Tamara J. Phillips - US grants

motivation; behavioral genetics; pregnane compound; corticosterone; ethanol; hormone regulation /control mechanism; hypothalamic pituitary adrenal axis; quantitative trait loci; restraint; psychological stressor; corticosteroid receptors; conditioning; alcoholic beverage consumption; genetic strain; linkage mapping; pharmacogenetics; neurohormones; neuropharmacology; preference; behavioral habituation /sensitization; reinforcer; chordate locomotion; phenotype; behavioral /social science research tag; behavior test; laboratory mouse;

DESCRIPTION: Quantitative Trait Locus (QTL) mapping has suggested that the gene coding for the serotonin 5-HT1B receptor subtype may influence several responses to ethanol. Preliminary data suggest that 5-HT1B knockout mice that lack this gene show excessive alcohol drinking, reduced sensitivity to ethanol-induced ataxia, possibly attenuated tolerance, but comparable acute ethanol withdrawal, relative to their wild-type counterparts. Mouse 5-HT1B receptors have been localized predominantly in axon terminals of the globus pallidus, substantia nigra, dorsal subiculum, raphe, and cerebellar deep nuclei. However, the specific neural 5-HT1B receptor population(s) that may be affecting these ethanol responses is unknown. We propose in Aims 1-3 to use congenic chimeras between 5-HT1B knockout and wild-type mice, to identify specific 5-HT1B receptor populations that subserve individual alcohol drinking and ataxia scores. In Aim 4, the 5-HT1B receptor will be reexpressed in specific parts of the brains of 5-HT1B knockout mice to test the hypothesis that the 5-HT1B heteroreceptors, rather than the autoreceptors, are responsible for the altered phenotype(s) of the knockout mice. It is expected that alcohol preference and ataxia will be altered by reinstatement of particular receptor populations. These studies will allow us to target specific aspects of neuropharmacology in functional studies to be performed later. In Aim 5, inducible knockouts will be produced, in which mice develop with normal levels of 5-HT1B gene expression that can be inhibited in adulthood. This will permit examination of the role that possible compensatory changes during development might play in determining alcohol drinking and ataxia. Finally, Aim 6 will continue the characterization of 5-HT1B and wild-type mice for additional motor and other behavioral responses to ethanol. These studies will serve as a preamble to future investigations using the approaches outlined above, with the intention of performing neurogenetic analyses with chimeras, tissue-specific and inducible knockouts for those phenotypes which prove to be associated with the 5-HT1B receptor.

The goal of this project is to explore the molecular mechanisms underlying the relationship between stress and alcohol's motivational and neuroadaptive consequences. We shall focus on the role of corticotropin-releasing hormone (CRH), the critical mediator of the stress response, in determining these alcohol effects. Relatively little is understood concerning alcohol consumption and relief from stress or the influence of stress hormones on alcohol reward neuroadaptation. Whereas there is substantial support for these associations, thus far there are no causal links. We postulate that CRH is a principle element, perhaps even the keystone, in stress-associated mechanisms of alcohol addiction, and that disruption or dysregulation of CRH pathways leading to altered stress reactivity thereby influences patterns of ethanol reinforcement, aversion and neuroadaption. We propose to test these postulates using mice with specific genetic alterations in CRH pathways. In particular, we will test mice deficient in CRH or the CRH receptors, CRH-R1 and CRH-R2 and mice that overexpress CRH in the brain. We predict that specific effects of these mutations will be seen on ethanol reinforcement, aversion, and neuroadaptation to ethanol. Specific aim 1 will determine whether dysregulation of CRH pathways alters the motivational effects of ethanol. We shall measure voluntary ethanol drinking behavior, and ethanol-induced conditioned place preference and taste aversion in the CRH mutant lines listed above. Specific aim 2 will determine whether dysregulation of CRH pathways alters neuroadaption to ethanol. We shall measure ethanol-induced sensitization and cross-sensitization between stress and ethanol, and severity of ethanol dependence. Specific aim 3 will determine whether specific changes in gene expression patterns are associated with behavioral patterns of neuroadaptation of ethanol. This research represents an important step toward the validation of CRH receptors as targets for future medications development in the treatment and prevention of alcoholism.

