Publications

2016
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.
Finegersh A, Homanics GE. Chromatin immunoprecipitation and gene expression analysis of neuronal subtypes after fluorescence activated cell sorting. Journal of Neuroscience Methods. 2016;263 :81–88.Abstract
BACKGROUND: With advances in cell capture, gene expression can now be studied in neuronal subtypes and single cells; however, studying epigenetic mechanisms that underlie these changes presents challenges. Moreover, chromatin immunoprecipitation (ChIP) protocols optimized for low cell number do not adequately address technical issues and cell loss while preparing tissue for fluorescence activated cell sorting (FACS). Developing a reliable FACS-ChIP protocol without the need for pooling tissue from multiple animals would enable study of epigenetic mechanisms in neuronal subtypes. METHODS: FACS was used to isolate dopamine 1 receptor (D1R) expressing cells from the nucleus accumbens (NAc) of a commercially available BAC transgenic mouse strain. D1R+ cells were used to study gene expression as well as histone modifications at gene promoters using a novel native ChIP protocol. RESULTS: Isolated cells had enrichment of the dopamine 1 receptor (D1R) mRNA and nearly undetectable levels of GFAP and D2R mRNA. ChIP analysis demonstrated the association of activating or repressive histone modifications with highly expressed or silent gene promoters, respectively. COMPARISON WITH EXISTING METHODS: The ChIP protocol developed in this paper enables characterization of histone modifications from ∼30,000 FAC-sorted neurons. CONCLUSIONS: We describe a one day FACS-ChIP protocol that can be applied to epigenetic studies of neuronal subtypes without pooling tissue.
Haass-Koffler CL, Henry AT, Melkus G, Simms JA, Naemmuddin M, Nielsen CK, Lasek AW, Magill M, Schwandt ML, Momenan R, et al. Defining the role of corticotropin releasing factor binding protein in alcohol consumption. Translational Psychiatry. 2016;6 (11) :e953.Abstract
The corticotropin releasing factor (CRF) exerts its effects by acting on its receptors and on the binding protein (CRFBP), and has been implicated in alcohol use disorder (AUD). Therefore, identification of the exact contribution of each protein that mediates CRF effects is necessary to design effective therapeutic strategies for AUD. A series of in vitro/in vivo experiments across different species were performed to define the biological discrete role of CRFBP in AUD. First, to establish the CRFBP role in receptor signaling, we developed a novel chimeric cell-based assay and showed that CFRBP full length can stably be expressed on the plasma membrane. We discovered that only CRFBP(10 kD) fragment is able to potentiate CRF-intracellular Ca(2+) release. We provide evidence that CRHBP gene loss increased ethanol consumption in mice. Then, we demonstrate that selective reduction of CRHBP expression in the center nucleus of the amygdala (CeA) decreases ethanol consumption in ethanol-dependent rats. CRFBP amygdalar downregulation, however, does not attenuate yohimbine-induced ethanol self-administration. This effect was associated with decreased hemodynamic brain activity in the CRFBP-downregulated CeA and increased hemodynamic activity in the caudate putamen during yohimbine administration. Finally, in alcohol-dependent patients, genetic variants related to the CRFBP(10 kD) fragment were associated with greater risk for alcoholism and anxiety, while other genetic variants were associated with reduced risk for anxiety. Taken together, our data provide evidence that CRFBP may possess both inhibitory and excitatory roles and may represent a novel pharmacological target for the treatment of AUD.
