Richard B. Simerly - US grants

The long-range goal of this work is to clarify the functional organization of neural circuitry underling reproductive physiology and behavior. Central to this goal is a better understanding of the organization of forebrain neural circuitry involved in these functions and the role that gonadal steroids play in their development and regulation. The proposed experiments represent a logical extension of past work and are a necessary first step toward identifying the cellular events involved in the modulation of specific neural systems by hormonal feedback. These experiments are designed to examine the hypothesis that circulating gonadal steroid hormones regulate tyrosine hydroxylase (THE) and/or proenkephalin (ENK) mRNA levels within two sexually dimorphic populations of cells in the anteroventral periventricular nucleus (AVPv). The AVPv appears to be a nodal point in the neural control of ovulation, and projects to the arcuate nucleus of the hypothalamus, a nucleus that is widely regarded to play an important role in gonadotropin secretion. Retrograde and anterograde axonal transport techniques will be used in combination with immunohistochemistry to determine whether THE and/or ENK are contained within this pathway. The influence of gonadectomy and various sex steroid treatments on The and ENK expression will be assessed in male and female rats at the level of single cells by using in situ hybridization histochemistry to detect possible changes in mRNA levels. The results of these experiments will gain added physiological relevance by comparing them with those obtained in normally cycling female rats at each stage of the estrous cycle. Finally, surgically lesioned animals will be studied with in situ hybridization in order to examine the possibility that any direct effects of gonadal steroids on THE and ENK expression may be augmented by neural influences from other steroid sensitive regions that project to the AVPv. The results of the proposed experiments should greatly contribute to what is currently known regarding the molecular mechanisms by which gonadal hormones control the neural events mediating ovulation. Knowledge of these mechanisms is essential to our emerging understanding of the processes that underlie infertility and fertility control.

The long range goal of this program of research is to clarify the molecular mechanisms by which neurons in the limbic system and hypothalamus integrate endocrine and sensory influences on reproductive function. The proposed project will utilize the anteroventral periventricular nucleus of the hypothalamus (AVPv) as a model system for this research because it is known to play a critical role in mediating hormonal feedback on gonadotropin secretion and receives neurotransmitter-specific afferents from hormone- sensitive regions of the forebrain that relay olfactory information to the hypothalamus. Moreover, it contains sexually dimorphic populations of tyrosine hydroxylase (TH), prodynorphin (PDYN), and proenkephalin (PENK) mRNA-containing neurons that show three distinct patterns of gene expression over the estrous cycle: TH expression is highest during metestrus, but PENK mRNA remains constant, while PDYN mRNA is selectively induced during estrus, indicating that progesterone may play a role in regulating this opioid peptide gene. Because neither the TH nor PDYN gene contains consensus estrogen-regulated enhancer sequences, the observed regulatory patterns are most likely due to indirect mechanisms. The overall objective of the proposed project is to identify molecular events that mediate the hormonal regulation of TH and PDYN in the AVPv by (1) identifying molecules involved in stimulus-transcription coupling that are differentially expressed in TH and PDYN mRNA-containing neurons, and (2) determining if the indirect regulation of TH and PDYN gene expression by estrogen and progesterone is mediated by the nuclear trans-acting factors CREB (cAMP regulated enhancer binding protein), c-fos and c-jun, or by hormone-sensitive, neurotransmitter-specific inputs to these cells. These broad goals will be accomplished by first, using a double in situ hybridization technique to determine if differences in the hormonal regulation of TH and PDYN mRNA levels correspond to differential patterns of co-expression of estrogen and progesterone receptor mRNA in these neurons (Specific Aim 1). Second, in situ and solution hybridization will be used to evaluate possible hormonally induced changes in the expression of CREB, c-fos and c-jun in the AVPv, and to determine whether TH and PDYN mRNA-containing neurons differentially express these trans-acting factors (Specific Aim 2). Third, because the AVPv receives strong SP- immunoreactive inputs from other hormone-sensitive regions, the hormonal regulation of substance P receptor mRNA and possible co-expression in female rats will be evaluated following treatment with substance P agonist or antagonist to determine if these peptidergic afferents trans- synaptically regulate gene expression in the AVPv (Specific Aim 4). The results of these experiments will contribute to our emerging understanding of neuroendocrine regulation of transmitter plasticity within forebrain circuits that mediate key reproductive events, and may provide vital clues about mechanisms that underlie hormone-dependent abnormalities in the neural control of ovulation and fertility.