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

short term memory

Psychostimulant drugs are addictive, and relapse to drug taking, even after prolonged periods of abstinence, is highly probable. The behavioral and neurochemical responses to psychostimulant drugs that change in concert with amount of drug exposure are thought to play an important role in addiction and relapse. A key hypothesis of Scientific Component 6 is that the seemingly diverse central nervous system alterations associated with addiction are coordinated in ways that are difficult to define by studying each in isolation. Two sets of selectively bred mice will be developed by the Animal Core Component 3 and used to help identify the genes and combinations of genes that influence methamphetamine (MA) self-administration and neuroadaptation. In Specific Aim 1, the genetic relationship between sensitization and self-administration will be examined in lines of mice bred for increased and reduced sensitivity to locomotor sensitization (a measure of neuroadaptation) produced by MA, and in separate lines bred for high and low oral MA self-administration; the operant intracranial MA self-administration model from Scientific Component 5 will be used to validate the oral self-administration model. In Specific Aim 2, full genome scans will be performed to identify genetic loci influencing each of the selected traits. In addition, data will be subjected to a search for epistatic (gene-gene) interactions to provide important insights into the genetic interplay that likely influences these complex traits. In Specific Aim 3, gene expression patterns will be examined in the brains of the selected lines using microarray methods on tissue from specific neuroanatomical locations to get a global picture of what gene expression differences may be associated with the differences in drug sensitivity. Neuroanatomic locations will be informed by Component 5 &pilot 8A. Finally, in Specific Aim 4, other traits that may be genetically correlated with the selection traits will be examined in the selected lines. For example, sensitivity to stress-induced reinstatement of MA-induced conditioned place preference will be examined;possible differences in impulsivity will be assessed using the Delay Discounting Procedure; neurochemical correlates will be examined in collaboration with Component 5. The preclinical genetic findings will be translated to our clinical investigators. Common implication of neuroanatomical pathways found across the Scientific Components could lead to the identification of coordinated pathways that influence addiction.

DESCRIPTION (provided by applicant): This program for pre- and post-doctoral training supports the training of specialists who are able to conduct basic research at levels ranging from the molecular to the cognitive/clinical, on the biological mechanisms underlying the etiology, treatment and prevention of alcohol use disorders. Twenty-three members of the graduate faculty of the Oregon Health & Science University (OHSU) serve as preceptors for predoctoral students and postdoctoral research fellows in two graduate programs at OHSU-Behavioral Neuroscience, and the Neuroscience Graduate Program. Major research interests represent five areas of common interest: (1) genetic bases for alcohol and responses and risk, (2) learned and unlearned determinants of alcohol and drug reward, (3) neurobiological bases for the rewarding and aversive effects of alcohol and other drugs, (4) neuroadaptive mechanisms associated with ethanol dependence and sensitization, and (5) effects of alcohol on memory and cognition. Technical strategies reflect four levels of analysis: I. Behavioral pharmacological/pharmacogenetic, II. Neurochemical/neurophysiological/ neuropharmacological, III. Cellular/molecular biological and IV. Cognitive neuroscience/social, including human/clinical level. Coordinated research efforts within the Portland Alcohol Research Center (PARC) and the Integrative Neuroscience Initiative on Alcoholism (INIA) have strengthened training by unifying investigators and creating multidimensional research projects. Training includes firm curricular grounding in the basic sciences, specific pharmacological training in alcohol and other abused drugs, and extensive and continuous participation in research. Six predoctoral trainees per year, beginning with 0-2 years of graduate experience, will be supported by the training grant for 2-3 years, and then by individual National Research Service Awards or their mentors' resources. Three postdoctoral trainees per year with 0-1 years of postdoctoral experience will be supported by the training grant for 2 years. We have a well developed plan for improving the diversity of our trainees and all trainees are expected to complete an initial intensive course in the Responsible Conduct of Research, as well as continuing education in this area. Ample opportunities exist for our trainees to be involved in public education and outreach.