Crabbe JC, Schlumbohm JP, Hack W, Barkley-Levenson AM, Metten P, Lattal KM. Fear conditioning in mouse lines genetically selected for binge-like ethanol drinking. Alcohol (Fayetteville, N.Y.). 2016;52 :25–32.Abstract
The comorbidity of substance- and alcohol-use disorders (AUD) with other psychiatric conditions, especially those related to stress such as post-traumatic stress disorder (PTSD), is well-established. Binge-like intoxication is thought to be a crucial stage in the development of the chronic relapsing nature of the addictions, and self-medication through binge-like drinking is commonly seen in PTSD patients. We have selectively bred two separate High Drinking in the Dark (HDID-1 and HDID-2) mouse lines to reach high blood ethanol concentrations (BECs) after a 4-h period of access to 20% ethanol starting shortly after the onset of circadian dark. As an initial step toward the eventual goal of employing binge-prone HDID mice to study PTSD-like behavior including alcohol binge drinking, we sought first to determine their ability to acquire conditioned fear. We asked whether these mice acquired, generalized, or extinguished conditioned freezing to a greater or lesser extent than unselected control HS/Npt mice. In two experiments, we trained groups of 16 adult male mice in a standard conditioned fear protocol. Mice were tested for context-elicited freezing, and then, in a novel context, for cue-induced freezing. After extinction tests, renewal of conditioned fear was tested in the original context. Mice of all three genotypes showed typical fear responding. Context paired with shock elicited freezing behavior in a control experiment, but cue unpaired with shock did not. These studies indicate that fear learning per se does not appear to be influenced by genes causing predisposition to binge drinking, suggesting distinct neural mechanisms. However, HDID mice are shown to be a suitable model for studying the role of conditioned fear specifically in binge-like drinking.
Herman MA, Contet C, Roberto M. A Functional Switch in Tonic GABA Currents Alters the Output of Central Amygdala Corticotropin Releasing Factor Receptor-1 Neurons Following Chronic Ethanol Exposure. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 2016;36 (42) :10729–10741.Abstract
The corticotropin releasing factor (CRF) system in the central amygdala (CeA) has been implicated in the effects of acute ethanol and the development of alcohol dependence. We previously demonstrated that CRF receptor 1 (CRF1) neurons comprise a specific component of the CeA microcircuitry that is selectively engaged by acute ethanol. To investigate the impact of chronic ethanol exposure on inhibitory signaling in CRF1+ CeA neurons, we used CRF1:GFP mice subjected to chronic intermittent ethanol (CIE) inhalation and examined changes in local inhibitory control, the effects of acute ethanol, and the output of these neurons from the CeA. Following CIE, CRF1+ neurons displayed decreased phasic inhibition and a complete loss of tonic inhibition that persisted into withdrawal. CRF1- neurons showed a cell type-specific upregulation of both phasic and tonic signaling with CIE, the latter of which persists into withdrawal and is likely mediated by δ subunit-containing GABAA receptors. The loss of tonic inhibition with CIE was seen in CRF1+ and CRF1- neurons that project out of the CeA and into the bed nucleus of the stria terminalis. CRF1+ projection neurons displayed an increased baseline firing rate and loss of sensitivity to acute ethanol following CIE. These data demonstrate that chronic ethanol exposure produces profound and long-lasting changes in local inhibitory control of the CeA, resulting in an increase in the output of the CeA and the CRF1 receptor system, in particular. These cellular changes could underlie the behavioral manifestations of alcohol dependence and potentially contribute to the pathology of addiction. SIGNIFICANCE STATEMENT: The corticotropin releasing factor (CRF) system in the central amygdala (CeA) has been implicated in the effects of acute and chronic ethanol. We showed previously that CRF receptor 1-expressing (CRF1+) neurons in the CeA are under tonic inhibitory control and are differentially regulated by acute ethanol (Herman et al., 2013). Here we show that the inhibitory control of CRF1+ CeA neurons is lost with chronic ethanol exposure, likely by a functional switch in local tonic signaling. The loss of tonic inhibition is seen in CRF1+ projection neurons, suggesting that a critical consequence of chronic ethanol exposure is an increase in the output of the CeA CRF1 system, a neuroadaptation that may contribute to the behavioral consequences of alcohol dependence.
