Grants Binge Domain

UOI: (Core)
Grant Number: 2U01AA013522-06
PI Name: BELL, RICHARD L.
PI Email:
PI Title:
Project Title: Rat Animal Models Core (RAMC)
Abstract: DESCRIPTION (provided by applicant): The long-range goals of the Rat Animal Models Core (RAMC) are to better understand the molecular neurobiological events underlying the development and maintenance of excessive ethanol (EtOH) drinking, and how these neurobiological events contribute to the long-range consequences of excessive EtOH drinking. The overall hypotheses to be tested are that (a) a number of neurobiological events, associated with excessive EtOH drinking, occur within the extended amygdala (E-AMYG); (b) the use of excessive drinking procedures, outlined below, in alcohol-preferring, P, and high alcohol-drinking, HAD-1 and HAD-2 rats enables the detection of these events; (c) site-specific lesioning of structures [accumbens-shell (ACBsh), central amygdala (Ce-AMYG), and bed nucleus of the stria terminalis (BNST)], within the E-AMYG, alter the development and/or maintenance of excessive drinking; and (d) experience with these excessive drinking procedures, by P and HAD rats, results in behavioral and/or physiological alterations associated with criteria for a valid animal model of alcoholism (i.e., expression of intoxication, tolerance, and withdrawal signs, and changes in the amount of ethanol consumed and/or pattern of ethanol consumption). Excessive drinking is defined as repeatable and sustainable blood EtOH concentrations (BECs) in the range of 100 to 150 mg% or higher over a chronic period. Three protocols of excessive drinking induction will be used to reflect (a) binge-like drinking during the dark cycle [drinking-in-the-dark-multiple scheduled access (DIDMSA)], with rats receiving three 1-hr access periods spaced 2 hrs apart across the dark cycle; (b) dependence-induced excessive drinking using a prolonged repeated alcohol deprivation (PRAD) procedure, with rats receiving 6 weeks of initial EtOH access followed by multiple cycles of 2 weeks of deprivation from and 2 weeks of re-exposure to EtOH access; and (c) withdrawal-induced, via EtOH vapor inhalation, (excessive) drinking, with a 3 bottle-choice test procedure (WID-3BC) used to measure intake after each cycle of exposure to and withdrawal from EtOH vapor inhalation. Overall, the results of this project will provide valuable information on the complex molecular neurobiological changes that contribute to the development and consequences of excessive alcohol drinking, and aid in the development of interventions for the prevention, and/or treatment of alcohol abuse and alcoholism.

U01:
Grant Number: 2U01AA013475-06
PI Name: BERGESON, SUSAN E.
PI Email: bergeson@mail.utexas.edu
PI Title: ASSISTANT PROFESSOR OF NEUROBIOLOGY
Project Title: Molecular Basis of Excessive Alcohol Drinking
Alcoholism, a disease of considerable morbidity, mortality and human suffering worldwide, is first and foremost characterized by excessive alcohol drinking. The "Two-Hit hypothesis" that both genetic and environmental factors contribute to excessive alcohol intake is the overarching focus of our current and proposed INIA research. Microarray results from our present UO1 funding have allowed substantial inroads to be made into the understanding of both the genetic predisposition to drink and the molecular consequences of alcohol exposure. Significant candidate genes have been identified though the overlap of several of our own studies and importantly across INIA collaborations. A large searchable, open source web-base database system containing over 30 million microarray data points from our studies was created to make sharing all data within and beyond the alcoholism research field facile (publicly announced in Bergeson et al., 2005). Our current objectives are to continue to build our array database to include expression analyses of six new INIA mouse models, focus us on characterizing candidate genes that fit our "two-hit hypothesis", and to use genetically altered mice and brain region specific viral mediated trans- gene and shRNA expression to test bi-directional expression (up and down regulation) effects on alcohol drinking. We will perform neurocirciutry specific microarray studies following viral-mediated gene changes in the presence and absence of alcohol drinking to better understand anatomical and molecular contributions to excessive alcohol drinking. Finally, initial studies focused on the role of miRNA and chromatin remodeling in alcohol drinking and/or consequences are proposed.

 

U01:
Grant Number: 2U01AA013520-06
PI Name: BLEDNOV, YURI A.
PI Email: yablednov@mail.utexas.edu
PI Title:
Project Title: Biochemical and Genetic Determinants of Differences in Alcohol Consumption
Abstract: DESCRIPTION (provided by applicant): The overall objective of this INIA grant is to define mechanisms of alcohol avoidance and provide expertise in behavioral testing to other INIA investigators to evaluate ethanol-related behaviors in new mutant mice. Based on our data from the current period of INIA funding, we propose that the avoidance of high concentrations of ethanol by rodents is caused by increased release and/or production of proinflammatory cytokines in liver and brain and that these cytokines cause long-lasting changes in gene expression in the brain. We also found that initial high acceptance of alcohol can decrease when alcohol is presented continuously with intervening weekly abstinence periods, and this decrease depends upon the genetic background. We developed two genetic mouse models which maintain consistent high levels of preference and consumption (FVBxB6F1 mice; Sustained Alcohol Preference, SAP) or develop avoidance of alcohol (NZBxB6F1 mice; Reduced Alcohol Preference, RAP). These animal models provide the opportunity to compare brain gene expression profiles formed by initial alcohol consumption, sustained consumption and development of alcohol avoidance. To test the cytokine hypothesis, we will study: 1) ethanol consumption in knockout mice lacking genes for some proinflammatory cytokines and/or their receptors; 2) the effect of systemic treatment with proinflammatory cytokines (particularly a-TNF) and antagonists of cytokine receptors on voluntary ethanol consumption; 3) brain gene expression profiles after cytokine treatment to allow comparison with array data obtained from mice with ethanol avoidance; 4) treatment of FVBxB6F1 and NZBxB6F1 hybrid mice via 3 cycles of alcohol consumption and abstinence and identification of key genes by microarray analysis; 5) generation of brain regional knock-down mice of key genes by administration of RNAi. This work will use 3 INIA Cores (Texas Array and Informatics Core, RA Harris -PI; Colorado RNAi core, W. Zawada - PI; California Mouse Animal Model Core, A. Roberts - PI) and collaborations with six INIA investigators (A. Alcantara - Austin, Texas; S. Bergeson - Austin, Texas; R. D. Mayfield - Austin, Texas; R. Davis - Houston, Texas; A. Ryabinin - Portland, Oregon; B. Tabakoff - Aurora, Colorado).