The goal of the histochemistry core is to provide technical expertise, help with experimental design, assistance with anatomical image analysis, and training opportunities in modern histochemical methodology. Histochemistry methods are an important part of projects 1-4 of this program project. A core laboratory will provide facilities and technical assistance in the histological and histochemical processing of animal tissues and obviate the purchasing of expensive equipment required for these procedures by each laboratory. The core will also provide the necessary expertise in trouble-shooting histochemical experiments, as well assist with the analysis and quantification of the results. By Centralizing the tissue processing and coordinating experimental protocols used by each laboratory this core will facilitate progress toward the stated goals of each project and enhance the process of interrelating results across projects. Dr. Chinkers (Project 1) will utilize the histochemistry core to evaluate the distribution of natriuretic peptides in the hypothalamus and study their possible co- expression in CRF neurons, as well as define the expression pattern of receptors for these peptides in the hypothalamus and pituitary. Dr. Low (Project 2) will need free access to the core facilities to characterize the development and adult organization of key aspects of hypothalamic and pituitary components of the HPA axis in transgenic mice that lack beta- endorphin. Dr. Thomas (Project 3) proposes studies that will required access to the histochemistry core for processing pituitary tissue from transgenic animals for immunohistochemistry and in situ hybridization. Dr. Cone (project 4) will require extensive use of the core facilities to study the expression pattern of novel melanocortin receptors in the brain of adult and developing normal and transgenic animals. In support of these projects the histochemistry core will: 1) Provide equipment, supplies and technical assistance for the acquisition of frozen tissue sections using both cryostat and sliding microtomes; 2) Store frozen tissue samples (-cT) and slide-mounted sections until they can be processed for histochemistry; 3) Supply detailed protocols for immunohistochemical (both double fluorescence and ABC staining) and in situ hybridization (using both non- and isotopically-labeled cRNA probes) histochemical techniques, as well as assist in the optimization of procedures for specific applications; 4) Assist in the experimental design and interpretation of all histochemical experiments; and 5) Provide image analysis facilities and assistance with anatomical and quantitative analysis of histochemical experiments.

The goal of this project is to develop and validate an in vitro organotypic primate model system for studying neural plasticity, and to initiate experiments that will clarify the cellular and molecular events underlying hormonal control of neural development in the cerebral cortex of the primate. Both steroid and thyroid hormones influence cognitive abilities and appear to exert profound effects on the structure and neurochemistry of the cortex. Moreover, there are significant differences between the organization and neurochemistry of the cerebral cortex in primates and other mammalian animal models. Thus, it is likely that unique mechanisms participate in regulating cortical development in humans and nonhuman primates. During the past project period, we processed a total of 6 fetuses and prepared over 500 explant cultures. Nearly all of these proved to be viable, and most appeared to remain healthy for at least 30 days in vitro. These cultures showed organotypic patterns of lamination characteristic of developing cortex in vivo and numerous neurons were immunoreactive for glutamate receptors. The presence of interlaminar connections was confirmed by making small implants of the fluorescent tracer DiI into localized regions of the cortical explants. Retrogradely labeled cells occupied positions that corresponded to cell layers, and labeled projection axons passed laterally in the explant through discrete zones as they do in mature tissue. In addition, the morphology of individual neurons appeared to be quite complex and generally was consistent with published reports. We have used this new model system to begin to study the role of sex steroid and thyroid hormones on cortical development. Our initial results suggest that estradiol promotes neurite outgrowth from cortical explants, which was not observed with testosterone. In preparation for an evaluation of the effects of sex steroids on neuronal function, we prepared cultures of primate cortex on glass coverslips using the roller-tube" culture method and used calcium imaging techniques to assess neuronal responsiveness to glutamate in these cultures. Neurons in the explants showed dose dependant changes in internal calcium concentrations in response to glutamate challenge. Future studies will exploit this novel experimental model system to further characterize hormone and neurotransmitter expression, as well as study hormonal regulation of neuronal signal transduction under defined, pharmacologically controlled conditions.

