Lisa M. Tarantino, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Vulnerability to develop addiction to psychostimulants, including cocaine and amphetamine, as well as other drugs, like alcohol, is influenced by both genetic and environmental factors. Genetic factors may modulate differing initial responses to drugs of abuse in humans and this has been linked to the propensity to develop a drug abuse disorder. Abnormal adaptations in stress response pathways have also been implicated in the development of drug dependence and while this relationship is firmly established, the underlying neurobiological mechanisms are not well understood. While no animal model exists that reproduces the entire spectrum of the drug abuse syndrome in humans, animal models do exist for certain drug-related behaviors including acute locomotor activation, behavioral sensitization and conditioned place preference in response to drug treatment, as well as rates of drug self-administration. We have identified an ENU-induced mutant, Highper, that shows hyperlocomotion in a novel environment, an exaggerated locomotor response to the psychostimulants cocaine and methylphenidate and a prolonged release of corticosterone following an acute stressor. We believe that the abnormal stress and drug responses exhibited by these animals are related; our hypothesis is that the exaggerated locomotor response to psychostimulants is exacerbated by previous exposure to stressful events. Using single nucleotide polymorphism (SNP) genotyping, we have mapped the Highper mutation to a 59 megabase region on chromosome 12. This region contains 411 genes, none of which has previously been implicated in both drug and stress responses. Thus, Highper may represent a novel model for studying the relationship between the stress and drug response behavioral domains. In this proposal, we outline an experimental strategy to address our three primary goals: 1) to further characterize the Highper line to better understand the abnormal stress and psychostimulant responses and the link between the two 2) to fine map and identify the causative mutation and 3) to characterize the functional disruption caused by the gene mutation both in vivo and in vitro. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The impact of cocaine addiction on the lives of those who suffer from it, their families and society as a whole is staggering. The specific factors that determine an individuals' risk of transitioning from initial cocaine use to abuse and addiction are largely unknown but a role for genetics has been established. Therefore, identification of specific genes that increase susceptibility to abuse cocaine has been an area of considerable effort in the research community. Attempts to identify genetic loci involved in addiction liabilityin human populations have met with some success but are complicated by lack of environmental control, genetic heterogeneity, the need for large sample sizes and ethical concerns. Genetically stable mouse populations have proven useful for the study of complex human diseases like addiction. While mice will never replicate the entire spectrum of traits that define the human disease state, there are commonly used behavioral models that are believed to faithfully represent key features of addiction. Most drugs of abuse cause psychomotor stimulation when administered acutely and initial sensitivity to a drug has been associated with increased risk for subsequent use and abuse. Therefore, locomotor activation in response to cocaine is often used as a model for addiction-related behavior in mice. It has been known for some time that inbred strains of mice differ widely in their locomotor response to cocaine proving a role for genetics in this complex behavior. More recently, a dense SNP panel has been developed that allows for determination of haplotype structure in inbred mouse strains. These SNP data, along with inbred strain behavioral data, can be used to conduct genome-wide association mapping (GWAS) to identify genomic loci that are associated with cocaine locomotor response. We have recently completed a 45 inbred strain survey of cocaine-induced locomotor response and have identified multiple genetic loci that influence the behavior. The goal of this application is to replicate these QTL in standard intercross (F2) populations and use haplotype information from inbred strains to narrow QTL intervals and identify candidate genes. We also propose to test a set of candidate genes by measuring behavior in knockout lines and by complementation testing. Finally, we propose to examine inbred strains at the extreme ends of the phenotypic distribution in an attempt to uncover the biological mechanisms that contribute to initial locomotor sensitivity to cocaine and may increase risk for addiction.

DESCRIPTION (provided by applicant): Behavior is affected by genetics, the environment, and a complex interplay between the two. Exposure to nutritional deficiencies at critical time points during development, particularly while in utero and during the postpartum period, increases the risk of psychiatric disease in humans, and alters behavioral outcomes in animal models of human psychiatric disease. Some of these effects have been linked to changes in methylation and gene expression, implying that epigenetic factors may contribute to the underlying disease process. Identifying specific genes that respond both to environmental variation and that influence behavior in animal models can reveal not only genetic pathways involved in brain development and function, but also how such pathways are sensitive to external factors in the environment. Such studies can thereby shed light on the etiology of human psychiatric disease, and provide new clues to prevention and treatment. The goal of this proposal is to identify genes whose actions are modulated by maternal nutrition and that influence behavior in adult offspring. The experimental design contains several innovative components, and makes use of a powerful new genetic resource for mouse studies, the Collaborative Cross (CC). The CC is a panel of recombinant inbred mouse lines, each of whose genomes comprises a balanced but randomized mosaic of genetic material from eight diverse founder strains. The proposed study uses the known haplotypic composition of the CC lines to generate sparse diallel cross of reciprocal matings between CC strains (CC recombinant inbred mice, or CC-RIX) that allows a genomewide survey of genetic effects that vary by parent of origin and maternal diet. Specifically, mating CC females will be exposed to nutritional deficiencies during gestation and in the postpartum period. CC-RIX offspring will then be subject to behavioral testing and assayed for gene expression by microarray. Novel statistical methodology developed during the project will be applied in order to identify and characterize strain- and diet-specific effects, as well as parent of origin effects due to allelic imbalance. Candidate genes will be assessed by direct behavioral examination of knockout mice and also by knockout complementation testing to validate the role of both diet and strain. The proposed research represents a novel application of a powerful new genetic platform in order to elucidate how genetics, in utero exposures, maternal diet and parent-of-origin combine to affect behavior.

