter NAR, Zheng CL, Searles RP, McWeeney SK, Grant KA, Hitzemann R. Chronic Voluntary Ethanol Drinking in Cynomolgus Macaques Elicits Gene Expression Changes in Prefrontal Cortical Area 46. Alcoholism: Clinical and Experimental Research [Internet]. 2020;44 (2) :470-478. Publisher's VersionAbstract

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 ( = 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.

Savarese AM, Ozburn AR, Metten P, Schlumbohm JP, Hack WR, LeMoine K, Hunt H, Hausch F, Bauder M, Crabbe JC. Targeting the Glucocorticoid Receptor Reduces Binge‐Like Drinking in High Drinking in the Dark (HDID‐1) Mice. Alcoholism: Clinical and Experimental Research [Internet]. 2020;44 (5) :1025-1036. Publisher's VersionAbstract

Background

Chronic alcohol exposure can alter glucocorticoid receptor (GR) function in some brain areas that promotes escalated and compulsive‐like alcohol intake. GR antagonism can prevent dependence‐induced escalation in drinking, but very little is known about the role of GR in regulating high‐risk nondependent alcohol intake. Here, we investigate the role of GR in regulating binge‐like drinking and aversive responses to alcohol in the High Drinking in the Dark (HDID‐1) mice, which have been selectively bred for high blood ethanol (EtOH) concentrations (BECs) in the Drinking in the Dark (DID) test, and in their founder line, the HS/NPT.

Methods

In separate experiments, male and female HDID‐1 mice were administered one of several compounds that inhibited GR or its negative regulator, FKBP51 (mifepristone [12.5, 25, 50, 100 mg/kg], CORT113176 [20, 40, 80 mg/kg], and SAFit2 [10, 20, 40 mg/kg]) during a 2‐day DID task. EtOH consumption and BECs were measured. EtOH conditioned taste and place aversion (CTA and CPA, respectively) were measured in separate HDID‐1 mice after mifepristone administration to assess GR’s role in regulating the conditioned aversive effects of EtOH. Lastly, HS/NPT mice were administered CORT113176 during DID to assess whether dissimilar effects from those of HDID‐1 would be observed, which could suggest that selective breeding had altered sensitivity to the effects of GR antagonism on binge‐like drinking.

Results

GR antagonism (with both mifepristone and CORT113176) selectively reduced binge‐like EtOH intake and BECs in the HDID‐1 mice, while inhibition of FKBP51 did not alter intake or BECs. In contrast, GR antagonism had no effect on EtOH intake or BECs in the HS/NPT mice. Although HDID‐1 mice exhibit attenuated EtOH CTA, mifepristone administration did not enhance the aversive effects of EtOH in either a CTA or CPA task.

Conclusion

These data suggest that the selection process increased sensitivity to GR antagonism on EtOH intake in the HDID‐1 mice, and support a role for the GR as a genetic risk factor for high‐risk alcohol intake.

Pozhidayeva DY, Farris SP, Goeke CM, Firsick EJ, Townsley KG, Guizzetti M, Ozburn AR. Chronic Chemogenetic Stimulation of the Nucleus Accumbens Produces Lasting Reductions in Binge Drinking and Ameliorates Alcohol-Related Morphological and Transcriptional Changes. Brain Sciences [Internet]. 2020;10 (2) :E109. Publisher's VersionAbstract
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.
Ozburn AR, Metten P, Potretzke S, Townsley KG, Blednov YA, Crabbe JC. Effects of Pharmacologically Targeting Neuroimmune Pathways on Alcohol Drinking in Mice Selectively Bred to Drink to Intoxication. Alcoholism: Clinical and Experimental Research [Internet]. 2020;44 (2) :553-566. Publisher's VersionAbstract

 

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.

Kirson D, Oleata CS, M R. Taurine Suppression of Central Amygdala GABAergic Inhibitory Signaling via Glycine Receptors Is Disrupted in Alcohol Dependence. Alcoholism: Clinical and Experimental Research [Internet]. 2020;44 (2) :445-454. Publisher's VersionAbstract

Background

Alcohol use disorder (AUD) increases brain stress systems while suppressing reward system functioning. One expression of stress system recruitment is elevated GABAergic activity in the central amygdala (CeA), which is involved in the excessive drinking seen with AUD. The sulfonic amino acid taurine, a glycine receptor partial agonist, modulates GABAergic activity in the rewarding effects of alcohol. Despite taurine abundance in the amygdala, its role in the dysregulation of GABAergic activity associated with AUD has not been studied. Thus, here, we evaluated the effects of taurine on locally stimulated GABAergic neurotransmission in the CeA of naïve‐ and alcohol‐dependent rats.

