Nancy G. Forger, PhD - US grants

DESCRIPTION (adapted from applicant's abstract): A large number of sex differences in the central nervous systems (CNS) of vertebrates have now been described. Such morphological dimorphisms may underlie well documented sex differences in behavior, in susceptibility to certain drugs, and in the incidence of some human mental disorders including autism, depression and schizophrenia. In many cases, neural sex differences have been shown to be due to differential exposure to gonadal steroid hormones in males and females. However, the cellular and molecular mechanisms governed by hormones in the nervous system are not well understood. The identification and cloning of several new neurotrophic molecules has fueled an explosion of research into the actions of trophic molecules in the CNS, and recent findings indicate a role for neurotrophic factors in sexually dimorphic development. Experiments in the first half of this proposal will test the idea that effects of gonadal steroids are mediated by trophic factors in a well-characterized model system. The spinal nucleus of the bulbocavernosus (SNB) and its target muscles constitute an anatomically simple system that is sexually dimorphic in many mammals. SNB motoneurons reside in the lumbar spinal cord and innervate striated perineal muscles attached to the phallus. Androgens regulate SNB motoneuron survival during perinatal development, and SNB cell size in adulthood. Recent observations suggest that some effects of androgens on this system are mediated by protein neurotrophic factors. Trophic factor antagonists will be administered to developing and adult rats in order to identify endogenously produced factors controlling SNB cell survival and morphological plasticity. In the second half of this proposal, the intracellular events regulated by hormones and neurotrophic factors will be explored. Specifically, a role for the death-regulatory protein, Bcl-2, in sexually dimorphic cell death will be tested in the SNB and in the anteroventral periventricular nucleus (AVPV) of the hypothalamus. Because the neurotrophic factors and death-regulatory proteins to be examined are expressed throughout the nervous systems of many vertebrates, including humans, information gained from this work will be relevant to our overall understanding of the extracellular and intracellular molecules mediating hormone regulated development and plasticity in neural tissues.

A large number of sex differences in the central nervous system of vertebrates have now been described. Such morphological dimorphisms may underlie well documented sex differences in behavior, in susceptibility to certain drugs, and in the incidence of some human mental disorders including autism, depression and schizophrenia. In many cases, neural sex differences have been shown to be due to gonadal steroid hormones acting early in development. However, the cellular and molecular mechanisms governed by hormones in the developing nervous system are not well understood. The long range objective of the candidate is to identify cellular and molecular mechanisms of sexual differentiation in the nervous system. A simple neuromuscular system which is sexually dimorphic in many mammals has proven to be a valuable model system for identifying basic principals underlying the development of neural sex differences. Motoneurons of the spinal nucleus of the bulbocavernosus (SNB) of rodents innervate striated muscles of the perineum. The number of SNB motoneurons and the survival of SNB target muscles depend on exposure to androgen during perinatal development. It has been demonstrated that a neurotrophic molecule, ciliary neurotrophic factor (CNTF), can mimic some of the effects of androgen in this system. Moreover, receptors to CNTF are present in the SNB system during the period of sexual differentiation. This raises the possibility that androgenic hormones and neurotrophic factors may interact to cause sex differences in the SNB and elsewhere. The identification of such a mechanism would represent a breakthrough in our understanding of the development of neural sex differences and would identify novel actions of neurotrophic factors. The candidate for this Independent Scientist Award is an associate professor in the Psychology Department at the University of Massachusetts; her teaching load is substantial. She is a member of the Neuroscience and Behavior program and enjoys the support of a very cohesive and active Neuroendocrine Group. The award would allow her time to interact more with students in the lab and with colleagues, to pursue new areas of research, and to develop more fully as a research scientist. In particular, the candidate will gain training in electron microscopy and in "differential display", which will allow the identification of genes regulated by hormones and trophic factors in the SNB neuromuscular system.

