John Godwin, Ph.D. - US grants

gonads; sex differentiation; sex behavior; neuroendocrine system; neuroanatomy; innervation; hormone regulation /control mechanism; steroid hormone; Osteichthyes; immunocytochemistry; high performance liquid chromatography;

gonads; sex differentiation; sex behavior; neuroendocrine system; neuroanatomy; innervation; hormone regulation /control mechanism; steroid hormone; alternatives to animals in research; Osteichthyes; immunocytochemistry; high performance liquid chromatography;

DESCRIPTION (Applicant's abstract): A long range goal of psychobiology is to understand how internal physiological processes and external environmental factors interact to influence neural function and individual behavior. Improving our understanding of this interaction has strong implications for basic and applied human behavioral biology. The objective of this application is to examine and differentiate social and gonadal hormone influences on the function of the arginine vasotocin/vasopressin (AVT/AVP) system in the brain. The central hypotheses to be tested are: 1) that AVT expression is primarily influenced by the social environment in an animal model system where behavioral sex is determined by dominance interactions rather than the gonads, and 2) that AVT stimulates dominant male-typical sexual and offensive aggressive behavior through central actions in the brain of this teleost fish. The rationale behind this research is that while we know much about how gonadal hormones influence the brain and behavior, much less is known about potential direct social influences on the brain. The AVT system is a logical focus for asking how social influences act on the brain since this family of protein hormones has been strongly linked to the display of sexual behavior in all major groups of vertebrates and to aggression behavior in important mammalian models. The subject of this study, the bluehead wrasse (Thalassoma bifasciatum) undergoes complete behavioral and gonadal female-to-male sex change as an adult animal. This sex change is accompanied by a four- fold increase in AVT-mRNA levels in the brain area which exerts primary control of sexual and aggressive behaviors. The AVT/AVP system is influenced strongly by gonadal steroid hormones in many species. Importantly, however, behavioral sex change can occur even in the absence of gonads in bluehead wrasses and is instead completely dependent on social environment. This species therefore appears to present a unique opportunity to directly examine social influences on this important neural system. To address the central hypotheses above, the PI will pursue three specific aims: 1) Use natural behavioral variation and experimental dissections of social and gonadal influences to examine the control of AVT-mRNA expression, 2) Determine whether AVT can induce sexual and aggressive behavior in this species and 3) Determine sites of AVT action in the wrasse brain by AVT receptor localization and cellular markers of neural activation. Direct influences of social environment on the expression and behavioral actions of AVT would have relevance to humans, where sexual and aggressive behaviors are also under environmental influences.

This project will investigate the detailed nature of brain evolution. Specifically, the part of the brain that mediates courtship behavior will be studied in contrasting populations of a well-studied fish species, the guppy of Trinidad. In nature, some guppy populations exist in the presence of other fish that are strong predators on adult guppies, while other populations above barrier waterfalls are free of those strong predators. In the presence of the strong predators, males are drably colored and court relatively little, but in the absence of these predators, males are more brightly colored and court more. Because of earlier field transplant experiments, these characteristics are known to evolve rapidly when guppies are moved from a high-predation to a low-predation environment, but the brain changes that cause the change in behavior are not known. To address the neural mechanism of this evolutionary change, the cells which produce a hormone (arginine vasotocin) that is critical for courtship in vertebrate animals, will be compared using molecular biological and neurobiological methods. The specific goal is to assess a possible role for arginine vasotocin in adapting courtship behavior to the local predator environment.

The general goal of this research is to understand the detailed nature of changes in the brain that underlie differences in animal behavior. The research approach taken here will help to link the progress in understanding the environmental factors shaping animal behavior with advances in our understanding of the functioning of the brain. Given rapid advances in our understanding of the genomic and neural bases of behavior, the knowledge generated by this project may also be useful in improving the reproductive performance of domestically grown fish.

Neuroendocrine mechanisms underlying rapid change in social behavior (0206677)

Principal Investigator: John Robert Godwin
Doctoral Student: Katharine Anne Semsar

