J Z, Chen C, Bell RL, H Q, Z L. Identification of HIVEP2 as a dopaminergic transcription factor related to substance use disorders in rats and humans. Translational Psychiatry [Internet]. 2019;9 (1) :247. Publisher's VersionAbstract
Playing an important role in the etiology of substance use disorder (SUD), dopamine (DA) neurons are subject to various regulations but transcriptional regulations are largely understudied. For the first time, we report here that the Human Immunodeficiency Virus Type I Enhancer Binding Protein 2 (HIVEP2) is a dopaminergic transcriptional regulator. HIVEP2 is expressed in both the cytoplasm and nuclei of DA neurons. Therein, HIVEP2 can target the intronic sequence GTGGCTTTCT of SLC6A3 and thereby activate the gene. In naive rats from the bi-directional selectively bred substance-preferring P vs -nonpreferring NP rat model of substance abuse vulnerability, increased gene activity in males was associated with the vulnerability, whereas decreased gene activity in the females was associated with the same vulnerability. In clinical subjects, extensive and significant HIVEP2-SLC6A3 interactions were observed for SUD. Collectively, HIVEP2-mediated transcriptional mechanisms are implicated in dopaminergic pathophysiology of SUD.
NM Z, KM P, M S, EV S, Pfefferbaum A. Hippocampal subfield CA2+3 exhibits accelerated aging in Alcohol Use Disorder: A preliminary study. Neuroimage: Clinical [Internet]. 2019;22 :101764. Publisher's VersionAbstract

The profile of brain structural dysmorphology of individuals with Alcohol Use Disorders (AUD) involves disruption of the limbic system. In vivo imaging studies report hippocampal volume loss in AUD relative to controls, but only recently has it been possible to articulate different regions of this complex structure. Volumetric analysis of hippocampal regions rather than total hippocampal volume may augment differentiation of disease processes. For example, damage to hippocampal subfield cornu ammonis 1 (CA1) is often reported in Alzheimer's disease (AD), whereas deficits in CA4/dentate gyrus are described in response to stress and trauma. Two previous studies explored the effects of chronic alcohol use on hippocampal subfields: one reported smaller volume of the CA2+3 in alcohol-dependent subjects relative to controls, associated with years of alcohol consumption; the other, smaller volumes of presubiculum, subiculum, and fimbria in alcohol-dependent relative to control men.

The current study, conducted in 24 adults with DSM5-diagnosed AUD (7 women, 53.7 ± 8.8) and 20 controls (7 women, 54.1 ± 9.3), is the first to use FreeSurfer 6.0, which provides state-of-the art hippocampal parcellation, to explore the sensitivity of hippocampal sufields to alcoholism. T1- and T2- images were collected on a GE MR750 system with a 32-channel Nova head coil. FreeSurfer 6.0 hippocampal subfield analysis produced 12 subfields: parasubiculum; presubiculum; subiculum; CA1; CA2+3; CA4; GC-ML-DG (Granule Cell (GC) and Molecular Layer (ML) of the Dentate Gyrus (DG)); molecular layer; hippocampus-amygdala-transition-area (HATA); fimbria; hippocampal tail; hippocampal fissure; and whole volume for left and right hippocampi. A comprehensive battery of neuropsychological tests comprising attention, memory and learning, visuospatial abilities, and executive functions was administered.

Multiple regression analyses of raw volumetric data for each subfields by group, age, sex, hemisphere, and supratentorial volume (svol) showed significant effects of svol (p < .04) on nearly all structures (excluding tail and fissure). Volumes corrected for svol showed effects of age (fimbria, fissure) and group (subiculum, CA1, CA4, GC-ML-DG, HATA, fimbria); CA2+3 showed a diagnosis-by-age interaction indicating older AUD individuals had a smaller volume than would be expected for their age. There were no selective relations between hippocampal subfields and performance on neuropsychological tests, likely due to lack of statistical power.

The current results concur with the previous study identifying CA2+3 as sensitive to alcoholism, extend them by identifying an alcoholism-age interaction, and suggest an imaging phenotype distinguishing AUD from AD and stress/trauma.

NM Z, KM P, Pfefferbaum A, EV S. Central Nervous System Correlates of “Objective” Neuropathy in Alcohol Use Disorder. Alcoholism: Clinical and Experimental Research [Internet]. 2019;43 (10) :2144-2152. Publisher's VersionAbstract


Among the neurological consequences of alcoholism is peripheral neuropathy. Relative to human immunodeficiency virus (HIV) or diabetes‐related neuropathies, neuropathy associated with alcohol use disorders (AUD ) is understudied. In both the diabetes and HIV literature, emerging evidence supports a central nervous system (CNS) component to peripheral neuropathy.