 

U01:
Grant Number: 2U01AA013519-06
PI Name: CRABBE, JOHN C.
PI Email: crabbe@ohsu.edu
PI Title: PROFESSOR
Project Title: Selective Breeding for Drinking in the Circadian Dark
Abstract: DESCRIPTION (provided by applicant): During the current funding period, we have developed a method for enhancing ethanol self-administration in mice. When C57BL/6J mice are offered access to ethanol for a short period during their circadian dark period, they will ingest significant quantities, reaching blood ethanol concentrations greater than 100 mg% (Rhodes et a/.,2005). The animals show signs of behavioral intoxication (Rhodes et al., in press). The current proposal is built around the production of replicated lines of mice selected for their propensity to high drinking in the dark (HDID). The experiments will further develop and characterize the model and provide selected lines for other INIA projects. HDID1 mice will be selected from HS/Npt control stock for their high (>150 mg-%) blood alcohol levels on the second day of 2 daily exposures to 20% ethanol for 2-4 hr/day, starting in hr 3 of their circadian dark cycle. A second replicate of this line (HDID2) will be started in the first renewal year, using the same selection index. If studies in the mean time (Aim 2) indicate that some modification of the test (e.g., multiple bottles) has resulted in a better selection phenotype, the HDID2 line will be started using the improved phenotype. A third replicate (HDID3) will be started in Renewal Year 3. Five phenotypes will be systematically evaluated in the selected lines vs the 8-way cross control lines as potential correlated responses to selection: Two-bottle EtOH preference, Scheduled access to ethanol (SHAG procedure), Prolonged access, The Alcohol Deprivation Effect, and Withdrawal induced Drinking after inhalation exposure. We will adapt the DID test to very young mice and study its developmental onset and time course. We will employ a chronic intermittent stress paradigm post-weaning, pre-pubertal mice of the HDID lines and controls and assess the DID phenotype in adulthood. Col Dr. Tamara Phillips will examine the effects of several test compounds on DID, using intracranial injection sites based on INIA targeted circuits. We will initially target the lateral septum and its afferent and efferent connections. Finally, we will breed additional naive mice and ship them to other interested investigators.

 

U01:
Grant Number: 2U01AA013479-06
PI Name: CUNNINGHAM, CHRISTOPHER L.
PI Email: cunningh@ohsu.edu
PI Title: PROFESSOR
Project Title: Dependence Driven Alterations in Ethanol Reinforcement
Abstract: DESCRIPTION (provided by applicant): This INIA Consortium U01 Project is focused on genetic differences and neuroadaptations in brain circuitry that are responsible for individual differences in vulnerability to excessive consumption of alcohol. We will extend our previous findings using an intragastric consumption (IGC) model in which several days of passive exposure to ethanol (or water) via a chronic intragastric (IG) cannula are followed by a self-infusion test procedure in which voluntary ingestion of a flavored solution is paired with IG ethanol. Previously, we found that IGC and preference for the ethanol-paired flavor (compared to a water-paired flavor) is enhanced by passive ethanol exposure and varies as a function of genotype in both rats and mice. We now propose to focus primarily on mice. Aim 1 will examine key parameters of the passive infusion phase in two inbred strains, C57BL/6J and DBA/2J. These parameters include: (a) dose per infusion and total daily dose, (b) number of daily ethanol infusions, and (c) number of days of passive ethanol exposure. Aim 2 will further address the hypothesis of genetic differences in sensitivity to dependence-driven ethanol reinforcement by extending the model to characterize IGC in 15 standard inbred strains, allowing examination of genetic correlations between IGC and a wide range of previously studied ethanol phenotypes. With support from the INIA Colorado Gene Array Core, we will also examine genetic correlations with whole brain gene expression. Aim 3 will test whether passive IG ethanol exposure produces changes in ethanol reinforcement/reward using the conditioned place preference procedure and limited access operant self-administration. Aim 4 will involve collaboration with other INIA projects by testing two mouse models that have been selectively bred for high blood ethanol concentrations in binge drinking procedures: (a) the SHAG and SLAC lines, and (b) the HDID line and its genetic control (HS/Npt). Finally, with support from the INIA Neurocircuitry Mapping and Genotyping Core, Aim 5 will use c-Fos immunohistochemistry and lesions to identify specific brain areas that influence the enhancement in IGC after passive ethanol exposure. The long-term goal of this project is to understand the genetic and neurobiological processes underlying the excessive drinking that contributes to alcoholism in humans. By improving our understanding of these processes, we can identify more effective treatment and prevention strategies.

U01:
Grant Number: 2U01AA013476-06
PI Name: DAVIS, RONALD L.
PI Email: rdavis@bcm.tmc.edu
PI Title: PROFESSOR
Project Title: Neuroadaptations to Ethanol in Drosophila
Abstract: DESCRIPTION (provided by applicant): The long-term goals of this research project are to elucidate the molecular and cellular mechanisms for neuroadaptations to ethanol. Previous studies have identified two new Drosophila mutants with a heightened sensitivity and impaired tolerance to ethanol. Preliminary spatial expression studies of one mutant, named homer, have suggested that the normal gene's function is sufficient in GABAergic neurons that innervate the ellipsoid body for normal behavioral responses to ethanol. These studies will be confirmed and extended. In addition, the directed expression of RNAi constructs and dominant negatives will be used to define where Corner expression is required in the adult brain for wild type behavioral responses to ethanol. Newly developed techniques that provide for experimenter control over transgene expression in time and space will be used to ascertain whether homer is required during development or whether it is required physiologically in the adult fly. Similar time and space expression studies using a normal transgene of the second mutant, named yps, will be performed to ascertain whether this gene's function is required in the same set of neurons and at the same time as homer for normal ethanol responses. RNA from neurons that require homer and/or yps will be isolated using newly developed affinity techniques and used to probe microarrays to define the changes in gene expression that occur with ethanol exposure in this specific set of neurons. The observed changes in gene expression with ethanol exposure will be used to predict which genes are functionally involved in behavioral responses to ethanol, and mutants in these genes will be assayed to further define the molecular genetic requirements for normal ethanol sensitivity and tolerance. These studies will further our understanding of the genetic basis for ethanol sensitivity and tolerance, important parameters for susceptibility to alcoholism.