To date, two mammalian bombesin-like peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB), have been characterized. These two peptides are widely distributed in mammalian nervous system and GI tract; but, of greatest clinical importance, GRP is expressed by many neoplasms and may stimulate the growth of these neoplasms. Recent studies suggest that there are a number of additional bombesin-like peptides that are yet to be identified and that these peptides likely play an important role in growth and development. Thus, we are attempting to identify new bombesin-like peptides by cross-hybridization with cDNAs encoding previously characterized bombesin-like peptides, as well as by using antibodies to amphibian bombesin-like peptides. Once new bombesin-like peptides are discovered, distribution studies and physiologic studies using the primate model will be established to determine the role of these new peptides in normal and neoplastic processes. To date , we have discovered 3 new amphibian bombesin-like peptides and two new amphibian bombesin receptors. Our new data suggests that the ligand for recently described bombesin BRS-3 receptor may turn out to be the long sought mammalian bombesin. FUNDING NIH CA39237 PUBLICATIONS Pradhan TK, Katsuno T, Ryan RR, Mantey SA, Akeson MA, Donohue PJ, Battey JF, Spindel ER. The bombesin receptor subtype 4 is coupled to phospholipase C and has a unique pharmacology. In Meeting of the American Gastroenterology Society (held in New Orleans, LA, May 16-20, 1998 (abstract).

The goal of the Imaging & Neuroanatomy Core is to provide technical expertise, assistance with confocal microscopy and image analysis, and training in modern histochemical methodology. The importance of imaging technology in modern biological research has undergone enormous advances that make the acquisition, processing, analysis and storage of digital images an important part of most research. In addition, a core laboratory will provide facilities and technical assistance for histochemical processing of animal tissues and obviate the purchasing of expensive equipment required for these procedures by each laboratory. By coordinating experimental protocols used by each laboratory this core will facilitate progress toward the state goals of each project and enhance the process of interrelating results across projects. The core will also provide the necessary expertise in trouble-shooting histochemical experiments and consult on the analysis and quantification of results. Therefore, the Imaging & Neuroanatomy Core will: (1) Provide equipment and training in the acquisition of frozen tissue sections using both cryostat and sliding microtomes; (2) Supply detailed protocols for immunohistochemical (both double fluorescence and ABC staining) and in situ hybridization (using both non- and isotopically-labeled cRNA probes) histochemical techniques, as well as assist in the optimization of procedures for specific applications. Training and assistance will also be provided for tract tracing studies; (3) Provide access to a 4 wavelength confocal microscope equipped with high N.A. objectives for acquisition of confocal images with exceptional Z axis resolution; (4) Provide facilities for image processing and analysis; (5) Provide training for project investigators in confocal microscopy, digital image capture, processing and analysis. Assistance will also be provided with specific experimental applications.

