David Pafford Crews - US grants

This is a request for an ADAMHA Research Scientist Award (RSA). My research objectives concern two important issues concerning the biological bases of behavior: (i) the evolution of behavior and the changes the underlying neuroendocrine mechanisms undergo in the process, and (ii) how genes and environment interact in the development of behavior. The behaviors measured are species-typical "innate" behaviors. Perhaps the greatest barrier to fruitful investigation of the first issue is that representatives of ancestral species usually no longer exist. The whiptail lizards are a rare case in which representatives of both the ancestral and the descendant species still exist. By comparing the neuroendocrine mechanisms that control sexual behaviors in the descendant species with those of the ancestral species, the evolutionary process can be examined directly. The fundamental problem of the second issue is that behavior is the combined result of both nature and nurture. Is it possible to relate gene expression during embryogenesis to the display of specific behaviors during adulthood? In other words, how are quantitative events like gene expression orchestrated into qualitative events like behavior? In many reptiles sex is determined by the temperature of the incubating egg. I am tracing how the temperature signal is transduced into a biological signal that acts ultimately at the genetic level to channel sexual development. This also has enabled me to begin examining the role of temperature-induced gene expression in the development of the adult phenotype. The perspective taken is multidisciplinary, combining the integrating molecular, cellular, physiological, and organismal phenomena within an evolutionary view of biological organization. Further, this research is conducted both in the field and in the laboratory, because the field is a valuable testing ground for the adaptive functions of behavior whereas the laboratory is the only possible arena for determining the physiological and developmental bases of behavioral phenomena observed in the field. This approach permits both the causal mechanisms and the functional outcomes of species-typical behaviors to become evident within each level of biological organization in a way that illustrates the relations among the level. A renewal of the RSA will enable me to continue collaborations with colleagues both at the University of Texas and other institutions. It will enable me to continue my field work, which usually occurs during the academic year. By freeing me from the usual course load and administrative duties, I can devote the majority of my time to research. It allows me to serve as mentor to postdoctoral, graduate, and undergraduate students working on independent research projects. The RSA has allowed me to participate in programs of continuing education for high school science teachers and to provide laboratory experience for high school students.

The specific objective of this proposal is to examine the causal mechanisms and functional outcomes of the two major annual reproductive tactics -- associated and dissociated -- exhibited by higher vertebrates. In many seasonally breeding vertebrates, gamete maturation and maximum secretion of sex steroid hormones immediately precede or coincide with courtship and copulatory (mating) behavior. This annual pattern may be termed the associated reproductive tactic, or prenuptial gametogenesis (Figure 1). A markedly different annual pattern is exhibited in many vertebrates, including some mammals, in which the gametes are produced only after the breeding season has ended; the gametes are then stored until the next breeding period. In these species, mating occurs when the gonads are not producing gametes and blood concentrations of sex steroid hormones are basal. This pattern may be referred to as a dissociated reproductive tactic, or postnuptial gametogenesis (Figure 1). I will fucus on one representative species of each reproductive tactic. The green anole lizard is similar to many laboratory and domesticated mammals and birds showing the associated tactic. In contrast, the red-sided garter snake shows the dissociated patterns. In many instances a direct comparison of these two species will be made, whereas in other instances gaps in our knowledge must be filled before conceptually valid comparisons can be made. Thus, some of the proposed experiments deal with only one species or tactic. Ultimately, however, my goal is to compare the two tactics at as many levels of organization as are feasible and resonable. Such a broad approach is crucial if important generalities underlying vertebrate reproductive processes are to emerge. My proposed studies will also contribute directly to our understanding of related areas of reproductive biology, including gamete storage and animal husbandry.

DESCRIPTION (provided by applicant): The goal of the proposed research is to elucidate the role of steroid hormones in controlling sexually dimorphic behaviors. Male-typical copulatory behaviors such as mounting and intromission are dependent on androgens in all vertebrates studied, but the mechanisms by which these hormones influence the brain and behavior are not well understood. Whiptail lizards of the genus Cnemidophorus enable a powerful comparative approach to understanding these mechanisms because some species are sexual, exhibiting the typical vertebrate pattern of sexually dimorphic behavior (estrogen-dependent receptivity in females and androgen-dependent mounting in males), and other species are all-female, reproducing clonally and exhibiting both male-like and female-like behavior, according to ovarian hormone levels. The experiments proposed involve C. inornatus, a typical sexual species, and C. uniparens, which is all-female. C. uniparens individuals, just like C. inornatus females, are receptive when their circulating estrogen levels are high, but unlike C. inornatus, they also exhibit the complete repertoire of male-typical copulatory behavior when presented with a receptive female during the periovulatory phase of the ovarian cycle when progesterone levels are high. This "pseudocopulatory" behavior can be evoked in the laboratory by administration of exogenous progesterone, and the operational goal of this research is to determine how progesterone is able to mimic the normal function of androgen in this way. Specific experiments will investigate how the progesterone receptor comes to be expressed in the preoptic area of the brain that is thought to mediate male-typical copulatory behavior, and in the process will yield information about the normal function of this area, as well as the developmental regulation of steroid hormone receptors. Other studies will elucidate the actions of androgens and progesterone on the neurotransmitters dopamine and nitric oxide, which are important components in the neural control of sexual behavior. This functional interchangeability of androgens and progestins has obvious fundamental implications for human health. All women normally experience fluctuating levels of progesterone, and millions of women use exogenous progestins. There is also a marked diurnal rhythm [in] progesterone levels in men, and progestins (usually in conjunction with testosterone) are beginning to be studied as a male contraceptive. The interaction of these progestins with the functions of other steroid hormones therefore warrants thorough investigation. From a scientific point of view, the hormonal control of sexual behavior is a particularly tractable model for elucidating the way in which particular genes, expressed in particular neural circuits, can affect specific behaviors, because the stimuli and behaviors involved are simple, the brain areas involved are well characterized, and the relationship between hormones and genes is comparatively well understood.

