Richard L. Bell, Ph.D. - US grants

DESCRIPTION (provided by applicant): The long-term objective of this research is to investigate neuroadaptations within the extended amygdala and its interconnections following excessive ethanol consumption in rats. One series of experiments will ensure the quality control and availabity of rats taken through the excessive ethanol drinking animal model. Towards this end, high-alcohol-consuming P and HAD (both replicate lines) rats will be taken through an ethanol drinking protocol involving repeated cycles of exposure to multiple ethanol concentrations followed by a period of deprivation. A second series of experiments will further characterize and refine the ethanol drinking protocol by evaluating the effects of altering the length of initial and subsequent 1) ethanol exposures and 2) deprivations and changing the available ethanol concentrations. A third series of experiments will examine the influence this experimental paradigm of cycles of ethanol availability and deprivation has in the drinking pattern of low-alcohol-consuming (e.g., NP, LAD-1, LAD-2 and Wistar) rats. The ethanol drinking protocol results in very high levels of ethanol intake in P and HAD rats (up to 16g/kg/day on the reinstatement of multiple concentrations of ethanol after three cycles of exposure and deprivation), suggesting that the reinforcing properties of ethanol may have been enhanced. The main hypothesis to be tested is that experience with excessive ethanol drinking results in neuroadaptive alterations in the extended amygdala and its interconnections. The rat lines that have been genetically selected for high alcohol drinking at Indiana University (i.e., P, HAD-1 and HAD-2) have been known to exhibit "loss of control" drinking when exposed to the ethanol drinking protocol proposed in this program. The results of this proposal will provide valuable information toward understanding the neural circuitry underlying excessive alcohol drinking and relapse of alcohol drinking. Such information would be important for developing pharmacotherapies for the treatment of alcoholism and alcohol abuse.

[unreadable] 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. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The overall objectives of this U01 research core application are to (a) provide selectively bred high ethanol (EtOH)-consuming rats that have experienced the drinking-in-dark (DID) EtOH binge-drinking protocol and their controls, or whole brains or brain sub-regions from these rats; and (b) use shRNAi's and other pharmacological tools to help identify genes, gene systems and receptors involved in the predisposition for, and development and maintenance of, EtOH binge-drinking. The overall hypothesis is that particular 'candidate' genes and their associated molecular networks within the extended amygdala and associated brain regions significantly contribute to the development and maintenance of, and a predisposition for, excessive EtOH drinking. The overall hypothesis will be tested by (a) providing alcohol-preferring (P) and high-alcohol-drinking (HAD) rats (or their whole brains or brain regions) that have been taken through the binge drinking protocol to other INIA U01 investigators; (b) using shRNAi's to reduce expression of 'candidate' genes within the extended amygdala and associated regions; and (c) examining the effects of ligands targeted for 'candidate' gene products and their molecular networks on, binge-drinking. Providing insight into the complex molecular and cellular events that lead to the development and maintenance of excessive alcohol drinking behavior in animal models is highly significant since these findings will provide the necessary foundation for developing novel treatment strategies targeting alcohol abuse and alcoholism. This is a highly innovative project since it will use state-of-the-art techniques to selectively reduce expression of 'candidate' genes in multiple genetically predisposed 'families' (i.e., the P, HAD1 and HAD2) of rats and in discrete CNS regions that are involved in regulating alcohol drinking. This U01 research core provides synergy with several INIA-West components by addressing the first two specific aims of INIA, i.e., confirm gene targets and identify drugable targets for medications focused on treating alcohol abuse and alcoholism.