The long range goal of this line of research is to clarify the organization and development of peptidergic neural pathways from the arcuate nucleus of the hypothalamus (ARH) to hypothalamic regions known to mediate neuroendocrine regulation of mammalian homeostasis have evolved to integrate the neural system that control gonadotropin secretion and ingestive behavior. The ARH represents a key neuroanatomical interface between such systems, and its importance for gonadotropin secretion and feeding behavior is well documented. However, the detailed neurological mechanisms underlying these essential functions remain unclear, and little is known about the nuclei of the hypothalamus play key roles in mediating gonadotropin section and feeding behavior and share strong connections with the ARH, as well as with the paraventricular nucleus of the hypothalamus (PVH), which may represent the final common pathway for hypothalamic regulation of energy balance. In addition, each of the these nuclei express receptors for melanocortin and opiatergic peptides, which have been implicated in the regulation of both ingestive behavior and neural development. The overall hypothesis of this proposal is that melanocortin and opiatergic peptides are expressed in projections from the RH to the AVPV, PVH, and DMH, and that these peptides directly influence the development of connections between these nuclei. Anterograde axonal transport and histochemical methods will be used, together with both in vivo and in vitro model systems, to address the following specific aims. Specific Aim 1. Anterograde axonal transport of the tracer PHA-L will be used together with immunohistochemistry to demonstrate the presence of beta- endorphin (betaEND) and alpha melanocyte stimulating hormone (alphaMSH) in projections from the ARH to the AVPV and DMH in adult mice (C57Bl/6J). Specific Aim 2. The fluorescent racer DiI will be used to demonstrate the development of projects from the ARH to the AVPV, PVH and DMH in neonatal male and female mice. Specific Im 3. In situ hybridization will be used to correlate the expression of MC4 and mu or delta opiate receptors in the AVPV, PVH and DMH of neonatal animals with the arrival of inputs to these nuclei from the ARH. Specific Aim 4. DiI will also be used to study the development of neural projections from the RH to the AVPV, PVH and DMH in transgenic mice that lack functional receptors for beta-endorphin or MC4. Specific Aim 5. Axonal transport and immunohistochemistry in transgenic mice that lack functional receptors for beta-endorphin and alpha MSH containing projections from the ARH to the AVPV, PVH or DMH in organotypic hypothalamic explant co-cultures maintained in vitro under defined conditions. These studies will provide novel insights into cellular mechanisms underling the development of hypothalamic neural systems regulating homeostasis, and may also provide clues about the ontogeny of neurological defects related to infertility and obesity.

DESCRIPTION ( Verbatim from the Applicant's Abstract): The long range goal of this work is to clarify the cellular and molecular events that underlie hormonal control of the development of sexually dimorphic neural pathways in the mammalian forebrain. Central to this goal is a clear understanding of how steroid hormones influence the development of connections between subpopulations of neurons that control sex specific behaviors and physiological responses. The proposed project will use the connections of the anteroventral periventricular nucleus of the preoptic region (AVPV) as a model system to study how sex steroid hormones specify sexually dimorphic patterns of afferent and efferent connections. The overall hypothesis is that the higher levels of estradiol present in males acts on the AVPV through the estrogen receptor alpha (ERa) during the first few days of life to differentiate its neurons and promote innervation by neurons of the principal nucleus of the bed nuclei of the stria terminalis (BSTp) while at the same time suppressing formation of projections from the AVPV to neuroendocrine neurons. Axonal labeling and histochemical methods, as well as both in vivo and in vitro experimental models, will be used to test this hypothesis.