This is a proposal for the continuation of a training grant at the University of Texas at Austin focusing on interdisciplinary training in the biological bases of behavior. A small select group of behavioral neuroscientists from the Psychology and Zoology departments of the University have joined together to provide training with special emphasis on integrating physiological, developmental, ecological, and evolutionary determinants of behavior. This broad-based approach combines unique, yet fundamental, training in the physiological and psychological sciences. The training program has three primary features. First, there is training in how to conduct investigations in both field as well as laboratory settings. Naturalistic field studies provide clues to the functional links between ecology, physiology, and behavior. Laboratory studies allow for the greater control that is required for the study of underlying mechanisms. From both come animal model systems that aid in the understanding of mental health disorders in humans. Second, we emphasize that only by using an integrative approach is it possible to discover the relationships between the development, physiology, ecology, and evolution of behavior. In this way, the relations between different levels of biological organization are illuminated. Third, students are encouraged to become proficient with contemporary analytic methods needed to exploit successfully different levels of scientific inquiry. All trainees are required to take three didactic courses (neuroscience, behavioral physiology, and human neuropsychology). Throughout their tenure (4 years for predoctoral trainees and 2 years for postdoctoral trainees), trainees attend weekly seminars in the Psychology and Zoology departments. These seminars include summaries of state-of-the-art knowledge in various areas, laboratory demonstrations of new techniques, and discussions of ethical issues in the conduct of research. Trainees are evaluated for their steady progress in scholarship and research in quarterly meetings of the training faculty. A qualifying and a dissertation examination are required of all predoctoral trainees. All of the research service facilities of the Psychology and Zoology departments (computer specialists and electronics and machine shops) are available to the trainees, as well as the well-equipped laboratories of the training faculty, all of whom are recognized authorities in their fields, as evident by sustained productivity in research, invitations to national and international symposia, continual research funding from federal agencies, and extensive experience in providing research training. All of the laboratories comply with NIH requirements.

Mammals, birds, and reptiles constitute the amniote vertebrates. In all mammals and birds, and in many reptiles, sex is determined at fertilization by genotype (genotypic sex determination or GSD). In some reptiles, however, the temperature at which the egg incubates determines whether the embryo hatches as a male or a female (temperature-dependent sex determination or TSD) Thus, in these species, embryos are initially bipotential and only later does channelization toward the male or female phenotype occur. The initial bipotentiality and later determination of sex by species exhibiting TSD offer a unique model system for studies of the mechanisms of sex determination and sexual differentiation in all amniote vertebrates, including man. We have discovered recently that the administration of estrogen to embryos incubating at male-producing temperatures causes ovarian development, over-riding any temperature effect. The proposed research will investigate (i) how this estrogen effect occurs, (ii) whether, under normal conditions, steroid hormones are present before the gonad is committed to testicular/ovarian development, and (iii) whether embryonic steroids have a similar role in the differentiation of non-gonadal phenotypes as in mammals and birds. We also have discovered that the temperature that an embryo experiences profoundly affects its adult morphology, physiology, and behavior. The mechanisms by which incubation temperature organizes adult sexuality will be studied through hormonal manipulations of embryos and neonates by castration and/or administration of steroid hormones, synthetic hormone agonists and antagonists, enzyme inhibitors, and steroid antisera. Together, these experiments will determine the extent to which GSD and TSD have similar physiological or biochemical bases in the control of gonad determination and in the biopsychology of sexual differentiation. The proposed studies are of fundamental importance to understanding, the development of sexuality in vertebrates, including the relation of different levels of sexuality within an individual. The studies will use multiple techniques, including behavioral testing, histology, -immunocytochemistry, monoclonal antibodies for hormone receptors, synthetic steroid agonists and antagonists, high-performance liquid chromatography, gas chromatography/mass. spectrometry, and radioimmunoassay.