In seeking a central substrate for AUD ‐related neuropathy, the current study was conducted in 154 individuals with AUD (43 women, age 21 to 74 years) and 99 healthy controls (41 women, age 21 to 77 years) and explored subjective symptoms (self‐report) and objective signs (perception of vibration, deep tendon ankle reflex, position sense, 2‐point discrimination) of neuropathy separately. In addition to regional brain volumes, risk factors for AUD ‐related neuropathy, including age, sex, total lifetime ethanol consumed, nutritional indices (i.e., thiamine, folate), and measures of liver integrity (i.e., γ ‐glutamyltransferase), were evaluated.


The AUD group described more subjective symptoms of neuropathy and was more frequently impaired on bilateral perception of vibration. From 5 correlates, the number of AUD ‐related seizures was most significantly associated with subjective symptoms of neuropathy. There were 15 correlates of impaired perception of vibration among the AUD participants: Of these, age and volume of frontal precentral cortex were the most robust predictors.


This study supports CNS involvement in objective signs of neuropathy in AUD.

You C, Savarese A, Vandegrift BJ, He D, Pandey SC, Lasek AW, Brodie MS. Ethanol acts on KCNK13 potassium channels in the ventral tegmental area to increase firing rate and modulate binge–like drinking. Neuropharmacology [Internet]. 2019;114 :29-36. Publisher's VersionAbstract
Alcohol excitation of the ventral tegmental area (VTA) is important in neurobiological processes related to the development of alcoholism. The ionotropic receptors on VTA neurons that mediate ethanol-induced excitation have not been identified. Quinidine blocks ethanol excitation of VTA neurons, and blockade of two-pore potassium channels is among the actions of quinidine. Therefore two-pore potassium channels in the VTA may be potential targets for the action of ethanol. Here, we explored whether ethanol activation of VTA neurons is mediated by the two-pore potassium channel KCNK13. Extracellular recordings of the response of VTA neurons to ethanol were performed in combination with knockdown of Kcnk13 using a short hairpin RNA (shRNA) in C57BL/6 J mice. Real-time PCR and immunohistochemistry were used to examine expression of this channel in the VTA. Finally, the role of KCNK13 in binge-like drinking was examined in the drinking in the dark test after knockdown of the channel. Kcnk13 expression in the VTA was increased by acute ethanol exposure. Ethanol-induced excitation of VTA neurons was selectively reduced by shRNA targeting Kcnk13. Importantly, knockdown of Kcnk13 in the VTA resulted in increased alcohol drinking. These results are consistent with the idea that ethanol stimulates VTA neurons at least in part by inhibiting KCNK13, a specific two-pore potassium channel, and that KCNK13 can control both VTA neuronal activity and binge drinking. KCNK13 is a novel alcohol-sensitive molecular target and may be amenable to the development of pharmacotherapies for alcoholism treatment.
Wolfe SA, H S, Patel RR, M K, D'Ambrosio SR, C C, M R. Molecular, Morphological, and Functional Characterization of Corticotropin-Releasing Factor Receptor 1-Expressing Neurons in the Central Nucleus of the Amygdala. eNeuro [Internet]. 2019;6 (3). Publisher's VersionAbstract

The central nucleus of the amygdala (CeA) is a brain region implicated in anxiety, stress-related disorders and the reinforcing effects of drugs of abuse. Corticotropin-releasing factor (CRF, Crh) acting at cognate type 1 receptors (CRF1, Crhr1) modulates inhibitory and excitatory synaptic transmission in the CeA. Here, we used CRF1:GFP reporter mice to characterize the morphological, neurochemical and electrophysiological properties of CRF1-expressing (CRF1+) and CRF1-non-expressing (CRF1–) neurons in the CeA. We assessed these two neuronal populations for distinctions in the expression of GABAergic subpopulation markers and neuropeptides, dendritic spine density and morphology, and excitatory transmission. We observed that CeA CRF1+ neurons are GABAergic but do not segregate with calbindin (CB), calretinin (CR), parvalbumin (PV), or protein kinase C-δ (PKCδ). Among the neuropeptides analyzed, Penk and Sst had the highest percentage of co-expression with Crhr1 in both the medial and lateral CeA subdivisions. Additionally, CeA CRF1+ neurons had a lower density of dendritic spines, which was offset by a higher proportion of mature spines compared to neighboring CRF1– neurons. Accordingly, there was no difference in basal spontaneous glutamatergic transmission between the two populations. Application of CRF increased overall vesicular glutamate release onto both CRF1+ and CRF1– neurons and does not affect amplitude or kinetics of EPSCs in either population. These novel data highlight important differences in the neurochemical make-up and morphology of CRF1+ compared to CRF1– neurons, which may have important implications for the transduction of CRF signaling in the CeA.