PROJECT SUMMARY PROJECT 2 ? COCAINE SENSITIVITY AND SENSITIZATION Addiction is a chronic, relapsing disease that begins with initial exposure, followed by escalating and uncontrolled use of the drug. However, not every individual who is exposed to drugs will develop addiction. In humans, initial sensitivity to psychostimulants has been shown to predict subsequent chronic drug use, and neuroadaptations following repeated use are thought to contribute to drug craving and risk of relapse. Acute locomotor activation and chronic locomotor sensitization to psychostimulants have been developed as animal models of initial sensitivity and neuroadaptive changes. Individual differences in these behaviors have been shown to be due, in part, to genetic differences. However, identifying specific genes has been a challenge using existing animal models. This Project utilizes two new mouse populations that have been designed to study complex traits, the Collaborative Cross (CC) and Diversity Outcross (DO). These populations have increased genetic and phenotypic variability and recombination that results in more precise mapping. Using a combination of behavioral phenotyping and gene expression analysis, the CC and DO will be used to identify genes and gene networks that contribute to phenotypic variation for both acute locomotor sensitivity and sensitization. CC lines that exhibit extreme phenotypes will be further characterized to determine whether pharmacokinetic variation is responsible for behavioral differences, or whether cocaine sensitivity correlates with dendritic spine density in the nucleus accumbens. The CSNA brings together a team of scientists with expertise in various aspects of addiction-related behavior, and a common goal ? identifying the underlying genetics of addiction-related phenotypes and advancing our understanding of the shared genetic etiology that underlies specific processes of addiction. Using an integrative experimental platform we will be able to uncover the biological basis of the relationship between initial drug response, sensitization, neuroadaptation and addiction related behaviors.

ABSTRACT Substance use disorders (SUD) result in increased risk of morbidity and mortality and impose substantial personal and financial burdens on affected individuals and society as a whole. There is considerable evidence that genetics increases the risk for SUDs, but identifying specific gene variants has been hampered by disease heterogeneity, genetic complexity and the inability to control or account for environmental exposures in human populations. Some of these issues can be overcome with addiction genetic studies in rodent models. However, standard methods of mapping in rodent populations often result in identification of large genomic regions that require considerable effort to narrow and thus delay or prevent identification of the causal gene variant. Recent technology and bioinformatic resources have made it possible to conduct genetic mapping studies using crosses between inbred mouse substrains (called Reduced Complexity Crosses or RCCs). Inbred mouse substrains diverge when inbred strain breeding colonies are transferred between collaborators or commercial entities. Polymorphisms segregate between these subpopulations at regions of the genome for which the parental strain has not yet become fixed, or from genetic drift, resulting in sets of strains that are genetically very closely related. Therefore, while standard inbred strains differ from each other at hundreds of thousands of loci, inbred substrains differ at thousands of loci ? only a fraction of which will be functional. These characteristics expedite identification of causal genes in substrains for which phenotypic differences exist. This approach has been used to identify a cocaine-sensitivity gene but has been limited to C57BL/6 substrains. Dozens of inbred substrains exist and opportunities for identifying genes that influence addiction-related behaviors are being missed. We have identified a striking phenotypic difference in cocaine locomotor sensitivity between the C3H substrains, C3H/HeJ and C3H/HeNTac. In this R21 application, we propose to use a RCC to identify the causal allele(s) for the phenotypic differences in these substrains. We will also examine substrain-specific pharmacokinetic and dose response phenotypes and examine the rewarding and reinforcing effects of cocaine using the drug self- administration paradigm. These studies will establish a drug response profile for each C3H substrain and allow us to begin exploring mechanisms by which the causal allele alters behavior.