U01: (Core)
Grant Number: 1U01AA016660-01
PI Name: EDENBERG, HOWARD J.
PI Email: edenberg@iupui.edu
PI Title: PROFESSOR
Project Title: Indiana Gene Array Core
Abstract: DESCRIPTION (provided by applicant): The overall aim of the Indiana Gene Array Core is to collaborate with INIA investigators to understand the differences in gene expression in the extended amygdala that are involved in the development of excessive drinking. Our hypothesis is that both innate differences in gene expression and differences in response to alcohol and to paradigms involving exposure to alcohol or its withdrawal (gene-environment interactions) contribute to the development of excessive alcohol drinking. The Indiana Gene Array Core will focus on the use of Affymetrix microarrays to measure gene expression in selected regions of rat brains and in Drosophila mutants after various treatments; primary collaborators will be McBride (U01), Ryabinin (U01) and Davis (U01). These studies build upon our work during the initial grant period; carrying out the follow-up studies in the same laboratory (with many of the same technical personnel) will reduce technical variations and allow more powerful metaanalyses. We will work with investigators on experimental design, extract and purify RNA from rat brain tissues provided by the collaborating investigators, carry out all of the quality control and biochemistry involved in GeneChip experiments, extract the data and assist with analyses and bioinformatics. This core will be housed in the Center for Medical Genomics, directed by the PI, which has all of the necessary equipment and carries out a high volume of Affymetrix GeneChip experiments with a focus on quality control. We will extend our services to include quantitative RT-PCR confirmation of key findings from the microarrays that we process, and will offer such services to other members of INIA. We will also provide members of INIA analyses of cis-acting elements that contribute to the differences in gene expression detected in the microarray experiments; we have developed model-based techniques that do not require prior knowledge of binding sites. This Core will provide high quality data in an efficient and economical way. We already have strong relatioinships with the users, which will benefit the overall project.

U01:
Grant Number: 5U01AA013483-05
PI Name: GRAHAME, NICHOLAS J.
PI Email: ngrahame@iupui.edu
PI Title: ASSOCIATE PROFESSOR
Project Title: Neural Basis of Ethanol Sensitization/Drinking in Mice
Abstract: DESCRIPTION (provided by applicant): Alcoholism is caused by a complex interaction of environmental, genetic, and physiological factors. A family history of alcoholism is a strong predictor of high alcohol seeking behavior in humans and in animal models of alcoholism, and the prognosis is worse when an individual has a both high genetic load for alcoholism and a personal history of alcohol use. This proposal seeks a better understanding of neural mechanisms that are altered by alcohol experience. The populations of mice that will be used in this project already show evidence of a link between behavioral sensitization to alcohol's locomotor stimulating effects and alcohol drinking. Specifically, these selectively bred, High Alcohol Preferring (HAP) mice are more likely to show locomotor sensitization (LMS) to ethanol following repeated administration than Low Alcohol Preferring (LAP) mice, indicating that LMS and drinking are likely to be genetically and physiologically linked. Investigating mechanisms underlying LMS may yield insight into the mechanisms of high alcohol drinking. This proposal directly seeks evidence about neural pathways involved in LMS and its relationship to excessive drinking by studying immediate early gene expression. Experiments will also assess whether exposure to alcohol sufficient to induce LMS increases either the incentive value of alcohol and/or alcohol drinking. Finally, because enduring LMS in HAP mice requires that they associate their test environment with previous alcohol injections, studies will seek to understand whether the memory of alcohol affects alcohol drinking and the incentive value of alcohol. One study will also test whether acamprosate, a treatment for alcoholism, can reverse changes in the rewarding value of alcohol caused by alcohol exposure. The hypothesis is that associative forms of neural and behavioral plasticity underlie both the acquisition of excessive drinking and LMS, and that the amygdala may lie at the heart of this interaction

U01: (Core)
Grant Number: 2U01AA013518-06
PI Name: HARRIS, ROBERT ADRON.
PI Email: harris@mail.utexas.edu
PI Title: PROFESSOR OF PHARMACOLOGY
Project Title: Texas Gene Array Core
Abstract: DESCRIPTION (provided by applicant): The overall objective of this INIA core is to continue to provide robust microarray analysis and informatics capabilities to INIA researchers. During the previous grant period, the INIA Microarray Core at UT Austin enabled the generation of large amounts of microarray data from INIA labs. In the next period, although we plan to generate microarray data, the emphasis of the Core will extend to carrying out novel analyses of consolidated datasets. At the same time we will enhance our microarray capabilities by enabling novel types of microarray profiling experiments such as microRNA (miRNA) profiling and chromatin immunoprecipitation (ChlP-chip) data analysis. The activities of this proposed INIA Core will include updating of our relational microarray database, the Longhorn Array Database (LAD), to accommodate these novel kinds of experimental platforms and datasets, and most importantly, developing completely new analysis capabilities that are not possible with the web browser model that is used by LAD. miRNAs provide a novel mechanism for changing function by changing the levels of brain proteins and we will enable INIA investigators to carry out pioneering neurobiological studies in this area. miRNAs have been shown to be important in post-transcriptionally regulating gene expression in cancer and miRNA profiling is likely to be valuable for understanding INIA models of excessive alcohol consumption. This core will give INIA researchers free access to the analyses tools for DNA microarray data that we have developed and are developing by making these accessible from a web browser. For example, generalized singular value decomposition (GSVD) and pseudoinverse projection, allow construction of predictive models from DNA microarray data. This website will enable researchers without in-depth expertise in mathematics and computer programming to concentrate on the scientific questions they set out to answer by analyzing cDNA, miRNA and oligonucleotide microarray data.

U01: (Core)
Grant Number: 2U01AA013484-06
PI Name: HITZEMANN, ROBERT J.
PI Email: hitzeman@ohsu.edu
PI Title: PROFESSOR
Project Title: Neurocircuitry Mapping and Genotyping Core
Abstract: DESCRIPTION (provided by applicant): Three behavioral models of excessive ethanol consumption (DID, SHAC and WID) are widely used throughout the INIA-West consortium. A fourth model intragastric consumption (IGC) is used at only one site but importantly provides a novel mouse model of what is likely to be dependence-induced drinking. A primary purpose of this core is to provide a standardized assessment of the circuits that are associated with each of these models and the changes in these circuits as the models are modified by genetic and/or pharmacologic procedures. The following aims have arranged the planned activities of the core in a systematic fashion, moving from the basic models to variations on the models. The advantage from a mapping perspective of the DID, SHAC and WID procedures is that the ethanol is consumed in a limited access period which allows one to access the circuits associated with both the initiation and continuation of the drinking episode. The proposed core has four aims/goals: 1. To map in C57BL/6J (B6) mice the circuits associated with DID, SHAC, WID and IGC using as appropriate a combination of in situ hybridization(ISH) and immunohistochemical (IHC) techniques. These studies will examine whether or not there are distinct circuits associated with the initiation and maintenance of a drinking episode. Given the widespread use of B6 mice throughout INIA-West, these data will serve as the primary reference circuits. 2. To map the relevant circuits in new genetic animal models. For DID and SHAC, replicate selected lines will be bred from heterogenous stock (HS/Npt) animals (Crabbe and Finn). The data obtained will be contrasted with the results obtained in specific aim 1. 3. To utilize the genetic and pharmacological variations on the four models to further refine the circuits associated with excessive ethanol consumption. Several UO1 applications plan to investigate variations on the basic behavioral models in order to refine at the anatomical and/or molecular level the relevant circuits. These data can be contrasted with the results obtained in aims 1 and 2. Importantly, aims 1-3 must be viewed as part of an iterative process. The core will begin to identify the relevant circuits, investigators will test the most promising targets which in turn will generate new models and thus, additional core activity. The core will also provide (aim 4) tissue for gene array and proteomics analyses and a high thoughput SNP genotyping facility for all INIA-West investigators.