Keywords: calcium binding proteins, corticotropin-releasing factor, dendritic spines, glutamatergic signaling, neuropeptides, stress and anxiety

Warden AS, Azzam M, Da Costa A, Mason S, Blednov YA, Messing RO, RD M, RA H. Toll-like receptor 3 dynamics in female C57BL/6J mice: Regulation of alcohol intake. Brain Behavior and Immunity [Internet]. 2019;77 :66-76. Publisher's VersionAbstract

Although there are sex differences in the effects of alcohol on immune responses, it is unclear if sex differences in immune response can influence drinking behavior. Activation of toll-like receptor 3 (TLR3) by polyinosinic:polycytidylic acid (poly(I:C)) produced a rapid proinflammatory response in males that increased alcohol intake over time (Warden et al., 2019). Poly(I:C) produced a delayed and prolonged innate immune response in females. We hypothesized that the timecourse of innate immune activation could regulate drinking behavior in females. Therefore, we chose to test the effect of two time points in the innate immune activation timecourse on every-other-day two-bottle-choice drinking: (1) peak activation; (2) descending limb of activation. Poly(I:C) reduced ethanol consumption when alcohol access occurred during peak activation. Poly(I:C) did not change ethanol consumption when alcohol access occurred on the descending limb of activation. Decreased levels of MyD88-dependent pathway correlated with decreased alcohol intake and increased levels of TRIF-dependent pathway correlated with increased alcohol intake in females. To validate the effects of poly(I:C) were mediated through MyD88, we tested female mice lacking Myd88. Poly(I:C) did not change alcohol intake in Myd88 knockouts, indicating that poly(I:C)-induced changes in alcohol intake are dependent on MyD88 in females. We next determined if the innate immune timecourse also regulated drinking behavior in males. Poly(I:C) reduced ethanol consumption in males when alcohol was presented at peak activation. Therefore, the timecourse of innate immune activation regulates drinking behavior and sex-specific dynamics of innate immune response must be considered when designing therapeutics to treat excessive drinking.

    Warden AS, Azzam M, Da Costa A, Mason S, Blednov YA, Messing RO, RD M, RA H. Toll-like receptor 3 activation increases voluntary alcohol intake in C57BL/6J male mice. Brain Behavior and Immunity [Internet]. 2019;77 :55-65. Publisher's VersionAbstract
    Many genes differentially expressed in brain tissue from human alcoholics and animals that have consumed large amounts of alcohol are components of the innate immune toll-like receptor (TLR) pathway. TLRs initiate inflammatory responses via two branches: (1) MyD88-dependent or (2) TRIF-dependent. All TLRs signal through MyD88 except TLR3. Prior work demonstrated a direct role for MyD88-dependent signaling in regulation of alcohol consumption. However, the role of TLR3 as a potential regulator of excessive alcohol drinking has not previously been investigated. To test the possibility TLR3 activation regulates alcohol consumption, we injected mice with the TLR3 agonist polyinosinic:polycytidylic acid (poly(I:C)) and tested alcohol consumption in an every-other-day two-bottle choice test. Poly(I:C) produced a persistent increase in alcohol intake that developed over several days. Repeated poly(I:C) and ethanol exposure altered innate immune transcript abundance; increased levels of TRIF-dependent pathway components correlated with increased alcohol consumption. Administration of poly(I:C) before exposure to alcohol did not alter alcohol intake, suggesting that poly(I:C) and ethanol must be present together to change drinking behavior. To determine which branch of TLR signaling mediates poly(I:C)-induced changes in drinking behavior, we tested either mice lacking MyD88 or mice administered a TLR3/dsRNA complex inhibitor. MyD88 null mutants showed poly(I:C)-induced increases in alcohol intake. In contrast, mice pretreated with a TLR3/dsRNA complex inhibitor reduced their alcohol intake, suggesting poly(I:C)-induced escalations in alcohol intake are, at least partially, dependent on TLR3. Together, these results strongly suggest that TLR3-dependent signaling drives excessive alcohol drinking behavior.
    Waeiss RA, Knight CP, Carvajal GB, Bell RL, Engleman EA, McBride WJ, Hauser SR, ZA R. Peri-adolescent alcohol consumption increases sensitivity and dopaminergic response to nicotine during adulthood in female alcohol-preferring (P) rats: Alterations to α7 nicotinic acetylcholine receptor expression. Behavioural Brain Research [Internet]. 2019;376. Publisher's VersionAbstract

