Jeanne M. Wehner - US grants

Numerous studies of learning ability have suggested that some genetic factor or factors are involved in the regulation of cognitive development, but the biological nature of such factors remains largely unknown. Dr. Wehner is searching for these factors using a clever animal model which encompasses both genetics and learning. Highly inbred strains of mice are trained to perform in a complex learning task, and their learning performances are then compared to the brain chemistry of several identifiable regions in the brain. Each individual within an inbred population is equivalent to every other (like identical twins). This provides an excellent population of subjects in which to examine how genetically determined components of brain chemistry might govern the processes of learning and memory. Dr. Wehner's studies are correlating the activity of the neurotransmitter acetylcholine with learning ability using both a spatial and a non-spatial learning task. It is well known, for example, that the hippocampus (a major structure within the forebrain) plays an important role in spatial learning, but that it is not involved in many forms of non-spatial learning. Thus, these investigations are providing information about the nature of the learning process itself, as well as the role of genetic determinants of brain chemistry in different forms of learning. She has already observed, for example, that specific enzymes within the hippocampus vary with the learning abilities of different mouse strains.

The proposed studies are designed to study the "tension-reduction hypothesis" for alcohol consumption by using animal models to examine the effects of stress and ethanol on brain neurochemistry. The animal models used will be the long-sleep (LS) and short-sleep (SS) mice which differ in CNS sensitivity to ethanol, recombinant inbreds of LS and SS mice, and various inbred strains of mice. Ethanol in humans and in animals produces a release of adrenal steroid hormones. It is known that ethanol differentially induces corticosterone release in various genetic stocks of mice, but the consequences of this release are unknown. Furthermore, it has been proposed that steroid hormones can modify GABAergic function in the CNS. We propose to examine the hypothesis that some of ethanol's acute actions are mediated via activation of corticosterone release either directly, or indirectly by an interaction with the GABAergic system. Ethanol's acute low-dose anxiolytic actions and high-dose sedative actions will be examined before and after mild stress. These studies will measure brain glucocorticoid receptors and GABAergic receptors. Animals will be adrenalectomized and steroid replacement studies will be performed to examine the role of corticosterone in CNS response to ethanol. Genetic correlational studies will be performed to examine the relationship between corticosterone release and behavioral actions of ethanol.

The proposed plan is to carry out ongoing research based on a previously funded RO-1 grant entitled "Ethanol-corticosterone-GABAergic interactions in LS and SS mice", and to broaden technical expertise in the area of molecular genetics. Participation in the ongoing research will be increased by a reduction of Dr. Wehner's current teaching load. Her immediate goal of understanding the role of stress in ethanol sensitivity will be pursued by engaging in current studies of stress/ethanol interactions regulating behavioral responses and examining the actions of steroids and ethanol at the GABA/Benzodiazepine receptor. The research plan includes examining the role of corticosterone in regulation of CNS sensitivity to ethanol using several behavioral measures. Long-sleep and short-sleep mice will be used because they differ in ethanol-induced corticosterone release and GABAergic receptors. Corticosterone levels will be manipulated by adrenalectomy, acute corticosterone treatments, and inhibition of corticosterone synthesis. The long-term goals of the research are to understand genetic regulation of ethanol sensitivity and changes occurring as a consequence of chronic stress and ethanol treatment. These goals will be pursued by acquiring training in molecular genetic techniques. These include quantitative trait loci mapping techniques, polymerase chain reaction techniques, and data analyses pertaining to mapping. These technologies are available in Dr. Thomas Johnson's laboratory at the University of Colorado. Thus, training in molecular genetics can be accomplished with- out leaving the candidate's home institution. Studies designed to understand the genetic regulation of differences in the above parameters in LS and SS mice will be pursued using recombinant inbred strains. Techniques for quantitative trait loci mapping will be applied as the technique are acquired by the candidate.