In contrast to the sex chromosome systems of mammals and birds, the sex of many reptiles is determined by the incubation temperature of the egg (temperature-dependent sex-determination, or TSD). Sex determination under TSD can also be controlled by the artificial application of specific steroid hormones, whereby the effects of temperature are overridden. The proposed work pursues this interplay in a turtle with TSD. The study has two specific aims. The first aim is to study further the physiological bases of TSD by (i) localizing key enzymes during the temperature-sensitive window, and (ii) capitalize on a recent discovery that exogenous androgen can cause male development. The second aim extends our line of research into the molecular biology of TSD/HSD to identify, map, and quantify steroid hormone receptors in the developing embryo. Together these studies will serve to generalize and unite anatomical, physiological, and molecular observations. The basic research proposed here has applications to the conservation of endangered reptiles.

This proposal requests continued support of a broadly based research program that seeks to understand (i) the evolution of hormone-brain-behavior mechanisms underlying sexual behaviors, and (ii) the dual neural circuits that subserve these behaviors. This goal is relevant to mental health since information on the brain mechanisms that control normal behavior provides the context in which to determine and evaluate psychopathology. The approach used is both comparative and multidiscipline. 'Me comparative method emphasizes the need for different model systems if we are to elucidate both the general rules which govern behaviors as when as their historical roots. Multidisciplinary studies spanning the molecular to the population levels of biological organization provide a multifaceted, yet integrated, perspective of individual behavior. The animal model systems to be used are parthenogenetic whiptail lizards and their sexual ancestors. The unisexual species is known to have descended directly from extant sexual species, thereby allowing direct ancestor-descendant comparisons. Because all individuals have ovaries, yet exhibit both male-like and female-like pseudosexual behaviors, the complication of having two gonadal sexes, each with their own particular hormonal milieu, is removed. This makes it possible to study the neural circuits that underlie mounting and receptive behavior in a manner not possible with more common laboratory animals. The studies proposed center on a comparison of the sexual ancestral species and their parthenogenetic descendants. They employ behavioral observation in both the field and the laboratory, functional neurology of specific behaviors as assessed by hormonal manipulation including intracranial implantation, hormone-receptor analysis, radioimmunoassay, immunocytochemistry, and autoradiography. Some experiments will exploit the fact that progesterone stimulates, rather than inhibits, the sexual behavior of males of the parental species and that progesterone activates male-like pseudosexual behavior in the unisexual descendant species. These studies will examine the mechanism of hormone action at the level of the receptor. Other studies will localize the sites of hormone action in the brain, show how different sexual behaviors are reflected in dimorphisms in brain nuclei, and identify and describe the dual neural circuits controlling mounting and receptive behavior.

ABSTRACT Proposal: #96-23546 PI: Crews & Rhen Title: Evolutionary origin of behavioral organization Organization of reproductive and aggressive behaviors by estradiol (via aromatization of testosterone in mammals) during early development is well documented in birds and mammals. There is a correlation between the organized and heterogametic sex in these groups; males are organized (lose the ability to display female-typical behavior) in mammals, whereas females are organized (lose the ability to display male-typical behavior) in birds. The opposite sex retains the capacity to display both female- and male-typical behavior. A similar correlation is observed in other vertebrates with genotypic sex determination. Alternate explanations are (1) that a developmental constraint results in organization of the heterogametic sex, or (2) that there is a functional relationship between organization and gonochorism. Reptiles with environmental sex determination do not fit into the constraint paradigm because they lack a heterogametic sex. In contrast, these species may be organized if organization is adaptive. In this study, manipulation of embryonic temperature (and thereby gonadal sex) and adult hormones will be performed to distinguish among hypotheses about organization. Log-linear, multivariate, and path analyses will be used to analyze reproductive and aggressive behaviors. Results will (1) illuminate the proximate cause of behavioral variation in an ESD species, and (2) for the first time delineate and test alternate hypotheses about the evolutionary causes of behavioral organization in the context of sex determining mechanisms.