Methods

We recorded intracellularly from CeA neurons of naïve‐ and alcohol‐dependent rats, quantifying locally evoked GABAA receptor‐mediated inhibitory postsynaptic potentials (eIPSP). We examined the effects of taurine and alcohol on CeA eIPSP to characterize potential alcohol dependence‐induced changes in the effects of taurine.

Results

We found that taurine decreased amplitudes of eIPSP in CeA neurons of naïve rats, without affecting the acute alcohol‐induced facilitation of GABAergic responses. In CeA neurons from dependent rats, taurine no longer had an effect on eIPSP, but now blocked the ethanol (EtOH)‐induced increase in eIPSP amplitude normally seen. Additionally, preapplication of the glycine receptor‐specific antagonist strychnine blocked the EtOH‐induced increase in eIPSP amplitude in neurons from naïve rats.

Conclusions

These data suggest taurine may act to oppose the effects of acute alcohol via the glycine receptor in the CeA of naïve rats, and this modulatory system is altered in the CeA of dependent rats.

Khom S, Steinkellner T, Hnasko TS, M R. Alcohol dependence potentiates substance P/neurokinin-1 receptor signaling in the rat central nucleus of amygdala. Science Advances [Internet]. 2020;6 (12) :eaaz1050. Publisher's VersionAbstract
Behavioral and clinical studies suggest a critical role of substance P (SP)/neurokinin-1 receptor (NK-1R) signaling in alcohol dependence. Here, we examined regulation of GABA transmission in the medial subdivision of the central amygdala (CeM) by the SP/NK-1R system, and its neuroadaptation following chronic alcohol exposure. In naïve rats, SP increased action potential–dependent GABA release, and the selective NK-1R antagonist L822429 decreased it, demonstrating SP regulation of CeM activity under basal conditions. SP induced a larger GABA release in alcohol-dependent rats accompanied by decreased NK-1R expression compared to naïve controls, suggesting NK-1R hypersensitivity which persisted during protracted alcohol withdrawal. The NK-1R antagonist blocked acute alcohol-induced GABA release in alcohol-dependent and withdrawn but not in naïve rats, indicating that dependence engages the SP/NK-1R system to mediate acute effects of alcohol. Collectively, we report long-lasting CeA NK-1R hypersensitivity corroborating that NK-1Rs are promising targets for the treatment of alcohol use disorder.
He D, Lasek AW. Anaplastic Lymphoma Kinase Regulates Internalization of the Dopamine D2 Receptor. Molecular Pharmacology [Internet]. 2020;97 (2) :123-131. Publisher's VersionAbstract

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.

Brenner E, Tiwari GR, Kapoor M, Liu Y, Brock A, RD M. Single cell transcriptome profiling of the human alcohol-dependent brain. Human Molecular Genetics [Internet]. 2020;29 (7) :1144–1153. Publisher's VersionAbstract
Alcoholism remains a prevalent health concern throughout the world. Previous studies have identified transcriptomic patterns in the brain associated with alcohol dependence in both humans and animal models. But none of these studies have systematically investigated expression within the unique cell types present in the brain. We utilized single nucleus RNA sequencing (snRNA-seq) to examine the transcriptomes of over 16 000 nuclei isolated from the prefrontal cortex of alcoholic and control individuals. Each nucleus was assigned to one of seven major cell types by unsupervised clustering. Cell type enrichment patterns varied greatly among neuroinflammatory-related genes, which are known to play roles in alcohol dependence and neurodegeneration. Differential expression analysis identified cell type-specific genes with altered expression in alcoholics. The largest number of differentially expressed genes (DEGs), including both protein-coding and non-coding, were detected in astrocytes, oligodendrocytes and microglia. To our knowledge, this is the first single cell transcriptome analysis of alcohol-associated gene expression in any species and the first such analysis in humans for any addictive substance. These findings greatly advance the understanding of transcriptomic changes in the brain of alcohol-dependent individuals.
Alhaddad H, Gordon DM, Bell RL, Jarvis EE, Kipp ZA, Hinds TD Jr, Sari Y. Chronic Ethanol Consumption Alters Glucocorticoid Receptor Isoform Expression in Stress Neurocircuits and Mesocorticolimbic Brain Regions of Alcohol-Preferring Rats. Neuroscience [Internet]. 2020;437 :107-116. Publisher's VersionAbstract
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.
Hamada K, Lasek AW. Receptor tyrosine kinases as therapeutic targets for alcohol use disorder. Neurotherapeutics. Neurotherapeutics [Internet]. 2020;17 (1) :4-16. Publisher's VersionAbstract
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.
Boulos LJ, Ben Hamida S, Bailly J, Maitra M, Ehrlich AT, Gavériaux-Ruff C, Darcq E, Kieffer BL. Mu opioid receptors in the medial habenula contribute to naloxone aversion. Neuropsychopharmacology [Internet]. 2020;45 (2) :247–255. Publisher's VersionAbstract
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.

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