Previous studies from our laboratory have indicated that genetic differences in spatial learning performance can be revealed using the Morris water task. C57BL/6 mice performed relatively well while DBA/2 mice perform poorly on the task. These strains and many recombinant inbred strains generated from a C57 X DBA/2 cross show variation in spatial learning performance and this variation is significantly correlated to hippocampal protein kinase C activity. Protein kinase C (PKC) is activated via generation of diacylglycerol during phosphatidyl inositol turnover, an important second messenger in brain. Because of the central role of PKC in the brain, it provides an important substrate for further investigation using a genetic approach. We purpose to examine the role of hippocampal PKC in determining genetic differences in learning performance from several perspectives. The nature of the learning deficit in DBA mice will be examined further using behavioral approaches. The question of whether protein kinase C activity changes as a function of training will also be investigated. Biochemical studies will be designed to elucidate the nature of the strain differences in hippocampal PKC activity by investigating the isozymic patterns in C57 and DBA mice. Molecular genetic studies will examine the DNA sequence of the major PKC genes from C57 and DBA mice; the expression of these genes in brain and potential effects of training on gene expression; and the regional localization of this expression will be examined by in situ hybridizations. The results of the proposed studies should allow a more complete assessment of the nature of spatial learning performance deficits, and provide information as to approaches for the future correction of such deficits. Furthermore, the role of hippocampal PKC in learning and memory processes should be delineated.

The goal of these experiments is to examine the memory component of behavioral tolerance to chronic administration of ethanol. We propose that learned tolerance involves activation of systems that may modulate complex learning and memory processes in mice. Current evidence suggests that brain protein kinase C may modulate some forms of learning and memory. To examine whether activation of protein kinase C is involved in the development of tolerance to ethanol, a genetic approach will be used. C57BL/6 mice which develop tolerance to the hypothermic effects of ethanol after intraperitoneal administration will be compared to DBA/2 mice which do not show pronounced tolerance to the hypothermic effects of ethanol after repeated administration. Previous experiments from this laboratory have shown that C57BL/6 and DBA/2 mice also differ in complex learning performance and hippocampal protein kinase C activity. Thus a relationship may exist between these parameters and the development of learned tolerance to ethanol. Learned tolerance to ethanol will be examined using two different routes of administration which differ in the complexity of environmental cueing and handling of the animals: 1) to maximize cueing, ethanol will be administered via intraperitoneal injections and body temperature will be monitored in mice handled daily and tested in different environments and 2) to minimize cueing, mice will receive intravenous administration of ethanol with constant monitoring of body temperature using minimitter systems. Dose-response and time-course experiments will be performed. Biochemical measurements of protein kinase C and phorbol ester binding in seven brain regions including hippocampus will be performed during and after ethanol treatment. After optimal conditions are selected, changes in potential protein kinase C phosphoprotein substrates will be measured as a function of ethanol tolerance in C57BL/6 and DBA/2 brain slices. Lastly, a BXD recombinant inbred strain study will be performed to assess whether common genes mediate the development of learned tolerance to ethanol, protein kinase C activity, or measurements of complex learning performance. These studies should provide new information concerning the role of learning and memory systems in the development of ethanol tolerance.

Previous studies from our laboratory have indicated that genetic differences in spatial learning performance can be revealed using the Morris water task. C57BL/6 mice performed relatively well while DBA/2 mice perform poorly on the task. These strains and many recombinant inbred strains generated from a C57 X DBA/2 cross show variation in spatial learning performance and this variation is significantly correlated to hippocampal protein kinase C activity. Protein kinase C (PKC) is activated via generation of diacylglycerol during phosphatidyl inositol turnover, an important second messenger in brain. Because of the central role of PKC in the brain, it provides an important substrate for further investigation using a genetic approach. We purpose to examine the role of hippocampal PKC in determining genetic differences in learning performance from several perspectives. The nature of the learning deficit in DBA mice will be examined further using behavioral approaches. The question of whether protein kinase C activity changes as a function of training will also be investigated. Biochemical studies will be designed to elucidate the nature of the strain differences in hippocampal PKC activity by investigating the isozymic patterns in C57 and DBA mice. Molecular genetic studies will examine the DNA sequence of the major PKC genes from C57 and DBA mice; the expression of these genes in brain and potential effects of training on gene expression; and the regional localization of this expression will be examined by in situ hybridizations. The results of the proposed studies should allow a more complete assessment of the nature of spatial learning performance deficits, and provide information as to approaches for the future correction of such deficits. Furthermore, the role of hippocampal PKC in learning and memory processes should be delineated.