In many egg-laying reptiles, the incubation temperature of the egg determines the sex of the offspring, a process known as temperature-dependent sex determination (TSD). How temperature both stimulates and inhibits genetic cascades to determine gonadal sex and channel sexual development is the focus of this application. The PI's working hypothesis is that incubation temperature modifies the endocrine microenvironment of the embryo such that steroid hormones serve as the proximate trigger for sex determination. This laboratory has developed the red-eared slider turtle as an animal model system and demonstrated that male and female gonadal development are separate pathways influenced by steroids, steroid-augmenting molecules, and steroid-interfering molecules. Female determination is caused by estrogens whereas male determination is caused by nonaromatizable androgens. Experiments are designed to determine if temperature accomplishes sex determination by acting on genes coding for steroid hormone receptors and aromafast, a key steroidogenic enzyme. The PI will continue the cloning and sequencing of the aromatase. To identify the patterns of expression and transcriptional regulation of selected specific genes during the critical periods of sex determination, the PI will utilize homologous antisense probes for estrogen receptor and aromatase of mENAs in in situ hybridization and ribonuclease protection assays at the beginning, during, and following the temperature-sensitive period. The ability to manipulate sex in TSD species by incubation temperature, exogenous hormones, and other agents provides unparalleled experimental control, thereby enabling more detailed analysis of the normal pattern of gene expression during sex determination than is possible with other amniote vertebrate species having sex chromosomes. Except for the trigger, the sex determining cascade appears to be similar between vertebrates having genotypic sex determination and those with TSD. The work on TSD draws attention t o the fact that temperature and steroid hormones can play a pivotal role in sexual development in an amniote vertebrate and is thus important for several reasons. First, it has long been assumed that steroid hormones of maternal or embryonic origin are not involved in gonad formation in mammals and birds. The work with TSD reptiles indicates that this conclusion may be premature. Second, since TSD may represent the evolutionary precursor to sex chromosomes, potential temperature and steroid effects in sex determination may be present, but partly or wholly masked, in homeotherms. Third, temperature has not been adequately investigated as a factor in steroid hormone action in homeotherms despite numerous studies documenting how both hormone responsiveness and hormone action are markedly dependent on temperature.

DESCRIPTION (Adapted from applicant's abstract): This research addresses the fundamental question of what determines behavioral variation. Many environmental variables can produce predictable effects on phenotype. One such variable is the incubation temperature of eggs in many reptiles. In the leopard gecko, embryos become male or female depending upon their temperature during development. In addition, between-sex as well as within-sex differences attributable to incubation temperature have been found in morphology, secretion of and sensitivity to steroid hormones, sociosexual behavior, reproductive success, and in the neuroanatomy and metabolic activity of brain areas that mediate sociosexual behaviors. These environmental effects are analogous to the effect of intrauterine environment in mammals, including humans. Given the homology of the endocrine and nervous systems across vertebrates, it is important to determine if homologous mechanisms underlie these analogous effects on behavior. If the mechanism underlying environmental effects on behavior in the leopard gecko is conserved (i.e., via sex steroids), this research will lend new insight into the evolution of sexual differentiation because temperature-dependent sex determination is thought to be the evolutionary precursor to genotypic sex determination (present in birds and mammals) and because reptiles are the ancestors of both birds and mammals. If the mechanism is different (i.e., direct temperature effects), this research would elucidate a novel process of sexual differentiation that may also be present in birds and mammals but, because of homeothermy, is masked. This latter possibility is especially important because young birds and mammals cannot regulate their body temperature as do adults. The proposed research is broadly categorized into three groups: thermoregulation and its relation to sociosexual behaviors (i.e., the degree to which the neural substrates mediating thermoregulatory and sociosexual behavior overlap), the development of the neural phenotypes of these brain regions and hormone milieus, and the effects of hormonal manipulations during development and neural manipulations in adulthood on thermoregulatory and sociosexual behaviors. The final category of experiments is particularly important as it will discern whether the incubation temperature effects are direct or indirect.

DESCRIPTION (Adapted From The Applicant's Abstract): This proposal requests continued support for a comparative, multidisciplinary research program that seeks to understand (i) the evolution of hormone-brain-behavior mechanisms underlying sexual behaviors, and (ii) the dual neural circuits that subserve these behaviors. The comparative method emphasizes the need for different model systems so as to elucidate the general rules which govern behaviors, as well as their historical roots. Multidisciplinary studies spanning molecular to population levels of biological organization provide a multifaceted, yet integrated, perspective of individual behavior. The animal models include parthenogenetic or all-female whiptail lizards and their sexual ancestors as well as transgenic mice. The unisexual species is known to have descended directly from the sexual species, thereby allowing ancestor-descendent comparisons. Further, because all parthenogenetic individuals have ovaries, yet exhibit both male-like and female-like pseudosexual behaviors, the complication of having two gonadal sexes, each with their own particular hormonal milieu, is removed. This makes it possible to study the neural circuits that underlie mounting and receptive behavior in a manner not possible with more common laboratory animals. The discovery that males can be created in this otherwise all-female species provides a unique opportunity to investigate the respective roles of genetics and gonadal hormones in the determination of adult sexual behavior and the underlying neural phenotype. Finally, the research based on the discovery that progesterone is an important modulator of male sexual behavior in both lizards and mammals will be extended. Recent studies with lizards and transgenic mice indicate that progesterone receptor is involved in the neuroendocrine control of male sexual behavior, and, in mammal, there is suggestive evidence that some of dopamine's effects on sexual behavior may be mediated by its interactions with the progesterone receptor. This grant aims to evaluate the generality of dopamine-progesterone receptor interactions across vertebrate taxa, to characterize the modulatory role of genotype on experiential effects, and to assess the independent contributions of genotypic sex vs. gonadal sex on sexual behavior and the brain. As work stemming from reptilian models has led to novel discoveries about mammalian systems, we anticipate that these studies will contribute important questions and hypotheses about the environmental and internal determinants of human sexual behavior.