U01:
Grant Number: 1U01AA016649-01
PI Name: HOFFMAN, PAULA L.
PI Email: paula.hoffman@uchsc.edu
PI Title: PROFESSOR
Project Title: Alcohol Drinking after Modulation of Differentially Expressed Genes in HAP and LA
Abstract: DESCRIPTION (provided by applicant): Voluntary alcohol consumption represents an endophenotype that can be modeled in rodents and that may reflect susceptibility to the development of alcohol dependence. Selective breeding of mice and rats for differences in this phenotype indicates a genetic influence on voluntary alcohol consumption. We have used mice selectively bred for high and low alcohol preference (HAP and LAP mice, respectively), as measured in a two-bottle choice paradigm, to identify candidate genes that contribute to alcohol preference drinking through their differential expression levels in brain. We now propose to confirm the differential expression of these genes, and localize the differential expression within brain regions, by quantitative reverse transcriptase real-time PCR (qRT-PCR) using a "voxelation" procedure, and by quantitative in situ hybridization, in brains of HAP and LAP mice. The expression of selected genes, prioritized based on function and proposed role in alcohol drinking behavior, will then be reduced by treatment of the mice with RNAi reagents. We will design siRNAs and shRNAs in collaboration with the INIA RNAi Core and Dharmacon. The efficacy and specificity of these reagents will be assayed in vitro. siRNAs will be delivered using osmotic minipumps or by site-specific infection of lentiviral vectors containing shRNAs. We will determine the time course, duration and extent of target gene down-regulation by qRT-PCR and specificity of effects by qRT-PCR and microarray analysis in collaboration with the INIA Colorado Gene Array Core. Once specific gene knockdown has been confirmed, mice will be tested for changes in alcohol preference drinking in the two-bottle choice paradigm. HAP and LAP mice will also be treated chronically with ethanol in the "withdrawal-induced drinking" procedures of the INIA Mouse Animal Models Core. Changes in brain gene expression that correlate with increases in voluntary alcohol consumption, following the chronic alcohol exposure, will be determined. These experiments will systematically investigate the role of identified candidate genes, alone and in combinations reflecting signal transduction pathways, in the modulation of voluntary alcohol consumption.

U01:
Grant Number: 1U01AA016658-01
PI Name: KIEFFER, BRIGITTE L.
PI Email: briki@igbmc.u-strasbg.fr
PI Title:
Project Title: Inactivation of mu opioid and CRF1 receptor genes in the extended amygdala
Abstract: DESCRIPTION (provided by applicant): Gene targeting in mice to study the role of neuropeptide receptors in ethanol dependence has recently yielded fascinating results. Knockout of the mu opioid receptor (MOP) gene blocks ethanol drinking and operant responding for ethanol. Knockout of the Corticotropin Releasing Factor receptor 1 (CRF1) gene reduces levels of anxiety under basal and alcohol withdrawal conditions. Altogether data demonstrate that blockade of these receptor systems reduce alcohol intake. Limitations of these "conventional" gene targeting studies are that (i) gene knockout occurs early, therefore compensatory mechanisms could take place during development, and (ii) knockout of the receptors occurs throughout the entire animal, therefore no information on the recruited neurocircuitry is provided. To address these issues, we will induce the knockout of MOP and CRF1 receptor genes specifically in the extended amygdala (EA) of adult animals, based on the overall hypothesis of INIA (Integrative Neuroscience Initiative on Alcoholism) regarding the role of the EA in excessive alcohol consumption. First, we will take advantage of two existing mutant mouse lines, one with a floxed MOP receptor gene (recently created in our laboratory), and another with a floxed CRF1 receptor gene (collaboration). Second, we will develop a novel transgenic mouse line expressing Cre recombinase in the EA. To do this, we will use a BAG promoter for the WFS1 (Wolfram syndrom 1) gene, that we have recently identified as an EA marker gene (Specific Aim 1). Third, we will breed floxed mice with the WFS1-Cre mouse to produce the conditional knockout of MOP (Specific Aim 2) and CRF1 (Specific Aim 3) receptor genes in the EA of adult mice. The two conditional lines will be fully characterized for receptor expression throughout the brain, for morphine responses (MOP) and for basal behaviors (Specific Aims 2 and 3). The two conditional lines will finally be extensively studied in behavioral models of excessive alcohol drinking, including the DID and WID models, as well as for acute ethanol responses and ethanol withdrawal (Specific Aim 4). Importantly, the WFS1-Cre transgenic mice generated in Specific Aim 1 will represent a unique tool for the conditional deletion of any other gene of interest in the extended amygdala, and will be generally useful in addiction research.

U01: (Core)
Grant Number: 2U01AA013517-06
PI Name: KOOB, GEORGE F.
PI Email: gkoob@scripps.edu
PI Title: DIRECTOR
Project Title: Integrative Neuroscience Initiative on Alcoholism
Abstract: DESCRIPTION (provided by applicant): This is a competing renewal application for a Consortium for the Integrative Neuroscience Initiative on Alcoholism (INIA)-West (Notice* NOT-AA-06-101) to identify the molecular, cellular, and behavioral neuroadaptations that occur in specific brain neurocircuitries that result in excessive alcohol consumption. More specifically, the focus of this multidisciplinary initiative will be on the molecular and cellular neuroadaptations in the brain reward circuits associated with the extended amygdala and its connections. The overall hypothesis for INIA-West is that genetic differences and/or neuroadaptations in this circuitry are responsible for the individual differences in vulnerability to the excessive consumption of alcohol. The overall goals of INIA-West are to identify the neurocircuitry and neurobiology responsible for excessive alcohol intake, and to provide the foundation to enable translation of the basic neuroscience findings to the human clinical condition. To accomplish these goals, the following Specific Aims are outlined: (1) To identify specific clusters of genes whose expression is regulated by alcohol and which are responsible for INIA-developed models of excessive alcohol consumption, (2) To confirm gene targets nominated by expression assays or other methods, by use of transgenic, knockout, inducible knockouts, site-specific knockouts, RNAi, and in situ hybridization, (3) To attract new and innovative investigators to the field of alcohol research. The structure of INIA-West is envisioned as two domains (Binge and Dependence) that integrate across three levels of analysis: molecular, cellular, neurocircuitry. Distributed Core facilities are proposed that transcend domains and include the Gene Array Cores, Animal Models Cores (rat and mouse) and the Neurocircuitry Cores. Each Domain is comprised of 10-12 U01 proposals and 1-2 Developmental U01 proposals. A Pilot Project program is proposed to identify exciting new areas of research and the continual recruitment of new investigators to the alcohol field. The INIA program will be directed by an Administrative Core in close cooperation with the Executive Committee, Steering Committee, and with the continual advice of the Scientific Advisory Board.