The reproductive state of lactation, which occurs in all mammals, is associated with an inhibition of reproductive cyclicity and ovulation due to a suppression of gonadotropin releasing hormone (GnRH), the hypothalamic neuroendocrine neurons regulating reproduction. The focus of our studies is to identify the afferent neuronal pathways activated during lactation by the suckling stimulus that are responsible for the suppression of GnRH neuronal function. This past year, we made the following observations 1) Using neuronal tract tracing techniques and confocal microscopy, we have shown that NPY neurons activated by the suckling stimulus project to areas containing GnRH neurons and make contact with GnRH neuronal processes. These results suggest a possible neuronal mechanism by which suckling inhibits GnRH neuronal activity. 2) The large increase in food intake during lactation most likely occurs in response to substances that signal changes in metabolic activit y, such as leptin (a satiety-inducing substance) and agouti-related transcript (ART, induces obesity). During lactation, blood leptin levels are greatly suppressed, whereas ART expression is increased in the hypothalamus. Current studies in the laboratory are examining the relationship between the changes in leptin and ART and the increase in NPY neuronal activity and food intake. In addition, we are exploring possible interactions between the regulation of food intake and of gonadotropin releasing hormone neuronal activity during lactation. It is well established that many causes of infertility are related to changes in energy homeostasis, such as in exercised-induced amenorrhea and anorexia nervosa. An understanding of the mechanisms by which the suckling stimulus imposes an inhibition on GnRH neuronal function provides information that is relevant to primates (including humans), in which the reproductive neuroendocrine axis regulating ovarian cyclicity is also inhibited. These studies have relevance to women's reproductive health as they will increase our understanding of hypothalamic causes of infertility and provide new approaches for contraception. FUNDING NIH HD14643 PUBLICATIONS Li C, Chen P, Smith MS. The acute suckling stimulus induces expression of Neuropeptide Y (NPY) in cells in the dorsomedial hypothalamus and increases NPY expression in the arcuate nucleus. Endocrinology 139:1645-1652, 1998. Li C, Chen P, Smith MS. Neuropeptide Y (NPY) neurons in the arcuate nucleus (ARH) and dorsomedial nucleus (DMH), areas activated during lactation, project to the paraventricular nucleus of the hypothalamus (PVH). Reg Peptides 75-76:93-100, 1998. Li C, Chen P, Smith MS. Neuropeptide Y (NPY) and tuberinfundibular dopamine (TIDA) are altered during lactation role of prolactin. Endocrinology 140 118-123,1999. Grove KL, Smith MS. Resistance of the hippocampus in the lactating rat to N-methyl-D-aspartate (NMDA)-mediated excitation is not due to a nonfunctional receptor system. Brain Res 814:157-163, 1998. Grove KL, Smith MS. 3?-hydroxysteroid dehydrogenase (3? -HSD) and mRNA distribution in the rat brain. In The Endocrine Society Program & Abstracts 80th Annual Meeting (held in New Orleans, LA, June 24-27, 1998), p 493 (abstract #P3-527). Brogan RS, Kuper J, Duncan JS, Trayhurn P, Smith MS. Serum leptin levels during lactation are related to the metabolic drain of milk projection lack of correlation to serum LH. In The Endocrine Society Program & Abstracts 80th Annual Meeting (held in New Orleans, LA, June 24-27, 1998), p 349 (abstract #P2-478). Chen P, Haskell-Luevano C, Cone RD, Smith MS. Coexpression of agouti-related transcript (ART) and neuropeptide Y (NPY) in the hypothalamic arcuate nucleus of female rats during the estrous cycle and lactation. In the Endocrine Society Program & Abstracts 80th Annual Meeting (held in New Orleans, LA, June 24-27, 1998), p 521 (abstract #P3-663). Li C, Chen P, Smith MS. Anatomical interactions between neuropeptide Y neurons from arcuate nucleus of the hypothalamus (ARH-NPY) and the gonadotropin-releasing hormone (GnRH) in the hypothalamus. In The Endocrine Society Program & Abstracts 80th Annual Meeting (held in New Orleans, LA, June 24-27, 1998), p 59 (abstract #OR5-5). Li C, Chen P, Smith MS. Neuropeptide Y neurons from the arcuate nucleus of the hypothalamus (ARH-NPY) directly project to corticotropin releasing hormone (CRF) neurons in the paraventricular nucleus (PVH). Soc Neurosci Abstr 24(pt 1):369, 1998 (abstract #146.8). Brogan RS, Grove KL, Smith MS. Leptin receptor expression during lactation. Society for the Study of Reproduction 58(suppl 1):96 (abstract #81).

Abstract not provided.