Greenberg GD, Crabbe JC. Gene Targeting Studies of Hyperexcitability and Affective States of Alcohol Withdrawal in Rodents. International Review of Neurobiology. 2016;126 :357–390.Abstract
Genetically engineered rodents can be used to examine the influence of single genes on alcoholism-related phenotypes. We review studies that employed gene targeting with a focus on ethanol withdrawal-associated behaviors. Earlier studies targeted the glutamate and GABA systems as contributors to the underlying hyperexcitable state of convulsions or similar signs of ethanol withdrawal. Over the past decade, many gene-targeting studies have continued to focus on the glutamatergic and GABAergic systems; however, an increasing number of these studies have focused on other withdrawal outcomes such as anxiety-like behavior and escalated ethanol consumption. Although negative affective states may drive escalated ethanol drinking, few reported studies examined the phenotypes together. However, there is significant overlap in the systems that were manipulated in relation to studying the phenotypes individually. These studies reveal common genetic influences on withdrawal-associated anxiety, convulsions, and escalated drinking that may contribute to relapse, setting the stage for the identification of novel medications to jointly target these effects.
Lalanne L, Lutz P-E, Trojak B, Lang J-P, Kieffer BL, Bacon E. Medications between psychiatric and addictive disorders. Progress in Neuro-Psychopharmacology & Biological Psychiatry. 2016;65 :215–223.Abstract
INTRODUCTION: Many epidemiological studies have revealed a frequent co-occurrence of psychiatric and substance use disorders. The term used in the literature to refer to this co-occurrence is dual diagnosis. The high prevalence of dual diagnosis has led physicians to observe the effects of medication prescribed to treat psychiatric disorders on the co-occurring substance use disorder and vice versa. The concept of medications between psychiatric and addictive disorders stems from these clinical observations, alongside which, however, it has developed from the observation that both psychiatric and substance use disorders share common neurobiological pathways and trigger common cognitive disorders. This has led researchers to develop medications on the basis of neurobiological and cognitive rationales. MATERIAL AND METHOD: In our article, we review peculiar medications based on neurobiological and cognitive rationales and that have an impact in both psychiatric and addictive disorders. RESULTS: We highlight how interesting these new prescriptions are for clinical observation and for the treatment of patients suffering from dual diagnosis. CONCLUSION: We then go on to discuss the interest in them from the perspective of clinical practice and clinical research, in that the development of medications to treat dual diagnosis helps to further our knowledge of both psychiatric and substance use disorders.
Deehan GA, Knight CP, Waeiss RA, Engleman EA, Toalston JE, McBride WJ, Hauser SR, Rodd ZA. Peripheral Administration of Ethanol Results in a Correlated Increase in Dopamine and Serotonin Within the Posterior Ventral Tegmental Area. Alcohol and Alcoholism (Oxford, Oxfordshire). 2016;51 (5) :535–540.Abstract
AIMS: Two critical neurotransmitter systems regulating ethanol (EtOH) reward are serotonin (5-HT) and dopamine (DA). Within the posterior ventral tegmental area (pVTA), 5-HT receptors have been shown to regulate DA neuronal activity. Increased pVTA neuronal activity has been linked to drug reinforcement. The current experiment sought to determine the effect of EtOH on 5-HT and DA levels within the pVTA. METHODS: Wistar rats were implanted with cannula aimed at the pVTA. Neurochemical levels were determined using standard microdialysis procedures with concentric probes. Rats were randomly assigned to one of the five groups (n = 41; 7-9 per group) that were treated with 0-3.0 g/kg EtOH (intraperitoneally). RESULTS: Ethanol produced increased extracellular DA levels in the pVTA that resembled an inverted U-shape dose-response curve with peak levels (\textasciitilde200% of baseline) at the 2.25 g/kg dose. The increase in DA levels was observed for an extended period of time (\textasciitilde100 minutes). The effects of EtOH on extracellular 5-HT levels in the pVTA also resembled an inverted U-shape dose-response curve. However, increased 5-HT levels were only observed during the initial post-injection sample. The increases in extracellular DA and 5-HT levels were significantly correlated. CONCLUSION: The data indicate intraperitoneal EtOH administration stimulated the release of both 5-HT and DA within the pVTA, the levels of which were significantly correlated. Overall, the current findings suggest that the ability of EtOH to stimulate DA activity within the mesolimbic system may be modulated by increases in 5-HT release within the pVTA. SHORT SUMMARY: Two critical neurotransmitter systems regulating ethanol reward are serotonin and dopamine. The current experiment determined that intraperitoneal ethanol administration increased serotonin and dopamine levels within the pVTA (levels were significantly correlated). The current findings suggest the ability of EtOH to stimulate serotonin and dopamine activity within the mesolimbic system.
