Lance J. Kriegsfeld - US grants

DESCRIPTION (applicant?s abstract): In the absence of environmental time cues, animals continue to exhibit daily cycles in behavior and physiology with a period of approximately 24 h. In mammals, these rhythms are endogenously generated by bilaterally symmetric nuclei situated above the optic chiasm, the suprachiasmatic nuclei (SCN). A direct retinohypothalamic tract (RHT) from retinal ganglion cells to the SCN is responsible for transmitting light information from the retina to the circadian pacemaker to allow animals to entrain (synchronize) these endogenous rhythms with the environmental light:dark cycle. An important question is what neurotransmitter(s) are involved in the signal transduction pathway from the retina to the SCN. The goal of the present proposal is to investigate the role of nitric oxide in photic entrainment and circadian organization. The present proposal has three specific aims: Specific Aim I: To determine the role of NO in the SCN in photic entrainment. Specific Aim II: To understand the neuroanatomical relationship of NO-positive cells to other known cell types / components of the SCN. Specific Aim III: To clarify the role of NO as an output signal, or as an intra-SCN coordination signal, to modulate circadian locomotor rhythms.

[unreadable] DESCRIPTION (provided by applicant): The timing and coordination of the secretion of myriad hormones is necessary for the maintenance of homeostasis and optimal body functioning. Traditionally, endocrinologists have focused on the role of negative feedback mechanisms and neuroendocrine pulse generators as the primary mechanisms regulating the temporal pattern of hormone secretion. However, it is becoming increasingly clear that endogenous timing systems play a crucial role in this regulation. We recently found that cells containing a novel inhibitory peptide, gonadotropin-inhibitory hormone (GnlH), are highly localized in the brains of Syrian hamsters and other rodents, with fibers projecting diffusely from the septum to the caudal hypothalamus. Importantly, our pilot work shows that brain or peripheral injections of GnlH inhibit LH in a dose-dependent manner. Furthermore, the GnlH system projects to gonadotropin-releasing-hormone (GnRH) neurons, permitting direct, inhibitory control of GnRH secretion. Fibers originating in the brain clock located in the suprachiasmatic nucleus (SCN) form close appositions with GnlH cell bodies, providing a potential means of temporal control. Finally, GnlH cells contain estrogen receptors and respond to estradiol stimulation, suggesting that estrogen acts on these cells to regulate steroid negative feedback. Together, these findings indicate that the GnlH system is organized to modulate reproductive function and the hypothalamo-pituitary- gonadal (HPG) axis. The present proposal is designed to further explore the neuroanatomical and functional means by which the GnlH system is integrated with well understood mechanisms of reproductive control of the HPG axis. This proposal will establish the precise means of communication 1) from the endogenous circadian clock to the GnlH system, 2) from the GnlH system to the reproductive axis, and 3) the functional consequences of the interactions between this novel system and the reproductive axis. Disruptions in the timing of hormone secretion have pronounced adverse effects on human health. For example, jet lag and shift work lead to a variety of reproductive disturbances and an increased risk of developing endocrine-responsive tumors. This proposal seeks to understand the neural mechanisms responsible for hormonal timing to help guide the treatment/prevention of these endocrine disorders. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Female reproductive functioning requires the precise temporal organization of numerous neuroendocrine events by a master circadian brain clock located in the suprachiasmatic nucleus (SCN). Across species, including humans, disruptions to circadian timing result in pronounced deficits in ovulation and fecundity. Our investigations focus on the circadian control of two key neuropeptides with opposing roles, gonadotropin-inhibitory hormone (GnIH, also known as RFRP-3) and kisspeptin. Across mammalian species, GnIH markedly inhibits the secretion of gonadotropin-releasing hormone (GnRH) and pituitary gonadotropin secretion, whereas kisspeptin is a pronounced stimulator of the GnRH neuronal network. Despite the well- established roles for both GnIH and kisspeptin in mammalian reproduction, as well as the knowledge that the circadian timing system is a crucial regulator of the female reproductive axis, the specific neurochemical pathways underlying these interactions are not well understood. The present proposal explores how a hierarchy of circadian oscillators interacts with the GnIH and kisspeptin signaling pathways to regulate GnRH secretion and the preovulatory GnRH/luteinizing hormone (LH) surge. Our work to date indicates that the SCN projects monosynaptically to the GnIH and kisspeptin systems to coordinate their activational states appropriately to allow for initiation of the GnRH/LH surge and ovulation. Additionally, our recent in vitro and in vivo findings point to a novel role for autonomous circadian clocks, operating in GnRH cells, in mediating daily responsiveness of the HPG axis to upstream neurochemical signaling, including kisspeptin. The present proposal combines system, circuit, molecular/genetic, and pharmacological approaches to investigate the interactions among the GnIH, kisspeptin, and circadian systems by exploring: 1) the specific neural loci at which kisspeptin and GnIH interact to regulate GnRH secretion, 2) the neurochemical means by which the SCN coordinates the timed secretion of kisspeptin and GnIH, and 3) the functional implications of these interactions for the GnRH/LH surge and ovulation. The proposed work addresses a classic question in regulatory biology, and has the potential for substantial translational impact in the development of safe/effective contraception, as well as the treatment of a host of reproductive disorders in humans, including precocious and delayed puberty, infertility, and polycystic ovarian syndrome. PUBLIC HEALTH RELEVANCE: Converging lines of evidence implicate a critical role for circadian timing in successful female reproduction across mammalian species, including humans. Women with irregular work or sleep cycles, for example, experience abnormal menstrual cycles, difficultly becoming pregnant, and an increased rate of spontaneous abortions. Given the necessity of proper hormonal timing in reproductive health, our studies examine the role of circadian brain clocks in the coordination of key positive and negative neurochemical regulatory systems critical for ovulation and reproductive success.

