Publications

2016
Zahr NM, Rohlfing T, Mayer D, Luong R, Sullivan EV, Pfefferbaum A. Transient CNS responses to repeated binge ethanol treatment. Addiction Biology. 2016;21 (6) :1199–1216.Abstract
The effects of ethanol (EtOH) on in vivo magnetic resonance (MR)-detectable brain measures across repeated exposures have not previously been reported. Of 28 rats weighing 340.66 ± 21.93 g at baseline, 15 were assigned to an EtOH group and 13 to a control group. Animals were exposed to five cycles of 4 days of intragastric (EtOH or dextrose) treatment and 10 days of recovery. Rats in both groups had structural MR imaging and whole-brain MR spectroscopy (MRS) scans at baseline, immediately following each binge period and after each recovery period (total = 11 scans per rat). Blood alcohol level at each of the five binge periods was \textasciitilde300 mg/dl. Blood drawn at the end of the experiment did not show group differences for thiamine or its phosphate derivatives. Postmortem liver histopathology provided no evidence for hepatic steatosis, alcoholic hepatitis or alcoholic cirrhosis. Cerebrospinal fluid volumes of the lateral ventricles and cisterns showed enlargement with each binge EtOH exposure but recovery with each abstinence period. Similarly, changes in MRS metabolite levels were transient: levels of N-acetylaspartate and total creatine decreased, while those of choline-containing compounds and the combined resonance from glutamate and glutamine increased with each binge EtOH exposure cycle and then recovered during each abstinence period. Changes in response to EtOH were in expected directions based on previous single-binge EtOH exposure experiments, but the current MR findings do not provide support for accruing changes with repeated binge EtOH exposure.
Thakore N, Reno JM, Gonzales RA, Schallert T, Bell RL, Maddox WT, Duvauchelle CL. Alcohol enhances unprovoked 22-28 kHz USVs and suppresses USV mean frequency in High Alcohol Drinking (HAD-1) male rats. Behavioural Brain Research. 2016;302 :228–236.Abstract
Heightened emotional states increase impulsive behaviors such as excessive ethanol consumption in humans. Though positive and negative affective states in rodents can be monitored in real-time through ultrasonic vocalization (USV) emissions, few animal studies have focused on the role of emotional status as a stimulus for initial ethanol drinking. Our laboratory has recently developed reliable, high-speed analysis techniques to compile USV data during multiple-hour drinking sessions. Since High Alcohol Drinking (HAD-1) rats are selectively bred to voluntarily consume intoxicating levels of alcohol, we hypothesized that USVs emitted by HAD-1 rats would reveal unique emotional phenotypes predictive of alcohol intake and sensitive to alcohol experience. In this study, male HAD-1 rats had access to water, 15% and 30% EtOH or water only (i.e., Controls) during 8 weeks of daily 7-h drinking-in-the-dark (DID) sessions. USVs, associated with both positive (i.e., 50-55 kHz frequency-modulated or FM) and negative (i.e., 22-28 kHz) emotional states, emitted during these daily DID sessions were examined. Findings showed basal 22-28 kHz USVs were emitted by both EtOH-Naïve (Control) and EtOH-experienced rats, alcohol experience enhanced 22-28 kHz USV emissions, and USV acoustic parameters (i.e., mean frequency in kHz) of both positive and negative USVs were significantly suppressed by chronic alcohol experience. These data suggest that negative affective status initiates and maintains excessive alcohol intake in selectively bred HAD-1 rats and support the notion that unprovoked emissions of negative affect-associated USVs (i.e., 22-28 kHz) predict vulnerability to excessive alcohol intake in distinct rodent models.