DESCRIPTION (Adapted from applicant's abstract): Individual differences in general and specific cognitive function in humans are regulated in part by genetic factors but little is known about the genes mediating this variation. Genetic regulation of complex learning can be addressed in animal models due to the highly conserved nature of brain structures. Contextual and noncontextual fear conditioning, a form of complex emotional learning, can be measured in both human and rodents. In contextual fear conditioning, learning the association of a mild shock with a sound in a particular chamber is shown as a fear response. Until recently, the genes regulating this type of complex phenotype have been difficult to map because of the polygenic nature of this regulation. The recent application of quantitative trait loci (QTL) analysis, a form of linkage analysis, allowed the identification of five QTLs on chromosomes 1, 2, 3, 10 and 16 that explain 70% of the genetic variation between C57BL/6 (B6) and DBA/2 (D2) for contextual fear conditioning. Some of these QTLs overlap with QTLs identified in other laboratories regulating emotionality or contextual fear conditioning in other genetic crosses. Thus, both QTLs that generalize across strains of mice and QTLs specific to the differences between B6 and D2 strains have been identified. The goal of the proposed studies is to narrow these QTL regions for further study. The specific aims of the proposal are: 1.) To create congenic mouse lines for QTLs on chromosomes 1 and 3 which regulate contextual fear conditioning and noncontextual fear conditioning identified previously in a cross of B6 and D2 mice. 2.) To examine whether the QTLs for fear conditioning also regulate genetic variation in other forms of complex learning or fear-related behaviors. 3.) To characterize differences in gene expression between interval-specific congenics and the B6 background strains in naive mice and after contextual fear conditioning. Our long-term goal is to identify the genes that regulate behavioral differences in mice with the hopes that applying this information to understand regulation of human learning. Studies of this form of learning are not only applicable to normal complex learning processes but also to certain psychopathologies like post-traumatic stress syndrome.

The goal of this RCA is to further the investigation and development of techniques for the investigation of the genetic basis of alcohol dependence. Short-term studies are designed to address the role of protein phosphorylation in regulating sensitivity and neuroadaptive processes. Protein phosphorylation is a basic regulator of cellular processes. One gene family the calcium/lipid activated protein kinase C (PKC) encodes at least 11 isotypes. Among these isotypes, the y-PKC isotype, is the only PKC solely expressed in neurons of the brain and spinal cord. Until the creation of null mutant mice ("knock-outs") that carry a disrupted y-PKC gene, it has been difficult to analyze the role of specific isotypes in behavioral, biochemical, and physiological processes. Y-PKC null mutants differ in their responses to ethanol but these responses are mediated by polygenic systems and it is unknown whether the impact of the null mutation in y-PKC is influenced by genetic background and varying gene-gene interactions. The goal of this proposal is to investigate the question of epistatic interactions by introgressing the null y-PKC or wild-type on to a variety of inbred strains which are known to differ in their responses to ethanol to create congenic lines. These backgrounds include: C57/BL6J, DBA/2J, and 129/Svev. Initial sensitivity to ethanol, the development of ethanol tolerance, and drinking of ethanol will be examined in these congenic lines as well as ligand-gated ion channel function. Long-term studies are designed to establish the molecular technique of RNA differential display to be used to address the functional nature of neuroadaptive processes that underlie tolerance development to ethanol and the lack of such tolerance in y-PKC null mutants.