    Adolescent alcohol drinking has been linked to increased risk for drug abuse during adulthood. Nicotine microinjected directly into the posterior ventral tegmental area (pVTA) stimulates dopamine (DA) release in the nucleus accumbens (NAc) shell. The α7 nicotinic acetylcholine receptor (nAChR) is a potent regulator of dopaminergic activity in the pVTA. The current experiments examined the effects of peri-adolescent ethanol (EtOH) drinking on the ability of intra-pVTA nicotine to stimulate DA release during adulthood and alterations in α7 nAChR expression within the pVTA. Alcohol-preferring (P) female rats consumed EtOH and/or water during adolescence (post-natal day [PND] 30–60) or adulthood (PND 90–120). Thirty days following removal of EtOH, subjects received microinjections of 1 μM, 10 μM, or 50 μM nicotine into the pVTA concurrently with microdialysis for extracellular DA in the NAc shell. Brains were harvested from an additional cohort after PND 90 for quantification of α7 nAChR within the pVTA. The results indicated that only adolescent EtOH consumption produced a leftward and upward shift in the dose response curve for nicotine to stimulate DA release in the NAc shell. Investigation of α7 nAChR expression within the pVTA revealed a significant increase in animals that consumed EtOH during adolescence compared to naïve animals. The data suggests that peri-adolescent EtOH consumption produced cross-sensitization to the effects of nicotine during adulthood. The interaction between adolescent EtOH consumption and inflated adult risk for drug dependency could be predicated, at least in part, upon alterations in α7 nAChR expression within the mesolimbic reward pathway.

      EV S, NM Z, M S, KM P, Pfefferbaum A. Convergence of three parcellation approaches demonstrating cerebellar lobule volume deficits in Alcohol Use Disorder. Neuroimage: Clinical [Internet]. 2019;24. Publisher's VersionAbstract
      Recent advances in robust and reliable methods of MRI-derived cerebellar lobule parcellation volumetry present the opportunity to assess effects of Alcohol Use Disorder (AUD) on selective cerebellar lobules and relations with indices of nutrition and motor functions. In pursuit of this opportunity, we analyzed high-resolution MRI data acquired in 24 individuals with AUD and 20 age- and sex-matched controls with a 32-channel head coil using three different atlases: the online automated analysis pipeline volBrain Ceres, SUIT, and the Johns Hopkins atlas. Participants had also completed gait and balance examination and hematological analysis of nutritional and liver status, enabling testing of functional meaningfulness of each cerebellar parcellation scheme. Compared with controls, each quantification approach yielded similar patterns of group differences in regional volumes: All three approaches identified AUD-related deficits in total tissue and total gray matter, but only Ceres identified a total white matter volume deficit. Convergent volume differences occurred in lobules I-V, Crus I, VIIIB, and IX. Coefficients of variation (CVs) were <20% for 46 of 56 regions measured and in general were graded: Ceres<SUIT<Hopkins. The most robust correlations were identified between poorer stability in balancing on one leg and smaller lobule VI and Crus I volumes from the Ceres atlas. Lower values of two essential vitamins—thiamine (vitamin B1) and serum folate (vitamin B9)—along with lower red blood cell count, which are dependent on adequate levels of B vitamins, correlated with smaller gray matter volumes of lobule VI and Crus I. Higher γ-glutamyl transferase (GGT) levels, possibly reflecting compromised liver function, correlated with smaller volumes of lobules VI and X. These initial results based on high resolution data produced with clinically practical imaging procedures hold promise for expanding our knowledge about the relevance of focal cerebellar morphology in AUD and other neuropsychiatric conditions.
      Roberts AJ, Khom S, Bajo M, Vlkolinsky R, Polis I, Cates-Gatto C, M R, DL G. Increased IL-6 expression in astrocytes is associated with emotionality, alterations in central amygdala GABAergic transmission, and excitability during alcohol withdrawalv. Brain Behavior and Immunity [Internet]. 2019;82 :188-202. Publisher's VersionAbstract