DESCRIPTION (provided by applicant): Accumulating evidence points to an intersection between binge drinking behavior, smoking, and the ongoing development of the medial prefrontal cortex, during adolescence. The concurrence of these events may have lasting detrimental effects on neurotransmission, drug use, and reckless and impulsive behaviors in adulthood. Glutamate is an amino acid neurotransmitter known to influence neuronal activity in mesolimbic and cortical areas and has been implicated in both ethanol and nicotine abuse. The prefrontal cortex both receives glutamate inputs and has glutamatergic projections to key sites involved in ethanol and nicotine self- administration. The current approach will test the hypothesis that concommittant voluntary ethanol and nicotine intake during peri-adolescence leads to profound effects on both ethanol and nicotine self-administration behavior, and mesocorticolimbic glutamate neurotransmission, in adulthood. The selectively bred alcohol- preferring (P) rat will be used in this part of the project because individual and concurrent voluntary intake of ethanol and nicotine has been demonstrated in this line. Control groups drinking ethanol, nicotine, or water alone will also be used to assess the impact of concurrent intake as compared to intake of each drug alone. Importantly, both male and female rats will be tested to identify sex differences in outcomes for all experiments in this application, and rats with initial ethanol and nicotine exposure as adults will be also be studied to identify changes specific to peri-adolescent initiation of drug taking. To assess the long-term consequences of concurrent ethanol and nicotine exposure during peri-adolescence, ethanol and nicotine self-administration (including measures of craving and relapse) will be examined after voluntary ethanol and nicotine intake in peri-adolescence. Concurrently, microdialysis-HPLC procedures will also be conducted in adult animals to measure how peri-adolescent ethanol and nicotine intake affects extracellular levels of glutamate in critical mesocorticolimbic brain circuits which have been implicated in alcohol and drug abuse. Together, these studies represent a novel approach to examine the impact of the co-use of ethanol and nicotine in the peri- adolescent period on neurobiology and the susceptibility to ethanol and nicotine in adulthood. It is anticipated that this application will provide important information for the development of early (adolescent) and late (adult) treatment and/or prevention strategies targeting the critical public health problems of alcoholism and nicotine addiction.

DESCRIPTION (provided by applicant): The major objective of this R24 Alcohol Research Resource nt is to supply to off-campus researchers at cost P/NP, HAD1-2/LAD1-2, and AA/ANA selectively bred rats, as well as HAP1-2-3/LAP2-3 and cHAP selected mice that show genetically high and/or low alcohol preference. (Specific Aims 2 and 3). The P/NP, HAD1-2/LAD1-2, HAP1-2-3/LAP2-3, and cHAP selected rodent lines were developed at Indiana University and this R24 is the sole funding source for maintaining the HAD1-2/LAD1-2 nucleus breeding colonies (Specific Aim 1). The HADl- 2/LAD1-2 lines from the N/Nih foundation stock are the only replicate rat lines selectively bred for divergent alcohol preference and they are phenotypically and genotypically different from the P/NP contrasting lines. In order to increase the value of this R24, a breeding colony ofAA/ANA rat lines will be transferred from Finland to Indiana as a new resource (Specific Aim 4). Neurochemically, AA rats are strikingly different from P rats, i.e., the mesolimbic dopamine pathway is not central either in the acquisition or maintenance of high alcohol preference in the AA rats; instead, innate neurocircuitries that involve endogeneous opioids and endocannabinoids appear to be the key. For the purpose of bringing further added value to this R24, Duke University will receive a subcontract to collaborate in creating a valid animal model of alcohol and nicotine co-abuse (Specific Aim 5). This approach is most expedient because Dr. Ting-Kai Li (the PI who created our P/NP, HAD1-2/LAD1-2, and HAP1-2/LAP1-2 selected rodent lines) is now a Professor of Psychiatry at Duke and Drs. Amir H. Rezvani and Edward D. Levin have active research programs at Duke that use iv nicotine self-administration routinely. This will be achieved by first comparing the five pairs of selectively bred rat lines with opposite alcohol preference (i.e., P/NP, HADl/LADl, HAD2/LAD2, AA/ANA, and sP/sNP) for their differences in willingness to self- administer nicotine by intraveneous route and to identify which high line has the highest proclivity to self- administer nicotine. This high line with both high alcohol drinking preference and high nicotine iv self- administration will then be used to investigate the effects of nondependent alcohol drinking and relapse-like alcohol drinking on nicotine self-administration. RELEVANCE (See instructions): Multiple alcohol-preferring rat lines from different genetic background are available, and together, they simulate behaviorally the distinct subtypes of alcoholics with high genetic load defined by a recent NESARC study. This R24 will supply to off-campus researchers the P/NP, HAD1-2/LAD1-2, and AA/ANA selectively bred rats as well as the HAP1-2-3/LAP2-3 and cHAP selected mice. Additionally, this R24 will create a new animal model of alcohol-nicotine co-abuse that will be extremely useful in basic preclinical research.