Many egg-laying reptiles lack sex chromosomes, depending instead upon the incubation temperature of the egg to determine the sex of their offspring, a process known as temperature-dependent sex determination (TSD). How temperature both stimulates and inhibits genetic cascades to determine the type of gonad and direct sexual differentiation is the focus of this application. This investigator and his colleagues have developed the red-eared slider turtle (Trachemys scripta elegans) as an animal model system to study TSD. These researchers have demonstrated that male and female gonadal development are the result of separate genetic cascades influenced by steroid hormones, and that specific enzymes activate or inhibit steroid hormone effects on sex determination. In the red-eared slider system, female (or ovary) determination involves estrogens whereas male (or testis) determination involves nonaromatizable androgens. Mammals and turtles share a common evolutionary history and recent findings indicate that despite a difference in the trigger (environmental temperature vs. sex chromosomes), the red-eared slider shares many characteristics of sexual development with the mammalian system, including the same genes involved along the sex determination pathway. Experiments are designed to identify the genes involved in testis formation and then to determine how temperature and sex hormones accomplish this feat. For example, relative levels of gene expression will be measured using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot analysis. Localization of gene expression will be analyzed with in situ hybridization. The ability to manipulate sex in a primitive vertebrate species by incubation temperature, exogenous hormones, and other agents provides unparalleled experimental control, thereby enabling more detailed analysis of the normal pattern of gene expression during sex determination than is possible with other amniotes (reptiles, birds and mammals are known as the amniote or higher vertebrates) having sex chromosomes. This work is important for several reasons. First, it has long been assumed that steroid hormones of maternal or embryonic origin are not involved in gonad formation in mammals and birds. The work with TSD reptiles indicates that this conclusion may be premature. Second, since TSD may represent the evolutionary precursor to sex chromosomes, potential temperature and steroid effects in sex determination may be present, but partly or wholly masked, in warm-blooded vertebrates. Third, temperature has not been adequately investigated as a factor in steroid hormone action at the genetic level in warm-blooded animals despite numerous studies documenting how both hormone responsiveness and hormone action are markedly dependent on temperature.

DESCRIPTION (provided by applicant): This is an application directed toward the NIMH Exploratory/Developmental Grant (R21) program. This research described herein addresses the fundamental question of what determines behavioral variation and its neural underpinnings, in particular the relative roles of genetic and epigenetic factors in infancy in shaping adult brain and behavior. Specifically, this project will examine how the genotype of the offspring and the gender composition of the litter alters the nature of maternal care and how, in turn, this early maternal environment influences the neural underpinnings of sexual, aggressive, and maternal behaviors of the offspring when they become adults. The present proposal combines the skills of three researchers each expert in their respective fields (Crews: metabolic mapping of brain activity; Fleming: rat sexual and maternal behavior; Ogawa: knockout mouse aggressive and maternal behavior) and will utilize two mammalian model systems (knockout mice and the rat) in a new way, thereby developing a novel perspective from which to approach studies of brain organization and behavior. The overall goal of the proposed research is to develop an innovative research program that will provide both a new perspective and a set of techniques and methods that will aid exploration of gene-environment interactions, in particular our understanding of how the environment influences the relationship between genotype and behavior during sensitive developmental periods. There are three specific aims: Specific Aim I. To test the hypotheses that the gender and genotype composition of the litter alter patterns of maternal care. Specific Aim II. To determine if the sociosexual behavior of the young in adulthood are due to this differential behavior of the mother toward her young. Specific Aim III. To establish if the different behavioral profiles exhibited in such animals in adulthood are reflected in different patterns of activity in a network of interconnected limbic nuclei.

Our understanding of the process by which individuality emerges is still rudimentary, but it is clear that the environment in which the individual develops, and its sociosexual interactions as an adult, are central to this process. This project addresses how the experiences passively acquired as an embryo interact with those of the adult, when the individual has a degree of behavioral regulation of its own environment. Behavioral and neural plasticity is at the root of these individual differences. Thus, the overall goal of the proposed research is to determine how embryonic experience and sexual experience later in life interact to affect adult male sexual behavior, and to reveal how this in turn effects the neural circuitry underlying behavior. Specifically, using the leopard gecko (Eublepharis macularius) as a model system, experiments will investigate the mechanisms involved in mediating the interaction between embryonic and adult experience. Obvious candidate mechanisms have two properties: they are influenced by the embryonic environment and they are capable of affecting sexual behavior and learning during adulthood. The mesolimbic dopamine system satisfies both these criteria and it is hypothesized that varying extent of embryonic hormone exposure organizes this system and the resulting differences mediate how the two morphs respond to experience. These studies are long-term and will involve the participation of undergraduate and graduate students for their execution. Students will be expected to present their findings at national meetings and be co-authors on papers published in peer-reviewed journals.