U01:
Grant Number: 1U01AA016648-01
PI Name: MAYFIELD, ROY D.
PI Email: dayne.mayfield@mail.utexas.edu
PI Title:
Project Title: Alcohol-responsive protein networks underlying excessive ethanol consumption
Abstract: DESCRIPTION (provided by applicant): Genomic and proteomic approaches offer distinct advantages in the search for novel ethanol sites of action because they allow large numbers of elements (RNA transcripts and/or proteins) to be examined simultaneously in an unbiased fashion. INIA West laboratories have performed numerous gene expression studies utilizing both Affymetrix and cDNA microarrays to identify genes that differ in animals bred for high and low ethanol drinking, as well as, those that change in response ethanol administration using drosophila, rodent, and human models of alcoholism. In a significant collaborative effort, INIA West investigators recently worked to consolidate these different microarray experiments and prioritize genes and functionally related groups of genes that were changed significantly in response to ethanol administration. The results of these studies indicate that ethanol alters the expression pattern of a number of genes known to be involved in synapse-related protein trafficking and synaptic transmission. Proteins encoded by the genes are important for a variety of synaptic events including neurotransmitter vesicle transport and targeting (synapsin, syntaxin, dynamin, and synaptotagmin), motor proteins involved in trafficking and targeting of synaptic proteins (kinesin family proteins (KIFs)), and scaffolding proteins (homer and discs-large homolog 2). Interaction proteomics will be used to identify novel protein complexes associated with each of these target proteins in C57BL/6 mice. The effect of excessive ethanol consumption (dependence model of withdrawal-induced drinking (WID)) on these protein complexes will be determined. The overall hypothesis is that excessive ethanol consumption alters protein complexes important for normal trafficking and targeting of proteins involved in synaptic transmission. The resulting changes in trafficking, targeting, and synaptic function underlie ethanol-related phenotypes.

U01: 
Grant Number: 1U01AA016652-01
PI Name: MCBRIDE, WILLIAM JOSEPH.
PI Email: wmcbride@iupui.edu
PI Title: PROFESSOR AND VICE-CHAIR (RESEARCH)
Project Title: Excessive alcohol drinking and CNS regional changes in genes expression
Abstract: DESCRIPTION (provided by applicant): The long-range goals of this proposal are to better understand the molecular neurobiological events that underlie the development of excessive alcohol drinking, and contribute to the long-range consequences of excessive alcohol drinking. The overall hypothesis to be tested is that changes in the expression of genes involved in neurotransmission, neuroplasticity, and intracellular signaling pathways within discrete regions of the extended amydala (E-AMYG) contribute to the development of excessive alcohol drinking. Excessive drinking is defined as sustainable blood alcohol concentrations (BACs) in the range of 100-150 mg% that are repeatedly attained over a chronic period. The overall hypothesis will be tested using selectively bred alcohol-preferring (P) and high-alcohol-drinking (HAD) rats. Micro-punch techniques will be used to obtain samples containing the nucleus accumbens shell (ACB-sh) and central nucleus of the amygdala (CeA). Changes in gene expression will be determined using Affymetrix microarrays. RT-PCR and in situ hybridization will be used to verify key findings from the microarray experiments. The excessive alcoholdrinking paradigm to be used will be the 'drinking in the dark multiple scheduled access' (DID-MSA) procedure. Time-course changes in gene expression will be determined following a drinking episode, and during the development of excessive alcohol drinking. The specific aims will be designed to determine changes in gene expression within the ACB-sh and CeA of P and HAD rats prior to, during initiation of, and following development of excessive alcohol drinking. The effects of chronic alcohol exposure and the development of excessive alcohol drinking are influenced by multiple genetic and environmental factors. This project will provide important molecular neurobiological information in discrete regions of the E-AMYG of genetically vulnerable subjects that could more comprehensively describe the complex inter- and intracellular events leading to the development, maintenance, and consequences of excessive alcohol drinking. Such information would be critically important for developing pharmacotherapeutic strategies to treat alcoholism and alcohol abuse.

U01:
Grant Number: 1U01AA016651-01
PI Name: MORRISETT, RICHARD A.
PI Email: ramorris@mail.utexas.edu
PI Title: ASSOCIATE PROFESSOR
Project Title: Accumbal Synaptic Regulation and Bistability In The WID Mouse Model
Abstract: DESCRIPTION (provided by applicant): Glutamatergic synaptic transmission in the nucleus accumbens (NAc) has been implicated in neuroadaptive alterations which underlie the development of addiction to alcohol. In an animal model adopted by the INIA-West consortium (Withdrawal-induced drinking mouse model, hereafter termed WID experience), C57/BL6J mice that experienced 3 intermittent 16 hrethanol vapor exposures displayed a 2-3 fold enhancement in both shell and core NAc cFos expression and a marked increase in their ethanol intake over mice that experienced constant ethanol exposure. Our preliminary data indicate conditioning stimuli which normally induces NMDA-receptor dependent synaptic depression in wild-type mouse shell Nac medium spiny neurons (MSNs) instead induces a remarkable conversion to synaptic potentiation in nondrinking mice that had undergone a single bout of WID experience. This form of synaptic plasticity in shell NAc MSNs has also been implicated in the expression of behavioral sensitization to cocaine and therefore may represent a fundamental cellular process involved in neuroadaptation to ethanol and other reinforcers. Changes in the level of excitatory drive onto MSNs have been implicated in sustained upstates of the membrane potential or bistability recorded under current clamp conditions. Such shifts in excitability of these neurons are thought to be critically involved in information processing in mesolimbic structures and thus we propose that MSN bistability is an important electrophysiological process altered by WID experience and therefore important to the development of ethanol dependence. Finally, alterations in AMPA and NMDA receptor synaptic drive are implicated in ethanol dependence, synaptic plasticity as well as membrane potential bistability. Therefore, our overarching hypothesis is that WID experience induces alterations in excitatory synaptic drive mediated by aberrant plasticity of AMPA/NMDA receptors and that these changes in plasticity impact MSN function (bistability). Thus, we propose to perform a comprehensive analysis of the alterations in synaptic plasticity, membrane potential bistability and AMPA/NMDA receptor mediated synaptic transmission and receptor levels induced by WID experience in medium spiny neurons of the shell of the NAc. This project uses electrophysiological, immunofluorescent, confocal microscopy and co-localization image analysis to investigate these hypotheses.