      Accumulating evidence from preclinical and clinical studies has implicated a role for the cytokine IL-6 in a variety of CNS diseases including anxiety-like and depressive-like behaviors, as well as alcohol use disorder. Here we use homozygous and heterozygous transgenic mice expressing elevated levels of IL-6 in the CNS due to increased astrocyte expression and non-transgenic littermates to examine a role for astrocyte-produced IL-6 in emotionality (response to novelty, anxiety-like, and depressive-like behaviors). Our results from homozygous IL-6 mice in a variety of behavioral tests (light/dark transfer, open field, digging, tail suspension, and forced swim tests) support a role for IL-6 in stress-coping behaviors. Ex vivo electrophysiological studies of neuronal excitability and inhibitory GABAergic synaptic transmission in the central nucleus of the amygdala (CeA) of the homozygous transgenic mice revealed increased inhibitory GABAergic signaling and increased excitability of CeA neurons, suggesting a role for astrocyte produced IL-6 in the amygdala in exploratory drive and depressive-like behavior. Furthermore, studies in the hippocampus of activation/expression of proteins associated with IL-6 signal transduction and inhibitory GABAergic mechanisms support a role for astrocyte produced IL-6 in depressive-like behaviors. Our studies indicate a complex and dose-dependent relationship between IL-6 and behavior and implicate IL-6 induced neuroadaptive changes in neuronal excitability and the inhibitory GABAergic system as important contributors to altered behavior associated with IL-6 expression in the CNS.

        EV S, Pfefferbaum A. Brain-behavior relations and effects of aging and common comorbidities in alcohol use disorder: A review. Neuropsychology [Internet]. 2019;33 (6) :760-780. Publisher's VersionAbstract

        Objective: Alcohol use disorder (AUD) is a complex, dynamic condition that waxes and wanes with unhealthy drinking episodes and varies in drinking patterns and effects on brain structure and function with age. Its excessive use renders chronically heavy drinkers vulnerable to direct alcohol toxicity and a variety of comorbidities attributable to nonalcohol drug misuse, viral infections, and accelerated or premature aging. AUD affects widespread brain systems, commonly, frontolimbic, frontostriatal, and frontocerebellar networks. Method and Results: Multimodal assessment using selective neuropsychological testing and whole-brain neuroimaging provides evidence for AUD-related specific brain structure-function relations established with double dissociations. Longitudinal study using noninvasive imaging provides evidence for brain structural and functional improvement with sustained sobriety and further decline with relapse. Functional imaging suggests the possibility that some alcoholics in recovery can compensate for impairment by invoking brain systems typically not used for a target task but that can enable normal-level performance. Conclusions: Evidence for AUD-aging interactions, indicative of accelerated aging, together with increasing alcohol consumption in middle-age and older adults, put aging drinkers at special risk for developing cognitive decline and possibly dementia. (PsycINFO Database Record (c) 2019 APA, all rights reserved)