Both environmental and internal physiological influences affect the expression of sexual and aggressive behavior by animals including humans. For species that live in unpredictable environments, the appropriateness of displaying sexual and/or aggressive behavior can change quickly. A good deal of research has focused on adaptations to changes in the physical environment, but social environments can be equally unpredictable and it is therefore important that animals be able to adapt their behavior quickly in the face of social change as well.
A large body of work over the past twenty years has documented important roles for neuropeptide hormones in mediating social behaviors ranging from mating and parental care to mate guarding and territorial defense. Two neuropeptide hormones found to play especially prominent roles are arginine vasotocin (AVT) in non-mammalian vertebrates and the very similar arginine vasopressin found in mammals (AVP). These hormones are released in the brain and act to alter neural function in areas known to be associated with social behavior. While many studies have shown effects of these hormones on behavior in the laboratory, there are still relatively few studies that have examined the detailed mechanisms by which AVT or AVP affect sexual and aggressive behavior in the full complexity of the natural environment.
This study would explore mechanisms by which AVT affects sexual and aggressive behavior and the social and hormonal conditions that determine the nature of the effects of this hormone on these behaviors. The experimental model is the bluehead wrasse (Thalassoma bifasciatum), a well studied sex- and role-changing coral reef fish. Bluehead wrasses exhibit socially controlled female-to-male sex change in which females can become functional males within approximately ten days of becoming socially dominant. This model species has two principal advantages for the type of study proposed. First, the dependence of the sex change process on cues from the social environment makes it a very good system for understanding social influences on neural function and the interaction of environmental and internal hormonal cues in controlling behavior. Second, because of large populations in shallow, protected waters and extraordinary ease of capture for marking and experimental manipulations, bluehead wrasses allow the mechanisms underlying neuropeptide actions to be studied in nature.
Previous work by John Godwin has shown that AVT expression increases in the hypothalamus as females begin to exhibit male behavior and their ovaries become testis during sex change. Studies by Katharine Semsar and John Godwin have shown that AVT can induce territorial aggression and courtship behavior in large males who do not hold territories while an AVT receptor blocker can reduce these behaviors in territory-holding males. The studies outlined in this proposal would determine: i) whether AVT is necessary for females to become males behaviorally and whether augmenting AVT can accelerate this process, ii) whether the ability to respond to AVT, as measured by receptors for this hormone, changes during sex change, and iii) what the effects of a potent androgenic hormone that rises during sex change (11-ketotestosterone) are on the expression of AVT and its receptor in the brain.
This work should improve our understanding of behavioral adaptation and the role of neuropeptide hormones in these processes. These mechanisms are of strong basic interest, but also important more generally because of the societal costs of aggressive behavior.

Differences between male and female brains and behavior result primarily from differences in the timing, level and location of gene expression. Even in species where sex is determined chromosomally and the gonads play central roles in the differentiation process, the exact nature of gene expression is influenced by a host of environmental influences ranging from nutrition to social interactions. Although we are building a solid understanding of how gonadal steroids influence the sexual differentiation of brain and behavior, the mechanisms by which social interactions affect this process remain poorly understood. This project develops an integrative approach to how gene expression in the brain changes during sexual differentiation, in a model system where it is possible to experimentally dissect gonadal and social influences that regulate behavior. A well-studied reef fish species, the bluehead wrasse, exhibits socially-controlled functional sex change. When local dominant males are removed, a large female changes into a male, and even gonadectomized females change to male behavioral phenotypes. The challenge is to identify those neural genes from the brain relevant to the behavior that are differentially expressed, between females that do not change sex and those that are induced to change sex in their natural environment. This project has a focus on developing the molecular technology to successfully approach that question, to first identify some of the relevant genes that then can be targeted to look for changes in expression during the behavioral change. Results will be important for further developing this system as a good genetic model to understand the neural basis of sexually dimorphic behavior.
The impact of this work will extend beyond neuroscience to animal behavior, to psychology, and to potential use in aquaculture and fish breeding. In addition, the project continues excellent field and laboratory training for students in an interesting project integrating animal behavior, endocrinology and molecular biology.

DESCRIPTION (provided by applicant): Anxiety disorders are among the leading causes of illness in the U.S., yet their origins and the way in which an individual's genotype interacts with the environment to influence disease remain poorly understood. The long-term goal of these studies is to better understand the genetic and neurobiological underpinnings of variation in anxiety and stress-responsiveness. This work will employ the zebrafish model, but with an innovative focus on fish recently derived from wild stocks. The value of these wild-derived individuals is in the heightened levels and greater variation in anxiety-related behaviors they display relative to an established laboratory stocks. The specific goals of this exploratory project are first to compare gene expression profiles in the brains of two wild-derived zebrafish lines divergent in the display of anxiety-related behavior (high and low respectively) to two established laboratory lines that also show behavioral variation. The wild-derived zebrafish lines exhibit what have been termed 'proactive'and 'reactive'coping styles in other model systems and also pronounced sex differences with females exhibiting higher levels of anxiety-related behavior. The second goal of these studies is to compare gene expression profiles in the brain between the two divergent wild-derived lines at time points ranging from early development until after maturation when sex differences in anxiety-related behaviors have emerged. The goal of this second aim is to begin exploring the developmental origins of coping style differences in adulthood. The benefits of this project should include behavioral and neurogenomic characterization of a zebrafish model system that will lead to mechanistic studies in zebrafish and potentially studies of identified candidate genes in human association studies. The zebrafish model system is also particularly well suited to studying the genomic and developmental underpinnings of anxiety-related behaviors and the ways in which these mechanisms are influenced by an organism's environment. PUBLIC HEALTH RELEVANCE: This project addresses the genetic and developmental origins of anxiety-related behaviors using the zebrafish as a model system. Anxiety disorders are prevalent and therefore represent a significant public health issue imposing significant illness and economic burdens on the U.S. population.