DESCRIPTION (provided by applicant): The hormonal control of cell death is currently the best established mechanism for creating sex differences in the nervous system. Nonetheless, very little is known about how hormones such as testosterone regulate neuronal cell death. This project will examine the cellular and molecular mechanisms underlying hormonally controlled cell death in three well-studied model systems: the spinal nucleus of the bulbocavernosus (SNB), the anteroventral periventricular nucleus (AVPv) of the hypothalamus, and the bed nucleus of the stria terminalis (BNST). Mice will be used throughout, to take advantage of the power of genetically manipulated strains. Members of the Bcl-2 family of proteins are crucial regulators of cell death in many neural areas. In Aim I we will ask whether Bcl-2 family members also regulate sexually dimorphic cell death by examining the expression and hormone regulation of pro-life (Bcl-2, Bcl-xL) and pro-death (Bax) molecules in the brain and spinal cord of neonatal mice. Testosterone decreases developmental cell death of the SNB and BNST, while increasing cell death in AVPv. We therefore predict that hormone treatments will differentially affect death-regulatory proteins in these neural areas. Bax knockout mice will be used in Aims 2 and 3 to test the hypotheses that Bax is required for the death of neurons in SNB, AVPv, and BNST; if so, then sex differences in neuron number in these regions will be eliminated in Bax knockouts. In addition to sex differences in overall cell number, AVPv and BNST exhibit large sex differences in the expression of neurotransmitters or neuropeptides. It is not known whether these differences are due to cell death, or the specification of neuronal phenotype. Cell death mutant mice will be used to discriminate between these hypotheses. Finally, in the SNB, testosterone likely regulates neuronal death indirectly, by controlling the availability of neurotrophic factors from target cells. Aim 4 will test whether a recently identified trophic factor rescues SNB cells of mice and, in so doing, alters the expression of Bcl-2 family members. Together, these studies will allow us to specify at a mechanistic level how hormones control neuronal cell death in neural systems. This work is relevant to understanding sex differences in susceptibility to human neurodevelopmental disorders and neurodegenerative diseases in adulthood.

DESCRIPTION (provided by applicant): A large number of sex differences have been described in the mammalian central nervous system. Such morphological dimorphisms may underlie well-documented sex differences in behavior, in susceptibility to certain drugs, and in the incidence of human neurological disorders. In many cases, neural sex differences have been shown to be due to gonadal steroid hormones acting early in development. However, the cellular and molecular mechanisms governed by hormones in the developing nervous system are not well understood. The long-range objective of this candidate for an Independent Scientist Award is to identify cellular and molecular mechanisms of sexual differentiation in the brain and spinal cord. Dr. Forger is a professor in the Psychology Department at the University of Massachusetts. She is a member of the Neuroscience and Behavior program and enjoys the support of a cohesive and very visible group of colleagues in the Center for Neuroendocrine Studies. The candidate will soon be directing several major research efforts. Her teaching load within the Psychology Department is substantial. The award will allow her time to intensively focus on funded projects, to interact more with trainees and colleagues, and to pursue new areas of research. In particular, this award will support training in confocal microscopy and molecular biological techniques. The research plan outlines strategies for investigating the mechanisms underlying hormonally controlled cell death in three well-studied model systems: the spinal nucleus of the bulbocavernosus, the anteroventral periventricular nucleus of the hypothalamus, and the bed nucleus of the stria terminalis. Mice will be used throughout, to take advantage of the power of genetically manipulated strains. An R01 proposal describing these studies was recently reviewed and received outstanding priority ratings. An Independent Scientist Award would ensure the success of this program.