DESCRIPTION (provided by applicant): Recently it has been discovered that early experiences can modify regulatory factors affecting gene expression in such a way that the DNA sequence itself is not changed but the individual's physiology and behavior are substantially influenced. In some instances these epigenetic effects can be transmitted across generations via incorporation into the germline where they become permanent and tend to be sex-linked, a relevant point as many affective disorders show gender bias. The proposed work will focus on how stress during a critical life history stage (peripubertal) might interact with the epigenome to influence aggressive and affiliative behaviors, metabolic activity in brain nuclei, and patterns of gene expression in specific brain nuclei. We will use an established male rat model of transgenerational epigenetic imprinting for early onset of multi- organ disease. There will be two groups: daily restraint stress for 6 hours for 21 days beginning at 22 days of age, and unstressed. Prior to sacrifice (120 days) animals will given a battery of behavioral tests. The brains will be sectioned in three alternating sets, the first set for cytochrome oxidase histochemistry, the second set for in situ hybridization, and the third set for the CA1 and CA3 of the hippocampus and basolateral amygdala for microanalysis (obtained using laser-capture microdissection). Specific Aim 1 will evaluate the behavioral phenotype of transgenerationally imprinted rats. Specific Aim II will determine if transgenerationally imprinted rats exhibit different patterns of metabolic activity in a defined network of interconnected limbic and forebrain nuclei known to be involved with agonistic and affiliative behaviors. Specific Aim III will determine if the distinct behavioral profiles exhibited by transgenerationally imprinted rats are reflected in unique patterns of gene expression in relevant brain regions. Within each Specific Aim, three hypotheses will be examined in three basic comparisons. Hypothesis 1: The transgenerational effects of vinclozolin exposure modifies both social and affiliative related behaviors and its related metabolic activity in specific brain nuclei as well as influencing the abundance of specific genes and altering the epigenome in the target brain areas. Hypothesis 2: Peripubertal stress potentiates both social and affiliative behaviors and its related metabolic activity in specific brain nuclei as well as influencing the abundance of specific genes and altering the epigenome in the target brain areas. Hypothesis 3: Peripubertal stress interacts with the environmentally induced, transgenerational epigenetic imprinting in a synergistic fashion to modify social behaviors as well as influencing the abundance of specific genes and altering the epigenome in the target brain areas. PUBLIC HEALTH RELEVANCE: How epigenetic modification can modulate how the environment and genetic constitution interact at the level of the brain, ultimately influencing agonistic and affiliative behaviors is the subject of the proposed work.

Two clear and present environmental impacts are affecting all life forms: climate change and anthropogenic chemical contamination. The red-eared slider turtle embryo has proven to be an exceptional model system to evaluate the role of each of these environmental factors and how they may interact. This species has temperature-dependent sex determination; that is, temperature (and hormones) acting during a narrow window of development, determine the sex of the individual. This research will reveal how the temperature signal is transduced into a molecular switch that results in an ovary or a testis. It will also evaluate the susceptibility of the embryo to anthropogenic environmental compounds, collectively known as endocrine disrupting chemicals (EDCs), which can cause disruptions in the normal steroid microenvironment causing developmental and reproductive abnormalities. This research integrates both areas of research and focuses on how the physical and chemical environments modify the molecular and cellular developmental pathways underlying gonad determination and sexual differentiation. Prior NSF-supported research established that two different "cassettes" of genes are involved in both processes. These cassettes are groups of functionally related genes that interact in a particular manner to cause an outcome at the cellular or tissue level. In this instance the first cassette is composed of a conserved core of gonad-determining genes that regulate gonadal differentiation in all vertebrates. The second cassette consists of the genes that code for the molecules that produce (steroidogenic enzymes) and sense (hormone receptors) steroid hormones. Understanding how these cassettes are engaged, how they interact with one another both temporally and spatially, and how the genes within each cassette interact under normal conditions as well as after exposure to EDCs are the long-term goals of this project.

The research program will provide interdisciplinary research opportunities for training graduate and undergraduate students. Regional mentoring activities will focus on undergraduate students from underrepresented groups. Sequence data generated will be deposited in GenBank.