Crabbe JC. Reproducibility of Experiments with Laboratory Animals: What Should We Do Now?. Alcoholism, Clinical and Experimental Research. 2016;40 (11) :2305–2308.
Hernandez RV, Puro AC, Manos JC, Huitron-Resendiz S, Reyes KC, Liu K, Vo K, Roberts AJ, Gruol DL. Transgenic mice with increased astrocyte expression of IL-6 show altered effects of acute ethanol on synaptic function. Neuropharmacology. 2016;103 :27–43.Abstract
A growing body of evidence has revealed that resident cells of the central nervous system (CNS), and particularly the glial cells, comprise a neuroimmune system that serves a number of functions in the normal CNS and during adverse conditions. Cells of the neuroimmune system regulate CNS functions through the production of signaling factors, referred to as neuroimmune factors. Recent studies show that ethanol can activate cells of the neuroimmune system, resulting in the elevated production of neuroimmune factors, including the cytokine interleukin-6 (IL-6). Here we analyzed the consequences of this CNS action of ethanol using transgenic mice that express elevated levels of IL-6 through increased astrocyte expression (IL-6-tg) to model the increased IL-6 expression that occurs with ethanol use. Results show that increased IL-6 expression induces neuroadaptive changes that alter the effects of ethanol. In hippocampal slices from non-transgenic (non-tg) littermate control mice, synaptically evoked dendritic field excitatory postsynaptic potential (fEPSP) and somatic population spike (PS) at the Schaffer collateral to CA1 pyramidal neuron synapse were reduced by acute ethanol (20 or 60 mM). In contrast, acute ethanol enhanced the fEPSP and PS in hippocampal slices from IL-6 tg mice. Long-term synaptic plasticity of the fEPSP (i.e., LTP) showed the expected dose-dependent reduction by acute ethanol in non-tg hippocampal slices, whereas LTP in the IL-6 tg hippocampal slices was resistant to this depressive effect of acute ethanol. Consistent with altered effects of acute ethanol on synaptic function in the IL-6 tg mice, EEG recordings showed a higher level of CNS activity in the IL-6 tg mice than in the non-tg mice during the period of withdrawal from an acute high dose of ethanol. These results suggest a potential role for neuroadaptive effects of ethanol-induced astrocyte production of IL-6 as a mediator or modulator of the actions of ethanol on the CNS, including persistent changes in CNS function that contribute to cognitive dysfunction and the development of alcohol dependence.
Contet C, Goulding SP, Kuljis DA, Barth AL. BK Channels in the Central Nervous System. International Review of Neurobiology. 2016;128 :281–342.Abstract
Large conductance Ca(2+)- and voltage-activated K(+) (BK) channels are widely distributed in the postnatal central nervous system (CNS). BK channels play a pleiotropic role in regulating the activity of brain and spinal cord neural circuits by providing a negative feedback mechanism for local increases in intracellular Ca(2+) concentrations. In neurons, they regulate the timing and duration of K(+) influx such that they can either increase or decrease firing depending on the cellular context, and they can suppress neurotransmitter release from presynaptic terminals. In addition, BK channels located in astrocytes and arterial myocytes modulate cerebral blood flow. Not surprisingly, both loss and gain of BK channel function have been associated with CNS disorders such as epilepsy, ataxia, mental retardation, and chronic pain. On the other hand, the neuroprotective role played by BK channels in a number of pathological situations could potentially be leveraged to correct neurological dysfunction.