Varodayan FP, Soni N, Bajo M, Luu G, Madamba SG, Schweitzer P, Parsons LH, Roberto M. Chronic ethanol exposure decreases CB1 receptor function at GABAergic synapses in the rat central amygdala. Addiction Biology. 2016;21 (4) :788–801.Abstract
The endogenous cannabinoids (eCBs) influence the acute response to ethanol and the development of tolerance, dependence and relapse. Chronic alcohol exposure alters eCB levels and Type 1 cannabinoid receptor (CB1 ) expression and function in brain regions associated with addiction. CB1 inhibits GABA release, and GABAergic dysregulation in the central nucleus of the amygdala (CeA) is critical in the transition to alcohol dependence. We investigated possible disruptions in CB1 signaling of rat CeA GABAergic transmission following intermittent ethanol exposure. In the CeA of alcohol-naive rats, CB1 agonist WIN 55,212-2 (WIN) decreased the frequency of spontaneous and miniature GABAA receptor-mediated inhibitory postsynaptic currents (s/mIPSCs). This effect was prevented by CB1 antagonism, but not Type 2 cannabinoid receptor (CB2 ) antagonism. After 2-3 weeks of intermittent ethanol exposure, these WIN inhibitory effects were attenuated, suggesting ethanol-induced impairments in CB1 function. The CB1 antagonist AM251 revealed a tonic eCB/CB1 control of GABAergic transmission in the alcohol-naive CeA that was occluded by calcium chelation in the postsynaptic cell. Chronic ethanol exposure abolished this tonic CB1 influence on mIPSC, but not sIPSC, frequency. Finally, acute ethanol increased CeA GABA release in both naive and ethanol-exposed rats. Although CB1 activation prevented this effect, the AM251- and ethanol-induced GABA release were additive, ruling out a direct participation of CB1 signaling in the ethanol effect. Collectively, these observations demonstrate an important CB1 influence on CeA GABAergic transmission and indicate that the CeA is particularly sensitive to alcohol-induced disruptions of CB1 signaling.
Schweitzer P, Cates-Gatto C, Varodayan FP, Nadav T, Roberto M, Lasek AW, Roberts AJ. Dependence-induced ethanol drinking and GABA neurotransmission are altered in Alk deficient mice. Neuropharmacology. 2016;107 :1–8.Abstract
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that is expressed in the brain and implicated in alcohol abuse in humans and behavioral responses to ethanol in mice. Previous studies have shown an association of human ALK with acute responses to alcohol and alcohol dependence. In addition, Alk knockout (Alk -/-) mice consume more ethanol in a binge-drinking test and show increased sensitivity to ethanol sedation. However, the function of ALK in excessive drinking following the establishment of ethanol dependence has not been examined. In this study, we tested Alk -/- mice for dependence-induced drinking using the chronic intermittent ethanol-two bottle choice drinking (CIE-2BC) protocol. We found that Alk -/- mice initially consume more ethanol prior to CIE exposure, but do not escalate ethanol consumption after exposure, suggesting that ALK may promote the escalation of drinking after ethanol dependence. To determine the mechanism(s) responsible for this behavioral phenotype we used an electrophysiological approach to examine GABA neurotransmission in the central nucleus of the amygdala (CeA), a brain region that regulates alcohol consumption and shows increased GABA signaling after chronic ethanol exposure. GABA transmission in ethanol-naïve Alk -/- mice was enhanced at baseline and potentiated in response to acute ethanol application when compared to wild-type (Alk +/+) mice. Moreover, basal GABA transmission was not elevated by CIE exposure in Alk -/- mice as it was in Alk +/+ mice. These data suggest that ALK plays a role in dependence-induced drinking and the regulation of presynaptic GABA release in the CeA.