DESCRIPTION (provided by applicant): Endocrine-disrupting chemicals (EDCs) are compounds in the environment that perturb endocrine systems. EDCs are ubiquitous in the modern world, and detectable in virtually all humans and wildlife. Many effects of EDCs are mediated by hormone receptors such as estrogen receptors (ERs) that are widely distributed in the brain, and thereby EDCs perturb neurobiological functions. The hypothalamus, hippocampus and amygdala are brain regions with high expression of ERs, and are proven targets for endocrine disruption. However, the cellular, molecular and physiological processes for these effects, and the functional behavioral implications for exposures of these brain regions to EDCs, are not well explored. Furthermore, the brain has a number of structural and functional sexual dimorphisms that develop early in life, a process that is sculpted by actions of estrogens on ERs, and disrupted by EDCs. Therefore, it is the overarching goal of this research to determine the molecular and cellular mechanisms by which exposure of developing fetuses to ecologically-relevant levels of EDCs perturb the sexual differentiation of specific brain areas, and the consequences on behaviors regulated by these regions. The proposed studies will fill a gap in knowledge by testing effects of perinatal exposure to a class of endocrine-disrupting chemicals, specifically polychlorinated biphenyls (PCBs), on sexually dimorphic neurobiological processes. PCBs are a ubiquitous and persistent environmental contaminant, and while banned for decades, they are still prevalent in soil and groundwater, leach into food and water, and are detectable in tissues of virtually all humans. The proposed studies will use a well-established rat model already in use in the PIs'labs to test effects of exposure to PCBs during a critical hormone-sensitive developmental window of late gestation, a critical period for brain sexual differentiation during which the developing nervous system is exquisitely sensitive to both endogenous and exogenous hormones, particularly estrogens. The experiments will quantify expression of a network of estrogen-regulated genes (Aim 1);elucidate some potential epigenetic mechanisms for regulation of identified targets (Aim 2);determine the manifestation of gene expression changes through protein immunohistochemistry (Aim 3);and ascertain the final outcome as a behavioral phenotype (Aim 4). These experiments have broad implications for humans as exposure to PCBs is universal, persistent, and has wide-ranging effects on health and disease. Therefore, understanding the latent effects of PCBs on neural development, and their underlying mechanisms, can inform public policy, medical interventions, and prevention. In addition, results on PCBs, used as a "model" EDC for decades, can help us better understand effects of other estrogenic EDCs still in common use such as those in plastics, pesticides, and beyond. PUBLIC HEALTH RELEVANCE: Exposures of humans to PCBs and other estrogenic EDCs are universal, and these compounds have wide- ranging effects on health and disease. Therefore, understanding the latent effects of PCBs on neural development, and their underlying mechanisms, can inform medical interventions and prevention, and guide public health policy. The rat model is highly conserved with humans, and it enables us to test the cause-and- effect relationship between prenatal PCBs, the development of adult disease, and the neural mechanisms underlying these biomedical processes.

DESCRIPTION (provided by applicant): Endocrine-disrupting chemicals (EDCs) are compounds in the environment that perturb endocrine systems. EDCs are ubiquitous in the modern world, and detectable in virtually all humans and wildlife. Many effects of EDCs are mediated by hormone receptors such as estrogen receptors (ERs) that are widely distributed in the brain, and thereby EDCs perturb neurobiological functions. The hypothalamus, hippocampus and amygdala are brain regions with high expression of ERs, and are proven targets for endocrine disruption. However, the cellular, molecular and physiological processes for these effects, and the functional behavioral implications for exposures of these brain regions to EDCs, are not well explored. Furthermore, the brain has a number of structural and functional sexual dimorphisms that develop early in life, a process that is sculpted by actions of estrogens on ERs, and disrupted by EDCs. Therefore, it is the overarching goal of this research to determine the molecular and cellular mechanisms by which exposure of developing fetuses to ecologically-relevant levels of EDCs perturb the sexual differentiation of specific brain areas, and the consequences on behaviors regulated by these regions. The proposed studies will fill a gap in knowledge by testing effects of perinatal exposure to a class of endocrine-disrupting chemicals, specifically polychlorinated biphenyls (PCBs), on sexually dimorphic neurobiological processes. PCBs are a ubiquitous and persistent environmental contaminant, and while banned for decades, they are still prevalent in soil and groundwater, leach into food and water, and are detectable in tissues of virtually all humans. The proposed studies will use a well-established rat model already in use in the PIs' labs to test effects of exposure to PCBs during a critical hormone-sensitive developmental window of late gestation, a critical period for brain sexual differentiation during which the developing nervous system is exquisitely sensitive to both endogenous and exogenous hormones, particularly estrogens. The experiments will quantify expression of a network of estrogen-regulated genes (Aim 1); elucidate some potential epigenetic mechanisms for regulation of identified targets (Aim 2); determine the manifestation of gene expression changes through protein immunohistochemistry (Aim 3); and ascertain the final outcome as a behavioral phenotype (Aim 4). These experiments have broad implications for humans as exposure to PCBs is universal, persistent, and has wide-ranging effects on health and disease. Therefore, understanding the latent effects of PCBs on neural development, and their underlying mechanisms, can inform public policy, medical interventions, and prevention. In addition, results on PCBs, used as a model EDC for decades, can help us better understand effects of other estrogenic EDCs still in common use such as those in plastics, pesticides, and beyond.