        GR R, Homanics GE. Intergenerational Effects of Alcohol: A Review of Paternal Preconception Ethanol Exposure Studies and Epigenetic Mechanisms in the Male Germline. Alcoholism: Clinical and Experimental Research [Internet]. 2019;43 (6) :1032-1045. Publisher's VersionAbstract
        While alcohol use disorder (AUD) is a highly heritable psychiatric disease, efforts to elucidate that heritability by examining genetic variation (e.g., single nucleotide polymorphisms) have been insufficient to fully account for familial AUD risk. Perhaps not coincidently, there has been a burgeoning interest in novel nongenomic mechanisms of inheritance (i.e., epigenetics) that are shaped in the male or female germ cells by significant lifetime experiences such as exposure to chronic stress, malnutrition, or drugs of abuse. While many epidemiological and preclinical studies have long pointed to a role for the parental preconception environment in offspring behavior, over the last decade many studies have implicated a causal relationship between the environmentally sensitive sperm epigenome and intergenerational phenotypes. This critical review will detail the heritable effects of alcohol and the potential role for epigenetics.
        Patel RR, Khom S, Steinman MQ, FP V, Kiosses WB, Hedges DM, Vlkolinsky R, Nadav T, Polis I, Bajo M, et al. IL-1β expression is increased and regulates GABA transmission following chronic ethanol in mouse central amygdala. Brain, Behavior and Immunity [Internet]. 2019;75 :208-219. Publisher's VersionAbstract
        The interleukin-1 system (IL-1) is a prominent pro-inflammatory pathway responsible for the initiation and regulation of immune responses. Human genetic and preclinical studies suggest a critical role for IL-1β signaling in ethanol drinking and dependence, but little is known about the effects of chronic ethanol on the IL-1 system in addiction-related brain regions such as the central amygdala (CeA). In this study, we generated naïve, non-dependent (Non-Dep) and dependent (Dep) male mice using a paradigm of chronic-intermittent ethanol vapor exposure interspersed with two-bottle choice to examine 1) the expression of IL-1β, 2) the role of the IL-1 system on GABAergic transmission, and 3) the potential interaction with the acute effects of ethanol in the CeA. Immunohistochemistry with confocal microscopy was used to assess expression of IL-1β in microglia and neurons in the CeA, and whole-cell patch clamp recordings were obtained from CeA neurons to measure the effects of IL-1β (50 ng/ml) or the endogenous IL-1 receptor antagonist (IL-1ra; 100 ng/ml) on action potential-dependent spontaneous inhibitory postsynaptic currents (sIPSCs). Overall, we found that IL-1β expression is significantly increased in microglia and neurons of Dep compared to Non-Dep and naïve mice, IL-1β and IL-1ra bi-directionally modulate GABA transmission through both pre- and postsynaptic mechanisms in all three groups, and IL-1β and IL-1ra do not alter the facilitation of GABA release induced by acute ethanol. These data suggest that while ethanol dependence induces a neuroimmune response in the CeA, as indicated by increased IL-1β expression, this does not significantly alter the neuromodulatory role of IL-1β on synaptic transmission.
        Otsu Y, Darcq E, Pietrajtis K, Mátyás F, Schwartz E, Bessaih T, Abi Gerges S, Rousseau CV, Grand T, Dieudonné S, et al. Control of aversion by glycine-gated GluN1/GluN3A NMDA receptors in the adult medial habenula. Science [Internet]. 2019;366 (6462) :250-254. Publisher's VersionAbstract
        The unconventional N-methyl-D-aspartate (NMDA) receptor subunits GluN3A and GluN3B can, when associated with the other glycine-binding subunit GluN1, generate excitatory conductances purely activated by glycine. However, functional GluN1/GluN3 receptors have not been identified in native adult tissues. We discovered that GluN1/GluN3A receptors are operational in neurons of the mouse adult medial habenula (MHb), an epithalamic area controlling aversive physiological states. In the absence of glycinergic neuronal specializations in the MHb, glial cells tuned neuronal activity via GluN1/GluN3A receptors. Reducing GluN1/GluN3A receptor levels in the MHb prevented place-aversion conditioning. Our study extends the physiological and behavioral implications of glycine by demonstrating its control of negatively valued emotional associations via excitatory glycinergic NMDA receptors.
        Mulligan MK, Abreo T, Neuner SM, Parks C, Watkins CE, Houseal MT, Shapaker TM, Hook M, Tan H, Wang X, et al. Identification of a functional non-coding variant in the GABA (A) receptor α2 subunit of the C57BL/6J mouse reference genome: major implications for neuroscience research. Frontiers in Genetics [Internet]. 2019. Publisher's VersionAbstract
        GABA type-A (GABA-A) receptors containing the α2 subunit (GABRA2) are expressed in most brain regions and are critical in modulating inhibitory synaptic function. Genetic variation at the GABRA2 locus has been implicated in epilepsy, affective and psychiatric disorders, alcoholism and drug abuse. Gabra2 expression varies as a function of genotype and is modulated by sequence variants in several brain structures and populations, including F2 crosses originating from C57BL/6J (B6J) and the BXD recombinant inbred family derived from B6J and DBA/2J. Here we demonstrate a global reduction of GABRA2 brain protein and mRNA in the B6J strain relative to other inbred strains, and identify and validate the causal mutation in B6J. The mutation is a single base pair deletion located in an intron adjacent to a splice acceptor site that only occurs in the B6J reference genome. The deletion became fixed in B6J between 1976 and 1991 and is now pervasive in many engineered lines, BXD strains generated after 1991, the Collaborative Cross, and the majority of consomic lines. Repair of the deletion using CRISPR-Cas9-mediated gene editing on a B6J genetic background completely restored brain levels of GABRA2 protein and mRNA. Comparison of transcript expression in hippocampus, cortex, and striatum between B6J and repaired genotypes revealed alterations in GABA-A receptor subunit expression, especially in striatum. These results suggest that naturally occurring variation in GABRA2 levels between B6J and other substrains or inbred strains may also explain strain differences in anxiety-like or alcohol and drug response traits related to striatal function. Characterization of the B6J private mutation in the Gabra2 gene is of critical importance to molecular genetic studies in neurobiological research because this strain is widely used to generate genetically engineered mice and murine genetic populations, and is the most widely utilized strain for evaluation of anxiety-like, depression-like, pain, epilepsy, and drug response traits that may be partly modulated by GABRA2 function.
        Most D, Salem NA, Tiwari GR, Blednov YA, RD M, RA H. Silencing synaptic MicroRNA‐411 reduces voluntary alcohol consumption in mice. Addiction Biology [Internet]. 2019;24 (4) :604-616. Publisher's VersionAbstract
        Chronic alcohol consumption alters the levels of microRNAs and mRNAs in the brain, but the specific microRNAs and processes that target mRNAs to affect cellular function and behavior are not known. We examined the in vivo manipulation of previously identified alcohol‐responsive microRNAs as potential targets to reduce alcohol consumption. Silencing of miR‐411 by infusing antagomiR‐411 into the prefrontal cortex of female C57BL/6J mice reduced alcohol consumption and preference, without altering total fluid consumption, saccharin consumption, or anxiety‐related behaviors. AntagomiR‐411 reduced alcohol consumption when given to mice exposed to a chronic alcohol drinking paradigm but did not affect the acquisition of consumption in mice without a history of alcohol exposure, suggesting that antagomiR‐411 has a neuroadaptive, alcohol‐dependent effect. AntagomiR‐411 decreased the levels of miR‐411, as well as the association of immunoprecipitated miR‐411 with Argonaute2; and, it increased levels of Faah and Ppard mRNAs. Moreover, antagomiR‐411 increased the neuronal expression of glutamate receptor AMPA‐2 protein, a known alcohol target and a predicted target of miR‐411. These results suggest that alcohol and miR‐411 function in a homeostatic manner to regulate synaptic mRNA and protein, thus reversing alcohol‐related neuroadaptations and reducing chronic alcohol consumption.
        Mittal N, Thakore N, Bell RL, Maddox WT, Schallert T, CL D. Sex-specific ultrasonic vocalization patterns and alcohol consumption in high alcohol-drinking (HAD-1) rats. Physiology and Behavior [Internet]. 2019;203 :81-90. Publisher's VersionAbstract