The Pilot Project Component is designed to provide a flexible means for developing and exploring new research activities or directions, and unique opportunities that can evolve into independently funded research projects. In addition, the Pilot Project Component provides a resource for attracting new investigators into drug abuse research. We propose to fund an average of three pilot projects per year with an average budget of $33K. The expected duration of these pilot projects will be 1-2 years. The Center Scientific Director will manage this Component. Pilot Project Component applications will be solicited from all OHSU- and Portland VA Medical Center-based investigators annually. Each proposal will be evaluated for scientific merit/innovation, thematic alignment and several other criteria by at least two members of the Center's Scientific Advisory Board, the Scientific Director, and the Center Director. Recommendations for funding will be considered for approval by the Center Executive Committee. Three pilot projects are proposed for funding in Year 1 of the Methamphetamine Abuse Research Center (MARC). Project 8A (W. Hoffman, PI) will use functional magnetic resonance imaging to study neuroanatomical associates of MA addiction and heightened impulsivity. Project 8B (S. Mitchell, PI) will examine in mice the notion that impulsivity is related to MA self-administration, using a delay discounting procedure. Project 8C (J. Raber, PI) will explore the hypothesis that histamine mediates the detrimental long-term effects of neonatal MA exposure on cognition. All of these pilots are related to a Center theme at least by virtue of their examination of MA effects. In addition, mice used in Project 8B and 8C will share genetic background with those used in Scientific Components 5 and 6, and will obtain mice from the Animal Core. Pilot project 8A also relates to the Center impulsivity, neuroadaptation and neuroanatomy themes;Pilot project 8B relates to the Center impulsivity theme;and Pilot project 8C relates to the Center stressor responsivity and neuroadaptation themes. It is expected that Pilot Component projects funded in years 2-5 of the Center will contribute to the study of MA abuse and to one or more of the additional themes of the Center: Impulsivity, stressor responsivity, neuroadaptation, and neuroanatomy, and to our goal of pursuing translational research in preclinical and clinical models.

The Animal and Phenotyping Core will provide services to all Components and Pilot Projects that require the use of mice. The main objective is to streamline animal production, distribution and testing of key phenotypes for Portland Alcohol Research Center (PARC)-related, and when possible, other projects. The Core will purchase mice for use in experiments or as breeders, assist in the creation or maintenance of animals of specific genotypes (e.g., knockouts, transgenics, selected lines), test mice for ethanol consumption, acute ethanol withdrawal, chronic ethanol withdrawal, impulsivity (using the Go/No-Go task), and withdrawal-induced drinking, and perform blood and brain ethanol concentration analyses as needed. The Phenotyping Division of the Core will optimize procedures for all traits for which it will oversee data collection. Members of the PARC with proven expertise have been included as key personnel to make certain that data are of the highest quality. The Core will determine animal distribution and phenotyping priorities, and maintain a database with individual animal information that can be communicated to investigators and to the Molecular and Bioinformatics Core (Component 3). As has been the case for the past and current years of the PARC, the breeding, production, procurement and supply of genetic animal models will remain under the direction of Dr. Tamara Phillips, Director. Dr. Phillips will also have responsibility for oversight of ethanol consumption data collection. Dr. Pamela Metten will continue as a Co-Director of the Core and will continue to take responsibility for oversight of dependence induction and training in withdrawal measurements, as well as blood and brain ethanol concentration measurement and analysis. Dr. Deborah Finn has been added as a Co-Director to provide expert advice for studies focused on ethanol withdrawal-induced drinking, since she has special expertise from her work associated with the Integrative Neuroscience Initiative on Alcoholism. Finally, Dr. Suzanne Mitchell is a newly added Co-Director who will oversee impulsivity data collection in mice, using a Go/No-Go task. Research results from the current 5-year period have led to newly proposed research across several components directed at more detailed analyses of relationships between ethanol drinking, withdrawal and impulsivity/response inhibition.