DESCRIPTION (provided by applicant): The world is contaminated, never to return to conditions that existed prior to the chemical revolution. Although some local remediation of contamination has occurred, at a global level this is simply not possible. A class of contaminants is known as Endocrine Disrupting Chemicals (EDC) because of their ability to perturb the body's hormone systems. Poor storage, spills, deliberate and accidental dispersal have had well-document effects on wildlife and human health. However, the field of EDC research remains highly controversial and polarized. Such compounds are now a permanent part of our environment and creating previously unknown evolutionary pressures. We must transcend traditional toxicological testing to develop new methods and perspectives if we are to anticipate and understand EDCs' impact on the future. Life in this new world is a combination of ancestral exposures due to heritable epigenetic modifications to DNA in germ cells (transgenerational), together with exposures experienced during the individual's own lifetime (body burden) that cause molecular epigenetic changes to that individual. These processes are an underappreciated force in driving evolutionary change in all species, including humans. The challenge is how to model the cumulative and progressive changes both across lifespans and within generations. We propose a unique study of multigenerational exposures to sequential environmental toxicants, each known to perturb hormones, brain and behavior. Our proposed model investigates interactions of ancestral and immediate epigenetic modifications, factors that have never been studied together. Over the course of 6 generations, we make the iconoclastic prediction that individuals will evolve in unique ways due to contemporary environmental driving forces (chemical contamination), with descendants responding differently to proximal chemical stimuli than their ancestors. To do this work, we propose to model two exposures separated by 3 generations; different EDC classes will be used at environmentally relevant levels, each with a different mode of action. Endpoints will be body weight, physiological parameters, neurobiological gene expression and molecular epigenetic assays, together with behavioral characterization of the animals in a suite of behaviors involved in social, anxiety, and cognitive function. Realistically simulating the nature of life challenges across and within generations will provide the framework for understanding and anticipating how environmental contamination will affect the evolution of all species.

ABSTRACT Every human has a body burden of endocrine-disrupting chemicals (EDCs), and levels of EDCs correlate with reproductive, endocrine, and neurobehavioral deficiencies. As environmental stressors, EDCs interact with other types of stressors to increase chronic disease and impair the quality of life. This proposal seeks to understand how the developmental trajectory of an individual is shaped by the interaction of behavioral stress during two life stages in the context of contamination. We focus on the social behavioral phenotype, as shifts in the reaction norms of social behavior can profoundly change an individual?s relationship to its community, his/her reproductive success, and mental health. We postulate that neurodevelopment in a contaminated world changes an individual?s baseline social phenotype, and that further life stressors overlaid upon the EDC phenotype create greater deflections from the behavioral norm. We will approach this question in several novel ways. First, we will model constant low-level exposure to a mixture of common-use EDCs throughout life, and assess emotional reactivity and sociality. Second, we will examine the effects of a typical life challenge (mild stress) during two critical life periods: to the mother (during pregnancy), to the individual during adolescence, or both. Third, we will compare males and females; this enables us to identify susceptibilities that may relate to well-established gender differences in disease and neurobehavioral dysfunction. Finally, by measuring changes in neuromolecular activity and neuroanatomical organization in a defined network of interconnected limbic and forebrain nuclei regulating the social phenotype, we can gain mechanistic insights into these processes. Thus, our overarching hypothesis is that each life stressor (lifelong EDC exposures, mild prenatal stress, and mild stress during adolescence) concatenates to shift the reaction norms for neurodevelopment and social behavior, and that the combination of stressors exacerbates adverse outcomes for neurobiological health. Mechanistically, we further propose that EDCs and stressors modulate this phenotype through perturbing the normal complementarity of estrogen and androgen signaling in the social decision-making network of the brain. There are 3 Specific Aims. Aim I will establish the sexually dimorphic behavioral phenotype of a lifetime of exposure to low levels of a mixture of common-use EDCs, upon which is superimposed mild stress during critical life stages (gestational, adolescent, or both). Aim II will determine underlying neuromolecular mechanisms for the changes caused by lifelong EDC exposures and gestational/adolescent stressors, focusing on estrogen/androgen-sensitive circuits. Aim III will identify neuroanatomical and cytoarchitectural substrates for the changes caused by EDCs and stressors, prioritizing estrogen-androgen signaling pathways. Proposed work has the potential to have a broad impact, ranging from societal and government policies, the health crisis caused by increasing chronic disease, and understanding fundamental biological principles at the molecular, cellular and organismal levels.