Sari Y, Toalston JE, Rao PSS, Bell RL. Effects of ceftriaxone on ethanol, nicotine or sucrose intake by alcohol-preferring (P) rats and its association with GLT-1 expression. Neuroscience. 2016;326 :117–125.Abstract
Increased glutamatergic neurotransmission appears to mediate the reinforcing properties of drugs of abuse, including ethanol (EtOH). We have shown that administration of ceftriaxone (CEF), a β-lactam antibiotic, reduced EtOH intake and increased glutamate transporter 1 (GLT-1) expression in mesocorticolimbic regions of male and female alcohol-preferring (P) rats. In the present study, we tested whether CEF administration would reduce nicotine (NIC) and/or EtOH intake by adult female P rats. P rats were randomly assigned to 4 groups: (a) 5% sucrose (SUC) or 10% SUC [SUC], (b) 5% SUC+0.07mg/ml NIC and 10% SUC+0.14mg/ml NIC [NIC-SUC], 15% EtOH and 30% EtOH [EtOH] and (d) 15% EtOH+0.07mg/ml NIC and 30% EtOH+0.14mg/ml NIC [NIC-EtOH]. After achieving stable intakes (4weeks), the rats were administered 7 consecutive, daily i.p. injections of either saline or 200mg/kg CEF. The effects of CEF on intake were significant but differed across the reinforcers; such that ml/kg/day SUC was reduced by ∼30%, mg/kg/day NIC was reduced by ∼70% in the NIC-SUC group and ∼40% in the EtOH-NIC group, whereas g/kg/day EtOH was reduced by ∼40% in both the EtOH and EtOH-NIC group. The effects of CEF on GLT-1 expression were also studied. We found that CEF significantly increased GLT-1 expression in the prefrontal cortex and the nucleus accumbens of the NIC and NIC-EtOH rats as compared to NIC and NIC-EtOH saline-treated rats. These findings provide further support for GLT-1-associated mechanisms in EtOH and/or NIC abuse. The present results along with previous reports of CEF's efficacy in reducing cocaine self-administration in rats suggest that modulation of GLT-1 expression and/or activity is an important pharmacological target for treating polysubstance abuse and dependence.
Smith ML, Li J, Cote DM, Ryabinin AE. Effects of isoflurane and ethanol administration on c-Fos immunoreactivity in mice. Neuroscience. 2016;316 :337–343.Abstract
Noninvasive functional imaging holds great promise for the future of translational research, due to the ability to directly compare between preclinical and clinical models of psychiatric disorders. Despite this potential, concerns have been raised regarding the necessity to anesthetize rodent and monkey subjects during these procedures, because anesthetics may alter neuronal activity. For example, in studies on drugs of abuse and alcohol, it is not clear to what extent anesthesia can interfere with drug-induced neural activity. Therefore, the current study investigated whole-brain c-Fos activation following isoflurane anesthesia as well as ethanol-induced activation of c-Fos in anesthetized mice. In the first experiment, we examined effects of one or three sessions of gaseous isoflurane on c-Fos activation across the brain in male C57BL/6J mice. Isoflurane administration led to c-Fos activation in several areas, including the piriform cortex and lateral septum. Lower or similar levels of activation in these areas were detected after three sessions of isoflurane, suggesting that multiple exposures may eliminate some of the enhanced neuronal activation caused by acute isoflurane. In the second experiment, we investigated the ability of ethanol injection (1.5 or 2.5g/kgi.p.) to induce c-Fos activation under anesthesia. Following three sessions of isoflurane, 1.5g/kg of ethanol induced c-Fos in the central nucleus of amygdala and the centrally-projecting Edinger-Westphal nucleus (EWcp). This induction was lower after 2.5g/kg of ethanol. These results demonstrate that ethanol-induced neural activation can be detected in the presence of isoflurane anesthesia. They also suggest, that while habituation to isoflurane helps reduce neuronal activation, interaction between effects of anesthesia and alcohol can occur. Studies using fMRI imaging could benefit from using habituated animals and dose-response analyses.
Truitt JM, Blednov YA, Benavidez JM, Black M, Ponomareva O, Law J, Merriman M, Horani S, Jameson K, Lasek AW, et al. Inhibition of IKKβ Reduces Ethanol Consumption in C57BL/6J Mice. eNeuro. 2016;3 (5).Abstract
Proinflammatory pathways in neuronal and non-neuronal cells are implicated in the acute and chronic effects of alcohol exposure in animal models and humans. The nuclear factor-κB (NF-κB) family of DNA transcription factors plays important roles in inflammatory diseases. The kinase IKKβ mediates the phosphorylation and subsequent proteasomal degradation of cytosolic protein inhibitors of NF-κB, leading to activation of NF-κB. The role of IKKβ as a potential regulator of excessive alcohol drinking had not previously been investigated. Based on previous findings that the overactivation of innate immune/inflammatory signaling promotes ethanol consumption, we hypothesized that inhibiting IKKβ would limit/decrease drinking by preventing the activation of NF-κB. We studied the systemic effects of two pharmacological inhibitors of IKKβ, TPCA-1 and sulfasalazine, on ethanol intake using continuous- and limited-access, two-bottle choice drinking tests in C57BL/6J mice. In both tests, TPCA-1 and sulfasalazine reduced ethanol intake and preference without changing total fluid intake or sweet taste preference. A virus expressing Cre recombinase was injected into the nucleus accumbens and central amygdala to selectively knock down IKKβ in mice genetically engineered with a conditional Ikkb deletion (Ikkb(F/F) ). Although IKKβ was inhibited to some extent in astrocytes and microglia, neurons were a primary cellular target. Deletion of IKKβ in either brain region reduced ethanol intake and preference in the continuous access two-bottle choice test without altering the preference for sucrose. Pharmacological and genetic inhibition of IKKβ decreased voluntary ethanol consumption, providing initial support for IKKβ as a potential therapeutic target for alcohol abuse.