DESCRIPTION (provided by applicant): We maintain a large number of unique genotypes of mice for alcohol research studies. These animals are used in research supported by multiple R01s, U01s, the Portland Alcohol Research Center (PARC) P60 grant, and several VA Merit Review Grants. These animals have been used for research by investigators in Portland, Oregon and at many other sites around the world. Partial support for all these genotypes is budgeted within the primary grants employing them, but cost inflation and the lack of synchrony between grant cycles and actual use has led us to fall further and further behind our ability to support these mice. Our ability to keep up is due partly to inflation exceeding the NIH COLA limits, and partly to budget caps on several of the principal supporting grants (P60, UOIs, and VA Merit Reviews). The proposed R24 seeks modest support to maintain these genotypes, in each case cost-shared with user fees and maintenance support from the parent grants. There are four aims: Aim 1. Help maintain core breeding colonies of long- term selected lines and their genetically heterogeneous control lines (WSP-1, WSP-2, WSR-1, WSR-2, FAST-1, FAST-2, SLOW-1, SLOW-2, WSC, HDID-1, HDID-2, and HS/Npt). Aim 2. Help maintain core breeding colonies of congenic strains and lines, and their progenitor inbred strains, for quantitative trait locus (QTL) gene mapping studies (17 genotypes covering parts of chromosomes 1, 6, and 19 and their progenitor inbred strains, C57BL/6J (86) and DBA /2J (D2), and two genotypes covering QTLs on chromosomes 1 and 11, iWSP2, iWSR-1). Aim 3. Help maintain core breeding colonies for the genetically heterogeneous stock HS-CC, derived by the Complex Trait Consortium for advanced intercross QTL mapping studies. Aim 4. Partially defray costs for cryopreservation of long term selected lines and congenic strains. Cryopreservation protects against catastrophic loss of crucial genotypes as well as providing access for potential future use [HOT-1, HOT-2, COLD-1, COLD-2, WSP-1, WSP-2, WSR-1, WSR-2, HDID-1, HDID-2, and 22 congenic strains (5 to be cryopreserved) for quantitative trait loci on chromosomes 1,4, 11 and 19]. All genotypes covered are freely available to interested investigators at cost. Funds to support distribution are not requested, as this support is provided under the primary grants supporting the various genotypes. Funds to support breeding and testing of additional animals are also not requested here, but rather under the parent grants. RELEVANCE: Genetic animal models, particularly with mice and rats, have been major contributors to our understanding of the pathophysiology of alcohol use disorders. The genetics and genomics resources available for mouse makes this species especially valuable for ascertaining genetic risk and developing novel pharmacotherapies. The models for which we seek support are unique.

Methamphetamine (MA) is remarkably addictive and relapse to excessive use is highly probable and poses serious health concerns. Genetic factors have been little studied with regard to their role in susceptibility to MA addiction or relapse. A key goal will be to utilize a validated animal model of genetically-determined high and low susceptibility to MA use to improve genetic mapping resolution and to study an already identified neuroimmune gene network that influences MA response in this genetic model. In Aim 1, in coordination with Animal Core 3, replicated sets of selected mouse lines bred for high and low voluntary consumption of MA will be produced and QTL mapping will be performed by the Biostatistics and Genetics Core 2. These mice will be used for studies proposed in Components 7, 8 and 9. In Aim 2, neurocircuitry will be examined in the selected lines, using cFos mapping after acute and repeated MA treatment, and these data will be used to identify brain regions for immune factor analysis, and will be compared to imaging results from Scientific Component 7 for brains from the MA consumption selected lines. In Aim 3, qPCR immunology arrays will be used to examine brain and peripheral blood mononuclear cell gene expression for immune specific genes using samples from selectively bred MA drinking line mice that have been acutely or repeatedly treated with MA or with saline, or are in remission. These data will be used in additional network analysis by the Biostatistics and Genetics Core 2 and compared to human peripheral gene expression results for controls, chronic MA users and user in remission. In Aim 4, cognitive, anxiety-like, and impulsivity-like traits will be examined in drug naive, acute, and repeated MA-exposed MADR mice, as well as mice in remission from MA exposure. Tissue from mice treated in the same way will be transferred to Translational Service Core 5 for analysis of immune factors and to Component 7 for imaging; half of each brain will be sent for each purpose to allow individual animal correlations to be performed. These data will also be examined for correspondence between behavior, neurocircuitry and immune system alterations. In addition, impulsivity-like measures in mice will be compared to similar measures in humans from Component 7. Finally, data collected in Component 9 will inform Component 8, with regard to traits and which of the selected lines to be studied for immunotherapeutic intervention. Cross-species analyses across components will identify key immune factors associated with chronic MA exposure (and remission) and MA-induced neuropsychiatric impairments, with the goal of ultimately identifying novel immunotherapeutic interventions.