Chesler EJ, Gatti DM, Morgan AP, Strobel M, Trepanier L, Oberbeck D, McWeeney S, Hitzemann R, Ferris M, McMullan R, et al. Diversity Outbred Mice at 21: Maintaining Allelic Variation in the Face of Selection. G3 (Bethesda, Md.). 2016;6 (12) :3893–3902.Abstract
Multi-parent populations (MPPs) capture and maintain the genetic diversity from multiple inbred founder strains to provide a resource for high-resolution genetic mapping through the accumulation of recombination events over many generations. Breeding designs that maintain a large effective population size with randomized assignment of breeders at each generation can minimize the impact of selection, inbreeding, and genetic drift on allele frequencies. Small deviations from expected allele frequencies will have little effect on the power and precision of genetic analysis, but a major distortion could result in reduced power and loss of important functional alleles. We detected strong transmission ratio distortion in the Diversity Outbred (DO) mouse population on chromosome 2, caused by meiotic drive favoring transmission of the WSB/EiJ allele at the R2d2 locus. The distorted region harbors thousands of polymorphisms derived from the seven non-WSB founder strains and many of these would be lost if the sweep was allowed to continue. To ensure the utility of the DO population to study genetic variation on chromosome 2, we performed an artificial selection against WSB/EiJ alleles at the R2d2 locus. Here, we report that we have purged the WSB/EiJ allele from the drive locus while preserving WSB/EiJ alleles in the flanking regions. We observed minimal disruption to allele frequencies across the rest of the autosomal genome. However, there was a shift in haplotype frequencies of the mitochondrial genome and an increase in the rate of an unusual sex chromosome aneuploidy. The DO population has been restored to genome-wide utility for genetic analysis, but our experience underscores that vigilant monitoring of similar genetic resource populations is needed to ensure their long-term utility.
Bell RL, Hauser SR, McClintick J, Rahman S, Edenberg HJ, Szumlinski KK, McBride WJ. Ethanol-Associated Changes in Glutamate Reward Neurocircuitry: A Minireview of Clinical and Preclinical Genetic Findings. Progress in Molecular Biology and Translational Science. 2016;137 :41–85.Abstract
Herein, we have reviewed the role of glutamate, the major excitatory neurotransmitter in the brain, in a number of neurochemical, -physiological, and -behavioral processes mediating the development of alcohol dependence. The findings discussed include results from both preclinical as well as neuroimaging and postmortem clinical studies. Expression levels for a number of glutamate-associated genes and/or proteins are modulated by alcohol abuse and dependence. These changes in expression include metabotropic receptors and ionotropic receptor subunits as well as different glutamate transporters. Moreover, these changes in gene expression parallel the pharmacologic manipulation of these same receptors and transporters. Some of these gene expression changes may have predated alcohol abuse and dependence because a number of glutamate-associated polymorphisms are related to a genetic predisposition to develop alcohol dependence. Other glutamate-associated polymorphisms are linked to age at the onset of alcohol-dependence and initial level of response/sensitivity to alcohol. Finally, findings of innate and/or ethanol-induced glutamate-associated gene expression differences/changes observed in a genetic animal model of alcoholism, the P rat, are summarized. Overall, the existing literature indicates that changes in glutamate receptors, transporters, enzymes, and scaffolding proteins are crucial for the development of alcohol dependence and there is a substantial genetic component to these effects. This indicates that continued research into the genetic underpinnings of these glutamate-associated effects will provide important novel molecular targets for treating alcohol abuse and dependence.