Warden A, Erickson E, Robinson G, Harris RA, Mayfield RD. The neuroimmune transcriptome and alcohol dependence: potential for targeted therapies. Pharmacogenomics. 2016;17 (18) :2081–2096.Abstract
Transcriptome profiling enables discovery of gene networks that are altered in alcoholic brains. This technique has revealed involvement of the brain's neuroimmune system in regulating alcohol abuse and dependence, and has provided potential therapeutic targets. In this review, we discuss Toll-like-receptor pathways, hypothesized to be key players in many stages of the alcohol addiction cycle. The growing appreciation of the neuroimmune system's involvement in alcoholism has also led to consideration of crucial roles for glial cells, including astrocytes and microglia, in the brain's response to alcohol abuse. We discuss current knowledge and hypotheses on the roles that specific neuroimmune cell types may play in addiction. Current strategies for repurposing US FDA-approved drugs for the treatment of alcohol use disorders are also discussed.
Ryabinin AE, Hostetler CM. Prairie Voles as a Model to Screen Medications for the Treatment of Alcoholism and Addictions. International Review of Neurobiology. 2016;126 :403–421.Abstract
Most preclinical studies of medications to treat addictions are performed in mice and rats. These two rodent species belong to one phylogenetic subfamily, which narrows the likelihood of identifying potential mechanisms regulating addictions in other species, ie, humans. Expanding the genetic diversity of organisms modeling alcohol and drug abuse enhances our ability to screen for medications to treat addiction. Recently, research laboratories adapted the prairie vole model to study mechanisms of alcohol and drugs of abuse. This development not only expanded the diversity of genotypes used to screen medications, but also enhanced capabilities of such screens. Prairie voles belong to 3-5% of mammalian species exhibiting social monogamy. This unusual trait is reflected in their ability to form lasting long-term affiliations between adult individuals. The prairie vole animal model has high predictive validity for mechanisms regulating human social behaviors. In addition, these animals exhibit high alcohol intake and preference. In laboratory settings, prairie voles are used to model social influences on drug reward and alcohol consumption as well as effects of addictive substances on social bonding. As a result, this species can be adapted to screen medications whose effectiveness could be (a) resistant to social influences promoting excessive drug taking, (b) dependent on the presence of social support, and (c) medications affecting harmful social consequences of alcohol and drug abuse. This report reviews the literature on studies of alcohol and psychostimulants in prairie voles and discusses capabilities of this animal model as a screen for novel medications to treat alcoholism and addictions.
Renteria R, Jeanes ZM, Mangieri RA, Maier EY, Kircher DM, Buske TR, Morrisett RA. Using In Vitro Electrophysiology to Screen Medications: Accumbal Plasticity as an Engram of Alcohol Dependence. International Review of Neurobiology. 2016;126 :441–465.Abstract
The nucleus accumbens (NAc) is a central component of the mesocorticolimbic reward system. Increasing evidence strongly implicates long-term synaptic neuroadaptations in glutamatergic excitatory activity of the NAc shell and/or core medium spiny neurons in response to chronic drug and alcohol exposure. Such neuroadaptations likely play a critical role in the development and expression of drug-seeking behaviors. We have observed unique cell-type-specific bidirectional changes in NAc synaptic plasticity (metaplasticity) following acute and chronic intermittent ethanol exposure. Other investigators have also previously observed similar metaplasticity in the NAc following exposure to psychostimulants, opiates, and amazingly, even following an anhedonia-inducing experience. Considering that the proteome of the postsynaptic density likely contains hundreds of biochemicals, proteins and other components and regulators, we believe that there is a large number of potential molecular sites through which accumbal metaplasticity may be involved in chronic alcohol abuse. Many of our companion laboratories are now engaged in identifying and screening medications targeting candidate genes and its products previously linked to maladaptive alcohol phenotypes. We hypothesize that if manipulation of such target genes and their products change NAc plasticity, then that observation constitutes an important validation step for the development of novel therapeutics to treat alcohol dependence.