According to the Centers for Disease Control and Prevention (CDC), alcohol use disorder (AUD) continues to be the third leading cause of death in the U.S. Multiple studies from the Collaborative Studies on Genetics of Alcoholism (COGA) and Study of Addiction: Genes and Environment (SAGE) confirm that being an individual with a family history positive (FHP) for alcoholism is a key, predictable determinant for the development and expression of AUD. The selectively bred alcohol-preferring P rat and, to a lesser extent, the high-alcohol- drinking HAD rat lines meet criteria put forth for a valid animal model of alcoholism and display certain genetic-, behavioral-, and physiological-related phenotypes observed in FHP individuals. This core is a Research Resource for the Integrative Neuroscience Initiative on Alcoholism (INIA-Neuroimmune [INIA-N]) consortium. The Specific Aims of this Research Resource will test the effects of (1) treatment with target small molecules on excessive ethanol (EtOH) drinking by P and HAD rats, (2) infusions of shRNA and/or cDNA (to downregulate or upregulate, respectively, target gene expression levels) into subregions of the extended amygdala (Ext-Amyg) and medial prefrontal cortex (mPFC) on excessive EtOH drinking by P and HAD rats, and (3) work closely with Dayne Mayfield?s, and others U01s, in identifying neuroimmune signaling, and/or their pathway, targets associated with a genetic predisposition to develop AUD. For this, we will use our previous and ongoing genomic and proteomic work for excessive alcohol drinking. Some of the targets observed so far match those of INIA-N as a whole including toll-like receptors [TLRs], interleukin receptors [ILRs], phosphodiesterase 4 [PDE4], and peroxisome proliferator-activated receptor [PPAR]. This core will work closely with U01 components and the U24 ?Electrophysiology Core? to address pertinent research questions raised by respective U01s and/or the Administrative Core. This is a significant core that will provide important verification and heuristic information on neuroimmune signaling in AUD in general, as well as in genetically predisposed subjects in particular. Moreover, this Core will evaluate compounds suggested by Mayfield?s LINCS analysis and manipulators of immune targets revealed in ongoing work of INIA-N. This is a highly innovative project that uses state-of-the-art techniques to selectively alter the expression of ?target? genes within discrete CNS subregions in multigenerational, genetically selected (P, HAD1 and HAD2) FHP rats. This U24 core provides synergy with a number of INIA-N investigators, including Blednov, Crabbe, Hitzemann, Kieffer, Lasek, Mason, Mayfield, Morrisett, Pfefferbaum, and Roberto, as well as Becker/Lopez of INIA-Stress.

Project Summary: Animal Production Core Alcohol and other substance abuse continue to be enormous public health issues with total costs in the US approaching a half billion dollars a year. Although there are major attempts at prevention, a better understanding of the genetic and environmental factors that increase the risk of alcohol use disorders, the neural circuits that underlie this risk, and the ability to regulate these circuits therapeutically are all still critically needed. In addressing this critical need, this Animal Production Core will continue to provide the P/NP and HAD/LAD rats, as well as the HAP/LAP and cHAP mice that have been selectively bred for a high vs low alcohol preference, to investigators in the Indiana Alcohol Research Center (IARC) or on the Indiana University?Purdue University at Indianapolis (IUPUI) campus. This Core is the outgrowth of over 30 years of experience with selective breeding, maintenance of breeding colonies, and solving problems arising with the logistics and nature of running an operation of this magnitude. We are therefore uniquely qualified in the coming funding period to continue to provide these animals to alcoholism and addiction researchers, to coordinate the use of the animals to avoid scientific overlap, and to manage issues that could affect their phenotype and associated behaviors. Addiction has become a disease of co-abuse, with the majority of individuals dependent on alcohol often being dependent on other licit or illicit substances as well. Given this, there are likely common environmental and genetic factors that result in a broad predisposition to the abuse of many substances, as well as alcohol specifically. The Animal Production Core will continue to characterize these lines (high vs low preference) as animal models of addiction, and promote their use in examining the genetic and neurobiological substrates of substance use, abuse and dependence.