        Ultrasonic vocalizations (USVs) have been established as an animal model of emotional status and are often utilized in drug abuse studies as motivational and emotional indices. Further USV functionality has been demonstrated in our recent work showing accurate identification of selectively-bred high versus low alcohol-consuming male rats ascertained exclusively from 22 to 28 kHz and 50–55 kHz FM USV acoustic parameters. With the hypothesis that alcohol-sensitive sex differences could be revealed through USV acoustic parameters, the present study examined USVs and alcohol consumption in male and female selectively bred high-alcohol drinking (HAD-1) rats. For the current study, we examined USV data collected during a 12-week experiment in male and female HAD-1 rats. Experimental phases included Baseline (2 weeks), 4-h EtOH Access (4 weeks), 24-h EtOH Access (4 weeks) and Abstinence (2 weeks). Findings showed that both male and female HAD-1 rats spontaneously emitted a large number of 22–28 kHz and 50–55 kHz FM USVs and that females drank significantly more alcohol compared to males over the entire course of the experiment. Analyses of USV acoustic characteristics (i.e. mean frequency, duration, bandwidth and power) revealed distinct sex-specific phenotypes in both 50–55 kHz FM and 22–28 kHz USV transmission that were modulated by ethanol exposure. Moreover, by using a linear combination of these acoustic characteristics, we were able to develop binomial logistic regression models able to discriminate between male and female HAD-1 rats with high accuracy. Together these results highlight unique emotional phenotypes in male and female HAD-1 rats that are differentially modulated by alcohol experience.

          Mittal N, Fleming SM, Martinez A, Thakore N, Bell RL, Maddox WT, Schallert T, CL D. Sex differences in cognitive performance and alcohol consumption in High Alcohol-Drinking (HAD-1) rats. Behavioural Brain Research [Internet]. 2019;381. Publisher's VersionAbstract
          Excessive alcohol (ethanol) consumption negatively impacts social, emotional, as well as cognitive function and well-being. Thus, identifying behavioral and/or biological predictors of excessive ethanol consumption is important for developing prevention and treatment strategies against alcohol use disorders (AUDs). Sex differences in alcohol consumption patterns are observed in humans, primates, and rodents. Selectively bred high alcohol-drinking rat lines, such as the “HAD-1” lines are recognized animal models of alcoholism. The present work examined sex differences in alcohol consumption, object recognition, and exploratory behavior in male and female HAD-1 rats. Naïve male and female HAD-1 rats were tested in an object recognition test (ORT) prior to a chronic 24 h intermittent ethanol access procedure for five weeks. Object recognition parameters measured included exploratory behavior, object investigation, and time spent near objects. During the initial training trial, rearing, active object investigation and amount of time spent in the object-containing section was significantly greater in female HAD-1 rats compared to their male counterparts. During the subsequent testing trial, time spent in the object-containing section was greater in female, compared to male, rats; but active object investigation and rearing did not statistically differ between females and males. In addition, female HAD-1 rats consumed significantly more ethanol than their male counterparts, replicating previous findings. Moreover, across all animals there was a significant positive correlation between exploratory behavior in ORT and ethanol consumption level. These results indicate there are significant sex differences in cognitive performance and alcohol consumption in HAD-1 rats, which suggests neurobiological differences as well.
          Meirsman AC, Ben Hamida S, Clarke E, de Kerchove d'Exaerde A, Darcq E, Kieffer BL. GPR88 in D1R- and D2R-Type Medium Spiny Neurons Differentially Regulates Affective and Motor Behaviors. eNeuro [Internet]. 2019;6 (4). Publisher's VersionAbstract