DESCRIPTION (provided by applicant): Neonatal factors that contribute to obesity are poorly understood. The long-range goal of this research is to clarify how the adipocyte-derived hormone leptin influences development of neuroendocrine pathways that regulate mammalian energy homeostasis. Central to this goal is determining how leptin affects development of neural projections from the arcuate nucleus of the hypothalamus (ARH), a key site for integration of information related to peripheral energy stores. Leptin signals are conveyed via the ARH to other brain regions involved in the regulation of body weight, such as the paraventricular nucleus of the hypothalamus (PVH), an important component of the final common pathway for regulation of energy metabolism. Evidence presented in the body of this proposal supports the concept that leptin functions as a developmental factor during neonatal life by directing formation of neural circuits involved in the control of feeding and energy balance. The overall hypothesis addressed in this proposal is that leptin acts directly on the ARH during a restricted neonatal critical period to promote formation of neural projections to the PVH involved in the regulation of body weight. We propose that the postnatal leptin surge has an enduring effect on ARH projections, and that developmental perturbations in leptin signaling cause permanent changes in neural pathways that transmit leptin signals in mature animals. Both physiological and in vitro experimental approaches will be used to test this hypothesis by addressing the following specific aims. Specific Aim 1. We propose to use axonal labeling methods and histochemical techniques to define the organization and development of ARH projections in obese mice that lack leptin (ob/ob mice) and in diabetic mice that lack a functional long form of its receptor (db/db mice). Specific Aim 2. We will also test the developmental activity of leptin by examining development of the ARH-PVH pathway in ob/ob and db/db mice treated with exogenous leptin, and determine if this developmental activity is restricted to a neonatal critical period. Specific Aim 3. To determine the site of action for leptin in directing development of the ARH-PVH pathway, we will utilize a new explant coculture assay, in addition to examining the direct effects of leptin on axon outgrowth from isolated ARH explants in vitro. Specific Aim 4. In addition, we will examine how nutritional manipulation of leptin levels impacts development of the ARH-PVH pathway. Specific Aim 5. Finally, we will determine if altered patterns of leptin dependent intrahypothalamic signaling accompany observed developmental changes in the ARH-PVH pathway. The results of the proposed research will expand our appreciation of leptin to include a profound developmental activity that promotes formation of leptin-responsive hypothalamic neural pathways. These studies may also provide essential clues about how the neonatal nutritional environment imposes enduring consequences on central regulation of feeding and energy balance throughout life.

ABSTRACT NOT PROVIDED

DESCRIPTION (provided by applicant): The long-range goal of this work is to clarify cellular mechanisms that underlie development of sexually dimorphic neural pathways in the mammalian forebrain. The proposed project will use a sexually dimorphic limbic-hypothalamic pathway as a model system to study how sex steroid hormones specify patterns of forebrain connections. During the second week of life, the anteroventral periventricular nucleus of the preoptic region (AVPV) receives a sexually dimorphic input from the principal nucleus of the bed nuclei of the stria terminalis (BSTp) that develops according to a directed mechanism. During the past project period we developed an organotypic co-culture model to demonstrate that sexual differentiation of this pathway is due to a target dependent, estrogen receptor alpha (ERalpha)-mediated, action of estrogen on the AVPV. Preliminary evidence presented in the body of this application indicates that the estrogen-regulated neurotrophin BDNF (brain derived neurotrophic factor), and the class 3 semaphorin Sema 3C, are expressed in the AVPV in sexually dimorphic patterns and appear to play important roles in promoting BSTp-AVPV projections. Our Overall Hypothesis is that E2 acts on the AVPV during the first few days of life to alter expression of neurotrophic and chemotropic factors that promote its innervation by BSTp neurons. Both in vitro and in vivo experimental models will be used to test two specific hypotheses: Specific Aim 1. During postnatal development, ERalpha mediates increased expression of BDNF in the AVPV, which acts on BSTp axons via TrkB receptors to promote neurite extension. Specific Aim 2. ERalpha dependent expression of semaphorin in the AVPV influences development of sexually dimorphic projections from the BSTp to the AVPV. In addition to characterizing expression of these molecules during development of the BSTp-AVPV pathway, we will utilize a loss of function/gain of function strategy to explore their role during growth and guidance of BSTp projections. The results of these studies will make a significant contribution to our emerging understanding of molecular events that mediate hormonal control of brain development, and will add to what is known about developmental regulation of axon guidance generally. These studies may also provide clues about mechanisms responsible for sex-linked aberrations in neural connectivity that contribute to a variety of hormone-sensitive neurological disorders.