Marballi K, Genabai NK, Blednov YA, Harris RA, Ponomarev I. Alcohol consumption induces global gene expression changes in VTA dopaminergic neurons. Genes, Brain, and Behavior. 2016;15 (3) :318–326.Abstract
Alcoholism is associated with dysregulation in the neural circuitry that mediates motivated and goal-directed behaviors. The dopaminergic (DA) connection between the ventral tegmental area (VTA) and the nucleus accumbens is viewed as a critical component of the neurocircuitry mediating alcohol's rewarding and behavioral effects. We sought to determine the effects of binge alcohol drinking on global gene expression in VTA DA neurons. Alcohol-preferring C57BL/6J × FVB/NJ F1 hybrid female mice were exposed to a modified drinking in the dark (DID) procedure for 3 weeks, while control animals had access to water only. Global gene expression of laser-captured tyrosine hydroxylase (TH)-positive VTA DA neurons was measured using microarrays. A total of 644 transcripts were differentially expressed between the drinking and nondrinking mice, and 930 transcripts correlated with alcohol intake during the last 2 days of drinking in the alcohol group. Bioinformatics analysis of alcohol-responsive genes identified molecular pathways and networks perturbed in DA neurons by alcohol consumption, which included neuroimmune and epigenetic functions, alcohol metabolism and brain disorders. The majority of genes with high and specific expression in DA neurons were downregulated by or negatively correlated with alcohol consumption, suggesting a decreased activity of DA neurons in high drinking animals. These changes in the DA transcriptome provide a foundation for alcohol-induced neuroadaptations that may play a crucial role in the transition to addiction.
Mechling AE, Arefin T, Lee H-L, Bienert T, Reisert M, Ben Hamida S, Darcq E, Ehrlich A, Gaveriaux-Ruff C, Parent MJ, et al. Deletion of the mu opioid receptor gene in mice reshapes the reward-aversion connectome. Proceedings of the National Academy of Sciences of the United States of America. 2016;113 (41) :11603–11608.Abstract
Connectome genetics seeks to uncover how genetic factors shape brain functional connectivity; however, the causal impact of a single gene's activity on whole-brain networks remains unknown. We tested whether the sole targeted deletion of the mu opioid receptor gene (Oprm1) alters the brain connectome in living mice. Hypothesis-free analysis of combined resting-state fMRI diffusion tractography showed pronounced modifications of functional connectivity with only minor changes in structural pathways. Fine-grained resting-state fMRI mapping, graph theory, and intergroup comparison revealed Oprm1-specific hubs and captured a unique Oprm1 gene-to-network signature. Strongest perturbations occurred in connectional patterns of pain/aversion-related nodes, including the mu receptor-enriched habenula node. Our data demonstrate that the main receptor for morphine predominantly shapes the so-called reward/aversion circuitry, with major influence on negative affect centers.
Lasek AW. Effects of Ethanol on Brain Extracellular Matrix: Implications for Alcohol Use Disorder. Alcoholism, Clinical and Experimental Research. 2016;40 (10) :2030–2042.Abstract
The brain extracellular matrix (ECM) occupies the space between cells and is involved in cell-matrix and cell-cell adhesion. However, in addition to providing structural support to brain tissue, the ECM activates cell signaling and controls synaptic transmission. The expression and activity of brain ECM components are regulated by alcohol exposure. This review will discuss what is currently known about the effects of alcohol on the activity and expression of brain ECM components. An interpretation of how these changes might promote alcohol use disorder (AUD) will be also provided. Ethanol (EtOH) exposure decreases levels of structural proteins involved in the interstitial matrix and basement membrane, with a concomitant increase in proteolytic enzymes that degrade these components. In contrast, EtOH exposure generally increases perineuronal net components. Because the ECM has been shown to regulate both synaptic plasticity and behavioral responses to drugs of abuse, regulation of the brain ECM by alcohol may be relevant to the development of alcoholism. Although investigation of the function of brain ECM in alcohol abuse is still in early stages, a greater understanding of the interplay between ECM and alcohol might lead to novel therapeutic strategies for treating AUD.