Pilot Core 6 is designed to provide a flexible means for developing and exploring new and innovative research activities or directions, and unique opportunities that can evolve into independently funded research projects. The pilot program is also expected to attract new investigators, and investigators new to methamphetamine (MA) research. During Years 6-10, we propose to fund an average of 3 projects/year with an average budget of $33K/project and an expected duration of 1-2 years. The Center Scientific Director, T. Phillips, will manage this Core, and applications will be solicited from all OHSU and Portland VA Medical Center-based investigators annually. Each proposal will be evaluated for scientific merit/innovation and for relatedness to the Center's overall goals by at least two members of the Center's Scientific Advisory Board (or in some cases, other external experts in the scientific field), the Scientific Director, and the Center Director. Recommendations for funding will be considered for approval by the Center Executive Committee. The Center Director, A. Janowsky, will submit written notification of the desire to initiate a new project to NIDA before implementing funding, and will await final approval from the NIDA Program Officer, as required. Occasionally, when a special opportunity arises, we may solicit an application for review outside of the cycle described above. For example, a faculty member could have an idea or possess a technique that would benefit the Center, and the Center might be able to provide a small amount of pilot funding (after scientific review and approval from our NIDA Program Officer) to help initiate these novel studies. The three new pilot projects funded in MARC Year 5, all from investigators new to MA research, will be considered for renewal in Year 6, along with other solicited applications. Project 6A (M. Ford, PI) will explore the ramifications of altered muscarinic receptor function on the discriminative stimulus effects of MA. Project 6B (E. Boudreau, PI) will examine the role of circadian period on effects of MA in mice using optical microangiography (OMAG) and other imaging methods. Project 6C (L. Ganzini, PI) will provide important demographic information for individuals with MA abuse problems, that will be important for interpreting clinical research.

DESCRIPTION (provided by applicant): The Portland Alcohol Research Center (PARC) focuses on the etiology and prediction of risk of alcohol abuse, alcoholism, and specific alcohol-related health problems (e.g., withdrawal seizures, excessive drinking). The genetic risk and protective markers and gene networks that we are studying will help us to develop strategies for the prevention of alcoholism. The first overarching theme of the PARC is to use behavioral genomics strategies, through studies of gene mapping and expression, and development of new genetic animal models, to identify genes underlying ethanol neuroadaptation. The other main PARC theme is exploring mechanisms underlying and traits related to ethanol neuroadaptation. Two specific hypotheses have emerged from the synthesis of PARC and related projects' findings. The first hypothesis is the intriguing idea that withdrawal and drinking are influenced by some of the same genes and gene networks. Many different studies and genotypes have found that high-withdrawal genotypes are genetically predisposed to drink less than low-withdrawal genotypes. We now also consider the effect of chronic ethanol exposure on alcohol consumption. The second hypothesis is that high trait impulsivity is a significant genetic risk factor for high alcohol drinking. Genetic dissociation of different aspects of impulsivity is supported by PARC findings, with high impulsivity on delay discounting tasks predicting greater and high impulsivity on go/no-go tasks predicting less non-dependent drinking. The latter also predicts greaten withdrawal severity following dependence. Five research components, three core components, and a pilot project component address these themes and hypotheses using mouse models and non-human primates. To the extent possible across species, we are making a concerted effort to integrate the components with a core circuit of brain structures that relate to drinking, withdrawal, and impulsivity. Our expanded bioinformatics effort has enabled expansion of a key strength of our group from the analysis of the contributions of individual genes on behavioral functions of the whole organism to include gene network identification. The PARC Is recognized as a leader in quantitative trait gene (QTG) Identification, successfully pursuing a genetic locus Influencing withdrawal to discover the first alcohol-related behavioral response QTG, Mpdz, and recent studies identifying high-quality QTG candidates including Kcnj9. An Education and Outreach component trains pre- and post-doctoral students in alcohol research, disseminates research findings to the public, and engages in a range of activities with elementary-to-high school students.