? DESCRIPTION (provided by applicant): Groundbreaking recent studies indicate that the community of microorganisms living in the digestive tract (the gut microbiota) plays a key role in psychiatric illnesses characterized by disordered emotional and social responses. Accelerated progress has also been made in understanding neural control of anxiety and social behavior, especially with regard to the roles of the `social neuropeptides,' vasopressin and oxytocin. To date nobody has linked these two exciting fields. Two established laboratories with complementary areas of expertise have joined forces to take on this task: one focused on sex differences in the brain and the neural basis of social behavior, the other on molecular pathways underlying the relationship between the microbiome and gut health. Together, these laboratories will develop mouse models to test the overall hypothesis that the gut microbiota acts early in life to permanently program vasopressin and oxytocin systems as well as anxiety- related and social behaviors controlled by these systems. In the first experiment, brain and behavior will be compared during development and in adulthood of germ-free mice after they have been colonized at birth by gut microbiota derived from either of two strains of mice that differ significantly in social and anxiety-related behaviors as well as composition of the microbiome. The second experiment takes advantage of a recent discovery in one of the two participating laboratories that commonly used food additives, i.e., emulsifiers, have surprisingly strong effects on physiology by acting on microbiota composition and its interaction with the host. Preliminary data suggest that these effects extend to the brain and behavior, which will be tested in the current project. Together, this project will address a crucial gap in our understanding of the gut-brain axis and its role in the development of anxiety-related and social behaviors, using physiologically relevant approaches. The findings will make possible subsequent identification of the specific microbiota affecting anxiety-related and social behaviors, and the signaling pathways involved. In addition, the payoff for studying effects of the microbiota on vasopressin and oxytocin expression will go beyond understanding microbiota effects on behavior, to include effects of the microbiota on autonomic functions, metabolic syndrome, and pain, all of which are affected by microbiota and modulated by these peptides.

This project addresses a deceptively simple question, yet one that has so far been largely overlooked: What are the consequences of birth (parturition) for brain development? Most mammals enter the world in a fairly dramatic fashion. A vaginal birth is accompanied by marked hormonal changes, mechanical stimuli associated with labor and delivery, and a transition from the sterile environment of the womb to one teeming with microorganisms. The fetus makes many physiological adjustments to survive outside the womb, and labor and delivery are the most reliable predictors of the upcoming challenges. This project examines the effect of birth on brain development, using mice as a model species. The investigators will look at effects of the timing of birth (within the normal range) and mode of birth (vaginal versus cesarean birth) on measures such as cell death and brain inflammation. Birth triggers major developmental switches for the lungs, heart, and other peripheral organs, and may play a similar role in brain development. If so, results from this project could fundamentally change the way we think about the role of birth in neural development. Both principal investigators are unusually active in education and outreach at the local, national, and international levels, and the project will involve undergraduates and graduate students. The home institution is ranked first in the nation among not-for-profit institutions in awarding bachelor's degrees to African-American and disadvantaged students, and the investigators' laboratories reflect this diversity.

The team of investigators will test the idea that birth alters brain development by varying the timing or mode of birth and examining effects on the patterning of neuronal cell death, and colonization of the brain by microglia. About 50% of the neurons that are initially produced are eliminated by apoptosis. The investigators recently found that cell death peaks just after birth in most forebrain regions of mice. In Aim 1, the investigators will vary the timing of parturition to test whether the association of birth with cell death is causal. Parturition also abruptly activates the peripheral immune system. The investigators hypothesize that this immune activation extends to the brain and will test that here. Microglia, the resident immune cells of the brain, are in an activated state perinatally, and increase in number soon after birth, but whether birth causes changes in microglia is unknown. The peripheral immune activation after delivery also varies by mode of birth: vaginal birth is associated with a greater increase of many immune markers compared to cesarean delivery. Interestingly, the investigators' preliminary data show that mode of birth affects cell death in several brain regions. Thus, to identify aspects of "birth" that influence brain development, Aim 2 will contrast effects of vaginal and cesarean delivery on cell death and neuroimmune activation. In Aim 3, the investigators will interfere with immune activation to determine whether effects of birth on microglia and cell death occur in parallel or are related (i.e., microglia may promote cell death).