McClintick JN, McBride WJ, Bell RL, Ding Z-M, Liu Y, Xuei X, Edenberg HJ. Gene Expression Changes in Glutamate and GABA-A Receptors, Neuropeptides, Ion Channels, and Cholesterol Synthesis in the Periaqueductal Gray Following Binge-Like Alcohol Drinking by Adolescent Alcohol-Preferring (P) Rats. Alcoholism, Clinical and Experimental Research. 2016;40 (5) :955–968.Abstract
BACKGROUND: Binge drinking of alcohol during adolescence is a serious public health concern with long-term consequences, including increased pain, fear, and anxiety. The periaqueductal gray (PAG) is involved in processing pain, fear, and anxiety. The effects of adolescent binge drinking on gene expression in this region have yet to be studied. METHODS: Male adolescent alcohol-preferring (P) rats were exposed to repeated binge drinking (three 1-hour sessions/d during the dark/cycle, 5 days/wk for 3 weeks starting at 28 days of age; ethanol intakes of 2.5 to 3 g/kg/session). We used RNA sequencing to assess the effects of ethanol intake on gene expression. RESULTS: Ethanol significantly altered the expression of 1,670 of the 12,123 detected genes: 877 (53%) decreased. In the glutamate system, 23 genes were found to be altered, including reduction in 7 of 10 genes for metabotropic and NMDA receptors. Subunit changes in the NMDA receptor may make it less sensitive to ethanol. Changes in GABAA genes would most likely increase the ability of the PAG to produce tonic inhibition. Five serotonin receptor genes, 6 acetylcholine receptor genes, and 4 glycine receptor genes showed decreased expression in the alcohol-drinking rats. Opioid genes (e.g., Oprk1, Oprm1) and genes for neuropeptides linked to anxiety and panic behaviors (e.g., Npy1r) had mostly decreased expression. Genes for 27 potassium, 10 sodium, and 5 calcium ion channels were found to be differentially expressed. Nine genes in the cholesterol synthesis pathway had decreased expression, including Hmgcr, encoding the rate-limiting enzyme. Genes involved in the production of myelin also had decreased expression. CONCLUSIONS: The results demonstrate that binge alcohol drinking during adolescence produces developmental changes in the expression of key genes within the PAG; many of these changes point to increased susceptibility to pain, fear, and anxiety, which could contribute to excessive drinking to relieve these negative effects.
Mayfield J, Arends MA, Harris RA, Blednov YA. Genes and Alcohol Consumption: Studies with Mutant Mice. International Review of Neurobiology. 2016;126 :293–355.Abstract
In this chapter, we review the effects of global null mutant and overexpressing transgenic mouse lines on voluntary self-administration of alcohol. We examine approximately 200 publications pertaining to the effects of 155 mouse genes on alcohol consumption in different drinking models. The targeted genes vary in function and include neurotransmitter, ion channel, neuroimmune, and neuropeptide signaling systems. The alcohol self-administration models include operant conditioning, two- and four-bottle choice continuous and intermittent access, drinking in the dark limited access, chronic intermittent ethanol, and scheduled high alcohol consumption tests. Comparisons of different drinking models using the same mutant mice are potentially the most informative, and we will highlight those examples. More mutants have been tested for continuous two-bottle choice consumption than any other test; of the 137 mouse genes examined using this model, 97 (72%) altered drinking in at least one sex. Overall, the effects of genetic manipulations on alcohol drinking often depend on the sex of the mice, alcohol concentration and time of access, genetic background, as well as the drinking test.