U01:
Neuroimaging In Animal Models of Alcoholism
Grant Number: 2U01AA013521-06
PI Name: PFEFFERBAUM, ADOLF
PI Email: dolf@synapse.sri.com
PI Title: DIRECTOR
Project Title: Neuroimaging in Animal Models of Alcoholism
Abstract: DESCRIPTION (provided by applicant): The brain damage resulting from excessive alcohol exposure may itself contribute to the self-perpetuating nature of human alcoholism. Our goals for this translational research project are twofold: 1) to demonstrate alcohol-induced brain damage in the rat when animals are exposed to high levels of alcohol and scheduled withdrawals and 2) to determine whether rats, selectively bred to drink high amounts of alcohol, drink in a pattern or quantity insufficient to produce neurotoxicity or, alternatively, have also inadvertently been selected for resistance to alcohol neurotoxicity. In the previous INIA funding cycle we found that substantial voluntary drinking by the P rat was only modestly neurotoxic as demonstrated with magnetic resonance (MR) imaging.MR-detectable brain abnormalities were limited to attenuated growth of the corpus callosum, with suggestion of effect in the hippocampus and cerebellum. We now propose to use the alcohol vapor chamber method to increase exposure and to schedule withdrawals, which we predict will enhance alcohol-induced brain damage, detectable with in vivo structural MRI of the whole brain and 2D J-resolved MR spectroscopy of ventromedial subcallosal gray matter. Hypothesis-guided postmortem histological and exploratory gene expression studies will confirm and extend in vivo observations. Specific Aims 1-3 will use two alcoholpreferring/non-preferring rat strains and their selection stock: P, NP, Wistar (W) and HAD-1, LAD-1, N/NIH rats. Our MR developments and expertise will be provided to other investigators through Specific Aim 4. Aim 1: Use in vivo MR to demonstrate structural and brain metabolite abnormalities in P and HAD-1 rats after vapor chamber exposure to and withdrawals from high levels of alcohol starting in peri-adolescence. Hypothesis: Alcohol treatment will result in attenuated growth trajectory of brain tissue, decreased size of selective brain structures, particularly the corpus callosum, frontal cortex, hippocampus, amygdala, and cerebellum, and decreased entromedial subcallosal NAA and choline. Aim 2: Use in vivo MR to determine if P and HAD-1 rats are more robust to deleterious effects of high binge and withdrawal alcohol exposure than their NP and LAD-1 counterparts and their W and N/NIH selection stock. Hypothesis: The magnitude of the MRI and MRS abnormalities will be NP > W> P and LAD-1 > N/NIH > HAD-1. Aim 3: Identify histological and explore gene expression correlates of alcohol-induced brain damage and determine if alcohol-induced brain damage occurs below the limits of detection of in vivo MR studies. Hypothesis: Compared with controls, alcohol-exposed animals will have reduced callosal size, thinner myelin sheathes, and lower neuron counts in gray matter regions predicted to sustain MR-detectable damage. Aim 4: As a resource, provide MR imaging expertise to other INIA-West and INIA-East investigators for in vivo studies of animal models of alcoholism.

 

U01: 
Grant Number: 1U01AA016655-01
PI Name: PHILLIPS, TAMARA J.
PI Email: phillipt@ohsu.edu
PI Title: PROFESSOR
Project Title: Mapping and Microarray Gene Expression Analysis in a Model of Excessive Drinking
Abstract: DESCRIPTION (provided by applicant): Ethanol drinking is a complex trait, and thus influenced by multiple genetic and environmental factors. Examining the influence of one gene at a time ignores the importance of possible interactions (e.g., epistasis, intra-allelic overdominance; see Phillips & Belknap, 2002). We propose to utilize a filial cross approach for the mapping of gene sets that result in excessive alcohol intake. C57BL/6J (B6) x FVB/NJ (FVB) F1 mice have been found to consume more ethanol than even the high ethanol preference B6 strain (Blednov et al., 2005). We will take advantage of this finding to resolve mode of inheritance for excessive drinking by combining quantitative trait locus (QTL) mapping with microarray gene expression analysis to identify colocalization of behavioral (bQTL) and expression QTL (eQTL). In Specific Aim 1, bQTL for excessive voluntary ethanol consumption will be mapped using the B6FVBF2. The F2 will be used to determine the mode of inheritance (additive, fully dominant, overdominant, epistatic) for each QTL taken singly and also in pairwise combinations. In Specific Aim 2, F3-F4 individuals will be used for brain region specific gene expression analyses. Individuals predicted by their genotype to be high or low drinking individuals will be selected for microarray profiling. These data will be subjected to eQTL analyses. This will allow us to identify bQTL and eQTL that are mapped to common chromosomal regions providing evidence of the specific gene(s) influencing the drinking trait. Brain regions to be studied will be selected from target tissues identified by the INIA Neurocircuitry group. In Specific Aim 3, possible genetically correlated responses will be measured to explore putative genetic relationships between the extreme drinking trait and others such as ethanol withdrawal, conditioned taste aversion, withdrawal induced drinking, drinking in the dark, ethanol acceptance, and ethanol conditioned place preference. As we obtain evidence for the specific locations of genes that influence the high drinking trait, other traits will be chosen for examination based on previous QTL mapping data that have identified associations of those traits with the locations we identify. Future work will also be focused on the most significant candidate genes implicated by our combined bQTL:eQTL analyses.

U01: (Core)
Grant Number: 2U01AA013523-06
PI Name: ROBERTS, AMANDA J.
PI Email: aroberts@scripps.edu
PI Title: ASSISTANT PROFESSOR
Project Title: Mouse Animal Models Core
Abstract: DESCRIPTION (provided by applicant): Alcoholism is characterized by increased ethanol intake, loss of control over ethanol intake, and compulsive ethanol taking. Progress in understanding the neurobiological basis of excessive ethanol drinking depends on the combined development and use of molecular and neuropharmacological tools for understanding the mechanisms of ethanol actions and animal models that allow interpretation of these advances in the context of ethanol addiction. The primary goal of the Mouse Animal Models Core is to provide well characterized and validated behavioral models of excessive ethanol consumption to INIA investigators. This Core has been completely designed around the specific needs of the INIA-West group and has developed from intense discussions and contact with everyone. Inherent in the goal of the Core is that it allows for both the standardization of these models and the pooling of resources. This Core will service twelve INIA investigators studying each of the two domains (Binge and Dependence, see below) and their three levels of analysis (neurocircuitry, cellular, and molecular). In this way, the Core shares the responsibility of making this consortium highly integrated and multidisciplinary and establishes economies of scale. Individual laboratories can focus on their own expertise and will not be required to establish these models in their own laboratories. One of the most innovative aspects of this Core is the application of its neuroadaptive models to the many genetic models (transgenics, knockouts, selectively bred mice) being studied and/or produced by INIA investigators. This will allow for a comprehensive study of the combined impact of genes and environment on the process of ethanol addiction and represents a major strength of the present application. In addition, these models will be extended to additional strains such as those used in the production of knockout and transgenic strains and those commonly used in ethanol research. Finally, this Core will examine phenotypic correlates of excessive alcohol consumption as this is critical to both our understanding of the susceptibility. Based on INIA investigators' needs the following Specific Aims have been formulated: Specific Aim 1. To provide established and refined neuroadaptive models to INIA investigators and Specific Aim 2. To combine neuroadaptive models with genetic models for INIA investigators.