DESCRIPTION (provided by applicant): This proposal, entitled, Developmental mechanisms of leptin resistance, is an application for competitive renewal of DK57768-09. The long-term outlook of our previous and proposed studies under this award is to understand molecular and neural mechanisms that impair leptin action and promote obesity and metabolic dysfunction. During the current funding period, we focused upon the mechanisms by which LepRb regulates hypothalamic physiology and may mediate feedback inhibition. Major conclusions from this work include the roles for Tyr985 (along with other LepRb signals) in the attenuation of LepRb action. Multiple lines of evidence now support the notion that cellular processes that attenuate LepRb signaling contribute to obesity and leptin resistance in vivo. Additional neural/developmental mechanisms contribute to the inception of obesity, however. Importantly, altered perinatal nutrition initiates a program that promotes obesity and metabolic syndrome in adulthood. Lack of leptin action or alterations in nutrient availability during this crucial perinatal window disrupts the development of projections from ARC neurons to their target nuclei, suggesting a potential role for leptin and the development of this ARC circuit in early metabolic programming. Many issues regarding these alterations in ARC projections remain to be addressed, however. As these issues are difficult to address with standard tools, we have generated a number of novel transgenic systems that will enable us to probe these issues in this application. The requirement for diverse intellectual and technical expertise in this proposal dictates an intimate collaboration between the Myers and Simerly labs. Together, we will: (1) Define the functional consequences of leptin deficiency on the development of leptin-regulated neural circuitry. (2) Examine the programming of leptin-regulated neural circuitry by perinatal undernutrition. (3) Determine the mechanisms that underlie the leptin-mediated developmental programming of the ARC neural circuitry. These studies will define the neural consequences of altered leptin and perinatal nutritional status within a core component of the neural circuitry that regulates energy homeostasis, as well as defining the mechanisms underlying these processes. The mechanistic insights derived from these studies will define processes that likely contribute to the inception of metabolic disease. PUBLIC HEALTH RELEVANCE: Leptin is a key regulator of body energy homeostasis and metabolism, and impaired leptin action may contribute to a variety of metabolic diseases. Understanding the mechanisms that may interfere with leptin action to mediate obesity and metabolic dysfunction is thus crucial. We have thus been working to define the mechanisms by which the leptin receptor, LepRb, mediates feedback inhibition of leptin action and to define the contribution of these processes to obesity in vivo. While these processes can contribute to the regulation of adiposity, they explain the propagation rather than the onset of obesity. In contrast, perinatal metabolic programming clearly underlies important aspects of the inception of obesity. Developmental alteration of hypothalamic leptin-responsive circuits represents a likely mediator of this perinatal programming. We have thus generated a number of novel mouse genetic models with which to analyze the mechanisms and consequences of perinatal hypothalamic programming by leptin and altered nutrition.

DESCRIPTION (provided by applicant): The long-term goal of this research project is to understand the molecular and neural mechanisms governing energy homeostasis. Brain-derived neurotrophic factor (BDNF) plays crucial roles in energy balance, as mutations in the genes for BDNF and its receptor TrkB lead to obesity in both mice and humans;however, the precise role of BDNF in the regulation of body weight remains unknown. The organization and activity of hypothalamic neural circuits plays a critical role in the control of energy balance and BDNF is a potent regulator of neuronal development and synaptic plasticity. This application proposes to test the hypothesis that BDNF controls body weight by regulating the formation of neural circuits in the mediobasal hypothalamus that are known to control energy balance. Defects in axonal growth, synaptogenesis, and spine development will alter the development of hypothalamic circuits, which will in turn impair hypothalamic integration of signals reflecting states of nutrition and fat stores. Aim 1 will determine if TrkB-expressing neurons in the arcuate nucleus (ARC) respond to changes in feeding status and investigate if BDNF regulates axonal growth of these neurons using both in vivo and in vitro approaches. Aim 2 will investigate if BDNF differentially regulates the formation of excitatory and inhibitory synapses in ARC neurons expresing either neuropeptide Y or proopiomelanocortin using both immunohistochemistry against presynaptic markers and whole-cell patch-clamp recordings. Aim 3 will determine if TrkB in the dorsomedial hypothalamus (DMH) is required for the control of energy balance and if the number and shape of dendritic spines on TrkB-expressing DMH neurons are altered in mutant mice that lack local BDNF synthesis and develop severe obesity. Findings from this proposed project would provide insights into the mechanism by which BDNF regulates energy balance as well as the role of structural changes in hypothalamic neural circuits in the development of obesity. PUBLIC HEALTH RELEVANCE: Obesity has become a leading health issue in this country due to its high prevalence and associated disorders. Despite the enormous economic cost of obesity, no effective treatments for obesity are currently available. Continuing research efforts to understand the molecular, cellular, and physiological processes regulating energy homeostasis are needed in order to develop effective and safe anti-obesity therapies.