Meirsman AC, Robé A, de Kerchove d'Exaerde A, Kieffer BL. GPR88 in A2AR Neurons Enhances Anxiety-Like Behaviors. eNeuro. 2016;3 (4).Abstract
GPR88 is an orphan G-protein-coupled receptor highly expressed in striatal dopamine D1 (receptor) R- and D2R-expressing medium spiny neurons. This receptor is involved in activity and motor responses, and we previously showed that this receptor also regulates anxiety-like behaviors. To determine whether GPR88 in D2R-expressing neurons contributes to this emotional phenotype, we generated conditional Gpr88 knock-out mice using adenosine A2AR (A2AR)-Cre-driven recombination, and compared anxiety-related responses in both total and A2AR-Gpr88 KO mice. A2AR-Gpr88 KO mice showed a selective reduction of Gpr88 mRNA in D2R-expressing, but not D1R-expressing, neurons. These mutant mice showed increased locomotor activity and decreased anxiety-like behaviors in light/dark and elevated plus maze tests. These phenotypes were superimposable on those observed in total Gpr88 KO mice, demonstrating that the previously reported anxiogenic activity of GPR88 operates at the level of A2AR-expressing neurons. Further, A2AR-Gpr88 KO mice showed no change in novelty preference and novelty-suppressed feeding, while these responses were increased and decreased, respectively, in the total Gpr88 KO mice. Also, A2AR-Gpr88 KO mice showed intact fear conditioning, while the fear responses were decreased in total Gpr88 KO. We therefore also show for the first time that GPR88 activity regulates approach behaviors and conditional fear; however, these behaviors do not seem mediated by receptors in A2AR neurons. We conclude that Gpr88 expressed in A2AR neurons enhances ethological anxiety-like behaviors without affecting conflict anxiety and fear responses.
Meirsman AC, Le Merrer J, Pellissier LP, Diaz J, Clesse D, Kieffer BL, Becker JAJ. Mice Lacking GPR88 Show Motor Deficit, Improved Spatial Learning, and Low Anxiety Reversed by Delta Opioid Antagonist. Biological Psychiatry. 2016;79 (11) :917–927.Abstract
BACKGROUND: GPR88 is an orphan G protein coupled receptor highly enriched in the striatum, and previous studies have focused on GPR88 function in striatal physiology. The receptor is also expressed in other brain areas, and here we examined whether GPR88 function extends beyond striatal-mediated responses. METHODS: We created Gpr88 knockout mice and examined both striatal and extrastriatal regions at molecular and cellular levels. We also tested striatum-, hippocampus-, and amygdala-dependent behaviors in Gpr88(-/-) mice using extensive behavioral testing. RESULTS: We found increased G protein coupling for delta opioid receptor (DOR) and mu opioid, but not other Gi/o coupled receptors, in the striatum of Gpr88 knockout mice. We also found modifications in gene transcription, dopamine and serotonin contents, and dendritic morphology inside and outside the striatum. Behavioral testing confirmed striatal deficits (hyperactivity, stereotypies, motor impairment in rotarod). In addition, mutant mice performed better in spatial tasks dependent on hippocampus (Y-maze, novel object recognition, dual solution cross-maze) and also showed markedly reduced levels of anxiety (elevated plus maze, marble burying, novelty suppressed feeding). Strikingly, chronic blockade of DOR using naltrindole partially improved motor coordination and normalized spatial navigation and anxiety of Gpr88(-/-) mice. CONCLUSIONS: We demonstrate that GPR88 is implicated in a large repertoire of behavioral responses that engage motor activity, spatial learning, and emotional processing. Our data also reveal functional antagonism between GPR88 and DOR activities in vivo. The therapeutic potential of GPR88 therefore extends to cognitive and anxiety disorders, possibly in interaction with other receptor systems.