U01:
Grant Number: 1U01AA016647-01
PI Name: RYABININ, ANDREY E.
PI Email: ryabinin@ohsu.edu
PI Title: ASSISTANT PROFESSOR
Project Title: Excessive drinking and urocortin 1 neurocircuit
Abstract: DESCRIPTION (provided by applicant): This proposal seeks to become a part of the INIA consortium "Neurobiologial Basis of Excessive Drinking", and focuses of the neuropeptide Urocortin 1 (Ucn1). Ucn1 is the most effective endogenous ligand of both corticotropin releasing factor (CRF) receptors CRF1 and CRF2. The main source of Ucn1 in the brain is the non-preganglionic Edinger-Westphal nucleus (npEW). One of the main projection areas of npEW is the lateral septum (LS). Recent evidence indicates that the Ucn1 system is extremely sensitive to alcohol, that differences in this system predispose animals to differences in alcohol consumption, and that manipulations of this system regulate alcohol intake. Based on this evidence we hypothesize that differences in Ucn1 activity are important determinants of excessive alcohol intake. In this project we propose to apply collaborative efforts to investigate three specific aims: (1) To identify genes showing consistently different expression in npEWand LS between selectively-bred high and low alcohol consuming animals using microarray technology. Following animal models will be explored: mice selectively bred for excessive drinking in the dark, mice selectively bred for excessive drinking in the scheduled fluid access procedure, mice selectively bred using the 2-bottle choice procedure, rats selectively bred using the 2-bottle choice procedure, and their respective control lines. Differences in gene expression will be confirmed using immunohistochemistry, in situ hybridization and quantitative RTPCR. (2) To test alcohol consumption in Ucn1 knockout mice using three behavioral models: DID - excessive drinking in the dark; SHAG - excessive drinking due to scheduled access; and the standard 2-bottle drinking procedure. We will also use microarray technology to investigate whether Ucn1 knockout mice developed compensations in genes identified in Specific Aim 1. (3) To test whether genes identified in animal models of excessive alcohol consumption in Specific Aim 1 and Specific Aim 2 are expressed in npEW and LS of human post-mortem brains, and whether they are differentially expressed between alcoholic subjects and controls. Human homologues of the identified genes will be tested by quantitative RT-PCR. Taken together, these studies will provide a thorough comprehensive analysis of the Ucn1 neurocircuit and its involvement in excessive alcohol consumption, and could provide groundwork for development of new approaches for Oregon Health treatments of alcoholism and alcohol abuse disorders.

 

U01:
Grant Number: 2U01AA013498-06
PI Name: SIGGINS, GEORGE R.
PI Email: geobob@scripps.edu
PI Title: PROFESSOR
Project Title: Electrophysiology of alcohol in extended amygdala
Abstract: DESCRIPTION (provided by applicant): This project is based on behavioral findings that the central amygdala nucleus (CeA) and locus coeruleus (LC) are key brain areas involved in stress reactions and the reinforcing properties of abused drugs, and that these behaviors may involve several transmitters (GABA, glutamate, norepinephrine) and neuropeptides (CRF, opioids and galanin). Both regions are implicated in motivated behaviors and anxiety states, and we hypothesize that these same neurochemical systems within the CeA and LC are involved in the excessive ethanol drinking seen in dependent animals. Therefore, we propose several sets of experiments: 1) T assess the role of CRF receptors in excessive drinking, by comparing the CeA cellular and network function in brain slices from control and excessively drinking mice (WID model) mice, with respect to the ethanol augmentation of GABAergic IPSCs or inhibition of glutamatergic EPSPs, combined with cytochemical localization of CRF and CRF receptors. 2) To determine the role of kappa opiate receptors (KORs) in excessive drinking, for comparison to our mu and delta receptor data, by examining CeA cellular function in brain slices from WID mice with a knockout (KO) for brain KORs. 3) To determine the role of galanin and its receptors in excessive drinking, by examining CeA and LC cellular in slices from WID mice and those with KOs for brain Gall and Gal2 receptors and with galanin over-expression, and by neurochemical and molecular biological measures in CeA and LC neurons. 4) To determine the effects on the largest WIDinduced changes from the results of Specific Aims 1-3, in the HDID mice selectively bred by the Crabbe and Finn groups for high drinking in the dark versus their controls, and for SHAG vs.SLAG lines, selected for scheduled high and low alcohol consumption. The electrophysiological studies will use CeA and LC brain slices and involve standard intracellular and whole-cell clamp methods. We will use a battery of measures to assess the pre- versus postsynaptic sites of action of ethanol and peptide effects. RIA, real-time PCR and receptor binding studies will be used in the galanin studies. This project should provide important new information on the possible squeal of ethanol intoxication at the cellular level, and, by comparisons of ethanol and peptide actions in control, excessively drinking, and knockout models, will also provide clues as to the synaptic, cellular and ion channel correlates of ethanol dependence.

U01: (Developmental)
Grant Number: 1U01AA016650-01
PI Name: SZUMLINSKI, KAREN K.
PI Email: szumlinski@psych.ucsb.edu
PI Title:
Project Title: mGluR-Homer interactions in excessive drinking
Abstract: DESCRIPTION (provided by applicant): Current theories of alcoholism posit that alcohol-induced neuroadaptations within limbic structures in the brain, including the nucleus accumbens (NAC), contribute to the transition from recreational alcohol drinking to excessive alcohol consumption. Recently, the Group 1 metabotropic glutamate receptor (mGluR) associated scaffolding protein Homer2 was identified as an active and necessary cellular mediator of alcohol-induced neural plasticity in mice. Constitutively expressed Homer proteins facilitate Group 1 mGluRstimulated intracellular signaling and cluster Group 1 mGluRs within the postsynaptic density, co-localizing these receptors with other proteins implicated in synaptic plasticity, such as PI3K (phosphatiylionsitol-3 kinase). Homer2 deletion reduces the function and the expression of Group 1 mGluRs in the NAC in vivo and the alcohol-avoiding and -intolerant behavioral phenotype of Homer2 knock-out (KO) mice resembles that produced by the pharmacological blockade of Group 1 mGluRs. Collectively, these observations suggest that Group 1 mGluR-Homer signaling is an important cellular mediator of excessive alcohol consumption. To test this hypothesis directly, this proposal will employ in vivo pharmacological and genetic approaches to characterize the role for Group 1 mGluR-Homer signaling within the NAC in regulating excessive alcohol consumption within the scheduled high alcohol consumption (SHAC) murine model (Aim 1). Immunohistochemical and immunoblotting approaches will be employed to determine the role for Homer2 n regulating the effects of sustained, excessive alcohol consumption upon the synaptic architecture of NAC neurons, as well as the formation, subcellular localization and function of mGluR-Homer signaling complexes (Aim 2). Finally, to relate genetic variance in excessive alcohol drinking to mGluR-Homer-PI3K expression and signaling within the NAC, immunoblotting the total protein content and membrane localization of members of the mGluR-Homer-PI3K signaling cascade will be compared between mouse lines selectively bred for high SHAC and SLAC (Scheduled Low Alcohol Consumption) phenotypes. The results of these studies will further our understanding of the cellular mechanisms involved in regulating the transition from recreational to excessive alcohol drinking and provide greater insight into the etiology of alcoholism and its treatment.