This institutional training grant is for training in the field of behavior genetics. The goals of behavior genetics are to elucidate the genetic and environmental components that regulate individual differences for a multitude of complex normal and abnormal phenotypes. Using multidisciplinary approaches, the genetic basis of psychopathologies, personality disorders, as well as normal cognition and personality, can be investigated. The application of biometrical genetic techniques and the development of quantitative trait loci methods allows the mapping of genes that regulate complex polygenic traits. Information from such analyses, along with neurochemical, neuropharmacological, and molecular genetic studies, will provide an understanding of gene function related to behavior. The Institute for Behavioral Genetics (IBG) at the University of Colorado has actively pursued the goals of behavior genetics for over 30 years. Its faculty is distinguished and active in research. Major research projects are now in progress in both human and animal behavior genetics. Facilities are available for gene mapping studies in human, mouse and nematode models, behavioral and biochemical studies in mice and nematodes, and biometrical analyses. Funds are requested to support five predoctoral and two postdoctoral trainees. Predoctoral trainees receive doctorate degree from a cooperating academic unit and certification in behavior genetics. Academic requirements in the training program include training in behavior genetics, quantitative and biometrical genetics, theoretical and computer-based statistics, molecular genetics, responsible conduct of research, and courses on behavioral and clinical phenotypes. Additional requirements vary according to the degree granting academic unit. Research experience is an integral part of training. Training for postdoctoral trainees is tailored to the individual, but acquisition of competence in all areas of the predoctoral program, as well as active participation in supervised research is emphasized.

DESCRIPTION (provided by applicant): Alcohol and nicotine are frequently used together. An extremely large fraction of alcoholics are also heavy smokers and studies in rodents have demonstrated that mice and rats selectively bred for differences in initial sensitivity to ethanol also differ in response to nicotine. The pharmacological and behavioral effects of ethanol and nicotine are both complex. Ethanol has multiple sites of actions in the brain including many ligand-gated ion channels. Nicotine?s actions in the brain are produced by the activation of nicotinic cholinergic receptors that are multimeric ion channels encoded by a large gene family. The goal of the proposed studies is to determine whether any of the nicotinic receptor subunits are involved in specific actions of alcohol by creating and testing null mutants with deletions in several of the important nicotinic receptor genes. Studies will investigate single-gene manipulations in the major, widely expressed subunits as well as other subunits with more limited but interesting expression patterns. A wide range of pharmacological and behavioral actions of ethanol will be assessed including: initial sensitivity, several forms of tolerance, anxiety, learning and memory, attention, ethanol consumption and impulsivity. The specific aims are: 1.) To examine the role of alpha4 containing brain nicotinic receptors in mediation of alcohol?s effects by creating constitutive and conditional alpha4 null mutants as well as those containing a site specific mutation in the alpha4 gene; 2.) To determine whether an alpha7 brain nicotinic receptor null mutation alters ethanol-related behaviors; and 3.) To examine the effects of alpha4 and alpha7 receptor null mutations on various genetic backgrounds. The results of these studies should provide a better understanding of the potential interactions and mechanisms underlying the co-administration of ethanol with nicotine.

[unreadable] DESCRIPTION (provided by applicant): This institutional training grant is for training in the field of behavior genetics. The goals of behavior genetics are to elucidate the genetic and environmental components that regulate individual differences for a multitude of complex normal and abnormal phenotypes. Using multidisciplinary approaches, the genetic basis of psychopathologies, personality disorders, as well as normal cognition and personality, can be investigated. The application of biometrical genetic techniques and the development of quantitative trait loci methods allow the mapping of genes that regulate complex polygenic traits. Information from such analyses, along with neurochemical, neuropharmacological, and molecular genetic studies, will provide an understanding of gene function related to behavior. The Institute for Behavioral Genetics (IBG) at the University of Colorado has actively pursued the goals of behavior genetics for over 35 years. Its faculty is distinguished and active in research. Major research projects are now in progress in both human and animal behavior genetics. Facilities are available for gene mapping studies in human, mouse and nematode models, behavioral and biochemical studies in mice and nematodes, and biometrical analyses. Funds are requested to support 4 predoctoral and 1 postdoctoral trainee. Predoctoral trainees receive doctorate degrees from a cooperating academic unit and certification in behavior genetics. Academic requirements in the training program include training in behavior genetics, quantitative and biometrical genetics, theoretical and computer-based statistics, molecular genetics, responsible conduct of research, and courses on behavioral and clinical phenotypes. Additional requirements vary according to the degree granting academic unit. Research experience is an integral part of training. Postdoctoral trainees also pursue a formalized program that emphasizes individual research as well as competence in molecular and quantitative behavior genetics. Other activities in their preparation include: supervision of students and/or technicians, hosting of seminar speakers, guest lecturing, and mandatory attendance in a course on the responsible conduct of research and a weekly journal club. [unreadable] [unreadable]