This research will provide new information about how hormones specifically control hunger. Decisions about how much to eat are affected by the amount of food previously eaten and the need to engage in other activities. Thus, motivation to eat will be carefully measured in either food-limited or food-unlimited laboratory rodents that will be provided with behavioral options, including the ability to interact with other members of their species. Experiments are designed to determine whether gonadotropin-inhibiting hormone (GnIH) increases the appetite for food, inhibits the reproductive system, or both, and to specify how GnIH orchestrates behavioral priorities. The activation of individual brain cells that secrete GnIH will be measured using immunohistochemistry and a map will be created of the GnIH-cell activation that occurs at the time hunger levels increase and decrease. Drugs that block GnIH binding to its receptors will be used to determine whether GnIH is necessary for food-restriction-induced changes in hunger and the reproductive system. Other experiments will examine whether hormones secreted by the ovary control neural activity in GnIH cells. This project will offer important insights into the high incidence of obesity and eating disorders in women and the explore links to the side effects of contraceptive and hormone replacement therapy. This research will have broad impact on the public's understanding of the appetite for fo, and its interaction with reproduction, fertility, exercise, health, lifestyle, drugs, and prescription hormones. The funding will be used to establish a collaboration with the Kriegsfled laboratory, to team-train graduate and undergraduate students, and to engage in K-12 outreach activities. The award will provide support for the PI's participation, as an NSF ADVANCE chair, in STEM women's seminars and a "writing boot camp". In addition to using traditional publication venues, both PI's will disseminate their work via their webpages and behavioral endocrinology blogs.

Project Summary Successful female reproduction requires the precise temporal coordination of numerous neuroendocrine events by a master circadian pacemaker in the suprachiasmatic nucleus (SCN). Across species, including humans, disruptions to circadian timing result in pronounced abnormalities in the estrous/menstrual cycle, reductions in fertility, and increased miscarriage rates. Our findings to date provide evidence for a network of ovulatory control in which the SCN temporally coordinates the activity of two, key neuropeptidergic systems with opposing actions, the RFamide-related peptide-3 (RFRP-3, the mammalian ortholog of avian gonadotropin-inhibitory hormone) and kisspeptin systems. Across mammalian species, RFRP-3 and kisspeptin are key inhibitory and stimulatory regulators of the reproductive axis, respectively. Despite the well-established role for these neuropeptides in mammalian reproduction, as well as the knowledge that the circadian timing system is a crucial regulator of the female reproductive axis, the specific means by which interactions among these systems appropriately coordinate the timing of neuroendocrine events necessary for ovulation remain unspecified. To understand how elements of this network synergize to produce the coordinated output to the gonadotropin-releasing hormone (GnRH) system required for ovulation, the present proposal uses transgenic mouse models, in combination with cell-specific viral approaches, to elucidate the cellular mechanisms and neurochemical signaling pathways by which the circadian clockwork interfaces with the RFRP-3 and Kp systems. Specifically, the present proposal will: 1) identify the neurochemical signaling pathways by which the SCN communicates with the RFRP-3 and kisspeptin systems, 2) examine the functional contribution of identified pathways systematically, and with cell-specific precision, and 3) determine the functional significance of autonomous, neuroendocrine-cell circadian timekeeping in this network of control through cell-phenotype-specific knockdown of essential clock genes. The proposed work not only addresses a classic question in reproductive biology, but also has the potential for substantial translational impact in the development of safe/effective contraception, as well as the treatment of a host of reproductive disorders in humans, including precocious and delayed puberty, infertility, and polycystic ovarian syndrome.