          The orphan receptor GPR88 is highly expressed in D1R- and D2R-medium spiny neurons (MSNs) and has been associated to striatum-dependent functions in rodents. The total deletion of Gpr88 in mice was shown to decrease anxiety-like behaviors, increase stereotypies and locomotion, and impair motor coordination and motor learning. Knowing the opposing role of D1R- and D2R-MSNs, we here investigated the respective roles of GPR88 in the two MSN subtypes for these behaviors. To do so, we compared effects of a conditional Gpr88 gene knockout (KO) in D1R-MSNs (D1R-Gpr88 mice) or D2R-MSNs (A2AR-Gpr88 mice) with effects of the total Gpr88 KO (CMV-Gpr88 mice). Overall, most phenotypes of CMV-Gpr88 mice were recapitulated in A2AR-Gpr88 mice, including reduced marble burying, increased social interactions, increased locomotor activity and stereotypies in the open field, and reduced motor coordination in the rotarod. Exceptions were the reduced habituation to the open field and reduced motor skill learning, which were observed in CMV-Gpr88 and D1R-Gpr88 mice, but not in A2AR-Gpr88 mice. D1R-Gpr88 mice otherwise showed no other phenotype in this study. Our data together show that GPR88 modulates the function of both D1R- and D2R-MSNs, and that GPR88 activity in these two neuron populations has very different and dissociable impacts on behavior. We suggest that GPR88 in D2R-MSNs shapes defensive and social behavior and contributes in maintaining the inhibition of basal ganglia outputs to control locomotion, stereotypies and motor coordination, while GPR88 in D1R-MSNs promotes novelty habituation and motor learning.

          Significance Statement GPR88, an orphan G-protein-coupled receptor, has been implicated in the regulation of striatum-dependent behaviors. In the striatum, GPR88 is most abundant in both medium spiny neurons expressing dopamine D1 and D2 receptors. We compared effects of a conditional Gpr88 gene knockout in D1R-MSNs or D2R-MSNs with effects of the total Gpr88 deletion. Our data suggest that GPR88 in D2R-MSNs shapes defensive and social behavior and contributes in maintaining the inhibition of basal ganglia outputs to control locomotion, stereotypies and motor coordination, while GPR88 in D1R-MSNs promotes novelty habituation and motor learning. Gpr88 therefore plays very distinct roles in modulating D1R- and D2R-type neurons function and the related behaviors.

          Kircher DM, Aziz HC, RA M, RA M. Ethanol Experience Enhances Glutamatergic Ventral Hippocampal Inputs to D1 Receptor-Expressing Medium Spiny Neurons in the Nucleus Accumbens Shell. Journal of Neuroscience [Internet]. 2019;39 (13) :2459-2469. Publisher's VersionAbstract
          A growing number of studies implicate alterations in glutamatergic signaling within the reward circuitry of the brain during alcohol abuse and dependence. A key integrator of glutamatergic signaling in the reward circuit is the nucleus accumbens, more specifically, the dopamine D1 receptor-expressing medium spiny neurons (D1-MSNs) within this region, which have been implicated in the formation of dependence to many drugs of abuse including alcohol. D1-MSNs receive glutamatergic input from several brain regions; however, it is not currently known how individual inputs onto D1-MSNs are altered by alcohol experience. Here, we investigate input-specific adaptations in glutamatergic transmission in response to varying levels of alcohol experience. Virally mediated expression of Channelrhodopsin in ventral hippocampal (vHipp) glutamate neurons of male mice allowed for selective activation of vHipp to D1-MSN synapses. Therefore, we were able to compare synaptic adaptations in response to low and high alcohol experience in vitro and in vivo. Alcohol experience enhanced glutamatergic activity and abolished LTD at vHipp to D1-MSN synapses. Following chronic alcohol experience, GluA2-lacking AMPARs, which are Ca permeable, were inserted into vHipp to D1-MSN synapses. These findings support the reversal of alcohol-induced insertion of Ca-permeable AMPARs and the enhancement of glutamatergic activity at vHipp to D1-MSNs as potential targets for intervention during early exposure to alcohol.