Despite decades of research, major questions remain about how environmental cues control the vertebrate reproductive system. The list of key biochemical "gatekeepers" for activation of the reproductive system from fishes to humans includes two important protein hormones, Gonadotropin-releasing hormone and kisspeptin, that function in the brain. This project would investigate how social cues affect these hormones in the brain of an animal model system that is particularly powerful for revealing connections between the social environment and the reproductive system. This animal model is the bluehead wrasse (Thalassoma bifasciatum), a fish species that has been the subject of many foundational studies of environmental influences on reproductive function. This project will use molecular neurobiology methods to map the gonadotropin-releasing hormone and kisspeptin signaling systems in the brain of the bluehead wrasse, in order to compare the expression of these hormones across different reproductive phases, and to test the effects of manipulating these hormones on reproductive function. A key element of the project is to provide undergraduates from North Carolina State University and Indian River State College in Florida with experience in behavioral field studies and molecular biological laboratory studies. A second benefit will be an increased understanding of the control of reproduction in fishes because the bluehead wrasse is a particularly useful animal for understanding the specific mechanisms of this control. This knowledge is valuable to the aquaculture industry because the control of reproduction represents a key barrier to the propagation of fishes in captivity. Finally, this project will lead to the development of educational materials focused on Florida marine environments in collaboration with the Center for Ocean Science Education Excellence. Gene sequences will be archived in Genbank and cDNAs will be provided to interested parties on a cost-of-transport basis.

The ability to respond and adapt to current environmental or social condition, depends to a large extent of the flexibility to make behavioral, physiological and neurological changes. An area of great interest is the plasticity of the brain to change in response to new challenges. One of the greatest examples of this ability is seen in fish that are capable of switching from female-to-male or male-to-female either behavioral or phenotypically. This occurs when changes in the population, loss of males or females increase the ability to reproduce by changing. In species with socially-controlled sex change and/or role change, the brain perceives alterations in the social environment that indicate it is favorable for these changes and therefore "permissive". This is observed in the bluehead wrasse (Thalassoma bifasciatum), a Caribbean coral reef fish with a lek-like mating system in which territorial-terminal phase (T-TP) males defend spawning territories for access to females and breed with multiple females. Removal of a T-TP male from a spawning territory creates a permissive environment that induces sex change in the largest female or male role change in the largest non-territorial male, either an initial phase (IP) male or non-territorial-terminal phase (NT-TP) male. This research will examine the role androgen hormones in response to changing social influences to promote behavioral and phenotypic changes. These types of studies are important as they have the ability to demonstrate the constraint of social interactions and the brains ability to change to when constraints are left or there are major changes in the environment.

Within minutes-to-hours of removing a bluehead wrasse T-TP male from its territory, changes in the brain of the largest female, IP male, or NT-TP male induce T-TP male-typical behaviors, such as courtship of females and agonistic behavior directed at other males. Changes in the brain also activate the hypothalamo-pituitary-gonadal (HPG) axis, causing transformation of female ovaries or IP male testes into TP male testes. Androgen production, particularly of 11KT, rises during gonadal transformation in both females and subordinate males during sex and role change. While previous studies show 11KT increases are not necessary for behavioral changes, other observations nevertheless suggest this androgen may play an important role. The neuropeptide vasotocin (homologue of vasopressin in mammals) appears responsible for early behavioral changes. However, could the presence of 11KT induce earlier onset of behavioral changes and increase the display of T-TP male-typical behaviors? NT-TP males have higher baseline levels of 11KT than IP males and females and appear to transition into the role of T-TP male faster. Is 11KT responsible for faster role change? The researchers hypothesize that 11KT promotes behaviors and gene expression profiles typical of T-TP phase males under permissive social conditions. Using a combination of capture/tag-and-release/recapture approaches, hormonal manipulations via gonadectomy and 11KT implantation, behavioral observations of tagged individuals, 11KT serum assays, and gene expression analysis via qRT-PCR, this study will test whether 1) significant differences exist in the timing, frequency, and duration of T-TP male-typical behaviors among females, IP males, and NT-TP males undergoing sex and role change under permissive conditions, 2) gonadal presence affects T-TP male-typical behaviors and gene expression of the vasotocin system and two other neuropeptide systems, kisspeptin and gonadotropin-releasing hormone, during sex and role change, and 3) 11KT promotes T-TP male-typical behaviors and gene expression of these neuropeptide systems during sex and role change. This study will help determine whether 11KT confers an advantage for individuals competing to become the territorial phenotype