PROJECT SUMMARY Despite a growing consensus that developmental programming of metabolic regulation contributes to the current obesity epidemic, our understanding of developmental mechanisms impacting this process remains rudimentary. The long range goal of this line of research is to define the developmental neurobiology of central pathways that contribute to developmental programming of metabolic phenotype. Maternal high fat diet (MHFD) exposure represents a developmental risk factor that contributes to metabolic dysregulation and impacts leptin sensitivity. MHFD also appears to impact leptin secretion, but it remains unknown whether it affects the developmental actions of leptin. For the proposed studies we will focus on neuronal pathways that connect the paraventricular hypothalamic nucleus (PVH) with two important components of the dorsal vagal complex (DVC): the dorsal motor nucleus of the vagus nerve (DMX) and the nucleus of the solitary tract (NTS), which regulate autonomic functions such as thermogenesis, energy expenditure and gastric motility. Inputs to the PVH from the NTS convey visceral sensory information, and inputs from the arcuate nucleus of the hypothalamus (ARH) and from the NTS convey hormonal signals. Integration of this information in the PVH impacts autonomic regulation through descending projections from the PVH to the DMX and NTS. However, it remains unknown if development of these connections is influenced by factors that program metabolic phenotype, such as MHFD and leptin. The overall hypothesis of the proposed research is that MHFD-L causes hyperleptinemia during a critical period of postnatal development that disrupts normal targeting of connections between the PVH and the DVC, and that the integrity of these connections is important for conveying signals that regulate distinct aspects of neuroendocrine physiology and autonomic function. Molecular genetic manipulation of leptin signaling, neuroanatomical methods, and physiological profiling will be used to address the following Specific Aims: 1) Define the impact of MHFD on formation of connections between the PVH and DVC in offspring, and document corresponding changes in postnatal leptin secretion and autonomic physiology; 2) Determine if leptin is required for development of bidirectional connections between the PVH and DVC; 3) Identify the site(s) of action, and physiological consequences, for developmental effects of leptin on formation of connections between the PVH and DVC. Completion of these aims will advance our understanding of how environmental signals program essential components of neural systems required to maintain normal metabolic physiology, and may identify novel therapeutic targets.

PROJECT SUMMARY Accumulating data indicate that environmental influences during prenatal and early postnatal development can affect individual susceptibility to obesity throughout life. The long-term goal of this line of research is to define molecular events that mediate environmental influences on development of neuronal circuitry involved in the regulation of mammalian energy homeostasis. The hypothalamus is a likely site for coupling early environmental signals to lifelong control of body weight, and neuronal projections from the arcuate (ARH) to the paraventricular (PVH) nucleus of the hypothalamus are essential in regulating energy balance. The adipocyte-derived hormone leptin directs formation of neuronal projections from the ARH to the PVH, and distinct aspects of body weight regulation are dependent on these developmental actions of leptin, specifically during a postnatal critical period. The mechanisms that restrict formation of neuronal pathways to discrete developmental periods are largely unknown. But DNA methylation, which appears to function as a major epigenetic mark stably modulating expression of genes that control development in a variety of neural systems, is an excellent candidate. The overall hypotheses of the proposed research are that 1) the critical developmental window during which ARH neurons are responsive to the neurotrophic actions of leptin is dictated by epigenetic mechanisms, and 2) cell type-specific methylation of genic and other regulatory regions is essential for accurate targeting of functionally distinct classes of neurons, with consequences for normal metabolic physiology. We will test these hypotheses by mapping cell type-specific epigenetic expression modules in the ARH during and after closure of this critical developmental window (Aim 1). In addition, we will identify leptin-mediated epigenetic changes within subpopulations of ARH neurons that display leptin- dependent patterns of projections to the PVH (Aim 2). We will also use transgenic mice with targeted disruption of the DNA methylation machinery to determine if DNA methylation is required for closure of the critical developmental period for the neurotrophic action of leptin on ARH projections and associated metabolic physiology (Aim 3). The proposed research will objectively and definitively assess whether epigenetic mechanisms specify critical periods of brain development when leptin and perhaps other environmental factors impact brain architecture and the regulation of energy balance throughout life.