U01: (Core)
Grant Number: 1U01AA016663-01
PI Name: TABAKOFF, BORIS
PI Email: boris.tabakoff@uchsc.edu
PI Title: PROFESSOR AND CHAIR
Project Title: Colorado Gene Array Core
Abstract: DESCRIPTION (provided by applicant): The Colorado Gene Array Core will focus on the creation of databases that can be used for developing expression QTL (eQTL) maps for mouse and rat brain. We are currently expanding the database of brain gene expression profiles for males and females of the BxD Rl mouse strains. The proposed studies will generate a similar database of gene expression profiles from brains of 31 Rl strains of BxH/HxB rats and the progenitor strains (males and females separately), and microsatellite and SNP genotyping of the rat strains will be performed in collaboration with Dr. Rob Williams, University of Tennessee. This information will allow for eQTL mapping for rat brain gene expression as well as mapping of QTLs for alcohol drinking in the rat strains. When combined with QTL analysis of complex traits, the generation of eQTLs facilitates the identification of candidate genes for these traits, and the Core will use this procedure and the generated data to identify candidate genes that contribute to anxiety and other alcohol related behaviors in the rats. The availability of brain gene expression data and eQTL mapping of the mouse and rat brain transcriptome will produce a novel and unique resource for use by alcoholism investigators. The Core will also perform gene expression analysis and quantitative RT-PCR for collaborating INIA investigators, and provide access to methodology for normalization, statistical analysis and eQTL searches of the generated data.

U01:
Grant Number: 1U01AA016653-01
PI Name: WU, CHRISTINE C.
PI Email: christine.wu@uchsc.edu
PI Title: ASSISTANT PROFESSOR
Project Title: Proteomic Dissection of Withdrawal-Induced Excessive Drinking
Abstract: DESCRIPTION (provided by applicant): Chronic exposure to alcohol results in neuroadaptive phenomena, including tolerance, sensitization, dependence, withdrawal, loss of control of drinking, and relapse that contribute to the development of excessive alcohol consumption. The goal of the INIA (Integrative Neuroscience Initiative on Alcoholism) Consortium is to identify the molecular, cellular, and behavioral neuroadaptations that occur in the brain reward circuits associated with the extended amygdala and its connections. It is hypothesized that genetic differences and/or neuroadaptations in this circuitry are responsible for the individual differences in vulnerability to the excessive consumption of alcohol. We propose to use quantitative proteomics to dissect the molecular mechanisms contributing to the behavioral phenotype of differential and excessive drinking (both baseline drinking and withdrawal induced drinking) in two animal models: 1) Adenylyl Cyclase 7 (AC7) transgenic animals - changes in the copy number of the AC7 gene produces changes in drinking phenotype using the Withdrawal Induced Drinking (WID) paradigm and 2) High Alcohol Preference (HAP)/Low Alcohol Preference (LAP) animals - animals selectively bred for differences in free-choice alcohol consumption by the 2 Bottle-Choice (2BC) paradigm - the selected genotype (changes in multiple genes) contributes to differential baseline drinking. Our goals are 1) to develop and optimize proteomic methodology for the quantitative analysis of enriched brain fractions, 2) to identify global differences in baseline protein expression between selected lines of the two animal models [AC7 transgenic model (AC7 transgenic versus wildtype) and HAP/LAP selective breeding model (HAP versus LAP)], and 3) to globally compare longitudinal changes in protein expression between animals (AC7 transgenic versus wildtype and HAP versus LAP) at selected time points during WID-2BC to identify proteins that contribute to the differential and excessive drinking behaviors.

U01: (Core)
Grant Number: 1U01AA016654-01
PI Name: ZAWADA, W MICHAEL.
PI Email: mike.zawada@uchsc.edu
PI Title: ASSISTANT PROFESSOR
Project Title: RNAi Core
Abstract: DESCRIPTION (provided by applicant): The original five-year INIA West has identified a large number of candidate genes that are differentially expressed in the brains of mice selectively bred for either high- or low-preference for alcohol. Additional conserved genes were identified in screens for alcohol tolerance in Drosophila. Manipulating the expression of these genes has naturally become a critical component of studies designed to further define their role in the development of alcohol preference or tolerance. The RNA Interference Core (RNAi Core) will provide a means for systematic modification of the target genes' expression in selected and precisely-defined brain areas. Two technological platforms for in vivo RNA interference will be employed: (1) small interfering RNA (siRNA) delivered by a direct injection or infusion into the CNS and (2) short hairpin RNA (shRNA) delivered via a lentiviral vector-based transduction. Initially, these methods will be standardized using transcripts identified in HAP and LAP mice as contributing to alcohol preference drinking behavior, and the functional consequences of the RNAi treatment will be assessed in these mice. The successful implementation of the methodology will provide an important resource for all INIA investigators, including those working with rat models. At the time of this submission, laboratories from California, Colorado, Indiana, Oregon and Texas are already collaborating in both Binge and Dependence Domains. The core will also perform RNAi treatments for INIA investigators, who will develop need for RNAi services with the emergence of additional target genes. Proposed projects will be evaluated for integration with INIA West goals by a Project Evaluation Committee composed of members of the INIA Steering Committee and an independent consultant. The core will capitalize on our ongoing collaboration with Dharmacon Corporation, a leader in the field of siRNA development. Dharmacon will provide many of the necessary reagents and work with us on improving the efficiency of gene silencing in the CNS. The core's goals will be accomplished by successful completion of the following aims: Aim 1. Perform high throughput in vitro screening of the RNAi sequences targeting the genes of interest. Aim 2. Silence expression of selected genes in vivo employing RNAi within precise neuroanatomical targets. Aim 3. Examine behavioral and transcriptional effects of gene silencing. The creation of the RNAi core is a logical extension of the work already completed by the INIA. The core will allow for systematic and high throughput manipulation of genes in the mammalian CNS, facilitating functional studies of these genes in alcohol preference.