Qiu B, Bell RL, Cao Y, Zhang L, Stewart RB, Graves T, Lumeng L, Yong W, Liang T. Npy deletion in an alcohol non-preferring rat model elicits differential effects on alcohol consumption and body weight. Journal of Genetics and Genomics = Yi Chuan Xue Bao. 2016;43 (7) :421–430.Abstract
Neuropeptide Y (NPY) is widely expressed in the central nervous system and influences many physiological processes. It is located within the rat quantitative trait locus (QTL) for alcohol preference on chromosome 4. Alcohol-nonpreferring (NP) rats consume very little alcohol, but have significantly higher NPY expression in the brain than alcohol-preferring (P) rats. We capitalized on this phenotypic difference by creating an Npy knockout (KO) rat using the inbred NP background to evaluate NPY effects on alcohol consumption. Zinc finger nuclease (ZNF) technology was applied, resulting in a 26-bp deletion in the Npy gene. RT-PCR, Western blotting and immunohistochemistry confirmed the absence of Npy mRNA and protein in KO rats. Alcohol consumption was increased in Npy(+/-) but not Npy(-/-) rats, while Npy(-/-) rats displayed significantly lower body weight when compared to Npy(+/+) rats. In whole brain tissue, expression levels of Npy-related and other alcohol-associated genes, Npy1r, Npy2r, Npy5r, Agrp, Mc3r, Mc4r, Crh and Crh1r, were significantly greater in Npy(-/-) rats, whereas Pomc and Crhr2 expressions were highest in Npy(+/-) rats. These findings suggest that the NPY-system works in close coordination with the melanocortin (MC) and corticotropin-releasing hormone (CRH) systems to modulate alcohol intake and body weight.
Rahman S, Engleman EA, Bell RL. Recent Advances in Nicotinic Receptor Signaling in Alcohol Abuse and Alcoholism. Progress in Molecular Biology and Translational Science. 2016;137 :183–201.Abstract
Alcohol is the most commonly abused legal substance and alcoholism is a serious public health problem. It is a leading cause of preventable death in the world. The cellular and molecular mechanisms of alcohol reward and addiction are still not well understood. Emerging evidence indicates that unlike other drugs of abuse, such as nicotine, cocaine, or opioids, alcohol targets numerous channel proteins, receptor molecules, and signaling pathways in the brain. Previously, research has identified brain nicotinic acetylcholine receptors (nAChRs), a heterogeneous family of pentameric ligand-gated cation channels expressed in the mammalian brain, as critical molecular targets for alcohol abuse and dependence. Genetic variations encoding nAChR subunits have been shown to increase the vulnerability to develop alcohol dependence. Here, we review recent insights into the rewarding effects of alcohol, as they pertain to different nAChR subtypes, associated signaling molecules, and pathways that contribute to the molecular mechanisms of alcoholism and/or comorbid brain disorders. Understanding these cellular changes and molecular underpinnings may be useful for the advancement of brain nicotinic-cholinergic mechanisms, and will lead to a better translational and therapeutic outcome for alcoholism and/or comorbid conditions.
Herman MA, Roberto M. Cell-type-specific tonic GABA signaling in the rat central amygdala is selectively altered by acute and chronic ethanol. Addiction Biology. 2016;21 (1) :72–86.Abstract
The central nucleus of the amygdala (CeA) is an important site for the reinforcing effects of ethanol and has been implicated in the development of alcohol dependence. The CeA GABAA receptor system is particularly vulnerable to the effects of acute and chronic ethanol exposure. Previous work in the CeA focused on ethanol and phasic GABAA receptor signaling, but tonic GABAA receptor signaling in the rat CeA remains understudied. In the present study, we found that the CeA contains two types of tonic conductance that are expressed in a cell-type-specific manner. Low threshold bursting (LTB) and some regular spiking (RS) neurons have an ongoing tonic conductance that is mediated by the α1-GABAA receptor subunit and is insensitive to acute ethanol exposure. Late spiking (LS) and a separate population of RS neurons do not display a persistent tonic conductance but have the potential for tonic signaling that is mediated by the δ-GABAA receptor subunit and can be activated by increasing the ambient GABA concentration or by acute ethanol exposure. Acute ethanol exposure differentially alters the firing discharge of different CeA cell types. Chronic ethanol exposure produces a switch in tonic signaling such that the tonic conductance in LTB and some RS neurons is lost and an ongoing tonic conductance is present in LS and a separate population of RS neurons. Collectively, these data demonstrate cell-type-specific tonic signaling in the CeA and provide new insight into how acute and chronic ethanol exposure differentially alter specific aspects of inhibitory circuitry in the CeA.

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