Bajo M, Montgomery SE, Cates LN, Nadav T, Delucchi AM, Cheng K, Yin H, Crawford EF, Roberts AJ, Roberto M. Evaluation of TLR4 Inhibitor, T5342126, in Modulation of Ethanol-Drinking Behavior in Alcohol-Dependent Mice. Alcohol and Alcoholism (Oxford, Oxfordshire). 2016;51 (5) :541–548.Abstract
AIMS: Several lines of evidence support a critical role of TLR4 in the neuroimmune responses associated with alcohol disorders and propose inhibitors of TLR4 signaling as potential treatments for alcoholism. In this work, we investigated the effect of T5342126 compound, a selective TLR4 inhibitor, on excessive drinking and microglial activation associated with ethanol dependence. METHODS: We used 2BC-CIE (two-bottle choice-chronic ethanol intermittent vapor exposure) paradigm to induce ethanol dependence in mice. After induction of the ethanol dependence, we injected T5342126 (i.p., 57 mg/kg) for 14 days while monitoring ethanol intake by 2BC (limited access to ethanol) method. RESULTS: T5342126 decreased ethanol drinking in both ethanol-dependent and non-dependent mice but T5342126 showed also dose-dependent non-specific effects represented by decreased animal locomotor activity, saccharine intake, and body core temperature. Six days after the last ethanol-drinking session, we examined the immunohistochemical staining of Iba-1 (ionized calcium-binding adapter molecule 1), a microglial activation marker, in the central nucleus of the amygdala (CeA) and dentate gyrus (DG) of the hippocampus. Notably, T5342126 reduced Iba-1 density in the CeA of both ethanol-dependent and non-dependent mice injected with T5342126. There were no significant differences in the DG Iba-1 density among the treatment groups. CONCLUSIONS: Collectively, our data suggest that T5342126, via blocking TLR4 activation, contributes to the reduction of ethanol drinking and ethanol-induced neuroimmune responses. However, the non-specific effects of T5342126 may play a significant role in the T5342126 effects on ethanol drinking and thus, may limit its therapeutic potential for treatment of alcohol dependence. SHORT SUMMARY: T5342126, an experimental TLR4 inhibitor, is effective in reducing ethanol drinking and inhibiting the activation and proliferation of microglia in both ethanol-dependent and non-dependent mice. However, T5342126's use as a potential candidate for the treatment of alcohol addiction may be limited due to its non-specific effects.
Bell RL, Hauser S, Rodd ZA, Liang T, Sari Y, McClintick J, Rahman S, Engleman EA. A Genetic Animal Model of Alcoholism for Screening Medications to Treat Addiction. International Review of Neurobiology. 2016;126 :179–261.Abstract
The purpose of this review is to present up-to-date pharmacological, genetic, and behavioral findings from the alcohol-preferring P rat and summarize similar past work. Behaviorally, the focus will be on how the P rat meets criteria put forth for a valid animal model of alcoholism with a highlight on its use as an animal model of polysubstance abuse, including alcohol, nicotine, and psychostimulants. Pharmacologically and genetically, the focus will be on the neurotransmitter and neuropeptide systems that have received the most attention: cholinergic, dopaminergic, GABAergic, glutamatergic, serotonergic, noradrenergic, corticotrophin releasing hormone, opioid, and neuropeptide Y. Herein, we sought to place the P rat's behavioral and neurochemical phenotypes, and to some extent its genotype, in the context of the clinical literature. After reviewing the findings thus far, this chapter discusses future directions for expanding the use of this genetic animal model of alcoholism to identify molecular targets for treating drug addiction in general.
Barkley-Levenson AM, Ryabinin AE, Crabbe JC. Neuropeptide Y response to alcohol is altered in nucleus accumbens of mice selectively bred for drinking to intoxication. Behavioural Brain Research. 2016;302 :160–170.Abstract
The High Drinking in the Dark (HDID) mice have been selectively bred for drinking to intoxicating blood alcohol levels and represent a genetic model of risk for binge-like drinking. Presently, little is known about the specific genetic factors that promote excessive intake in these mice. Previous studies have identified neuropeptide Y (NPY) as a potential target for modulating alcohol intake. NPY expression differs in some rodent lines that have been selected for high and low alcohol drinking phenotypes, as well as inbred mouse strains that differ in alcohol preference. Alcohol drinking and alcohol withdrawal also produce differential effects on NPY expression in the brain. Here, we assessed brain NPY protein levels in HDID mice of two replicates of selection and control heterogeneous stock (HS) mice at baseline (water drinking) and after binge-like alcohol drinking to determine whether selection is associated with differences in NPY expression and its sensitivity to alcohol. NPY levels did not differ between HDID and HS mice in any brain region in the water-drinking animals. HS mice showed a reduction in NPY levels in the nucleus accumbens (NAc) - especially in the shell - in ethanol-drinking animals vs. water-drinking controls. However, HDID mice showed a blunted NPY response to alcohol in the NAc core and shell compared to HS mice. These findings suggest that the NPY response to alcohol has been altered by selection for drinking to intoxication in a region-specific manner. Thus, the NPY system may represent a potential target for altering binge-like alcohol drinking in these mice.

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