Martin J. Kelly - US grants

neurophysiology; neuroendocrine system; neurons; estrogens;

Although it has been known for decades that opiates inhibit the reproductive cycle of the mammal via direct actions on the hypothalamus, little research has been done on the cellular mechanism of their action. The present proposal will evaluate firstly the physiological effects of the endogenous opioids on hypothalamic neurons, and secondly, the effects of chronic morphine on these same cells. Hypothalamic slices will be prepared from cycling female guinea pigs, and single electrode voltage clamp experiments will be done in order to elucidate the acute and chronic effects of opioids on the membrane properties of arcuate and cell-poor zone (ARC-CPZ) neurons. Dose response curves will be generated based on the changes in membrane current caused by specific opioid agonists. Schild analysis will be utilized to characterize the specific receptor(s) involved in the direct actions of the opioids, and the specific K+ and/or Ca+2 conductance(s) coupled to the receptor(s) will be ascertained. LHRH release from hypothalamic slices will be measured and Schild analysis of the inhibition of peptide release by specific agonists will be correlated with the voltage clamp data on single ARC-CPZ neurons. The effects of 17beta-estradiol (E2) on the acute actions of the opioids will be ascertained by using slices prepared from ovariectomized females and ovariectomized females that have received E2 replacement. Dose response curves will be established for specific opioid agonists mesuring ion conductances. LHRH release will also be measured in these same slices and shifts in the dose response curve and any changes in receptor affinity ascertained. Once the physiological role of the endogenous opioids in regulating the activity of ARC-CPZ neurons has been assessed, then studies will be initiated to determine the effects of chronic morphine on the electrophysiological properties of these cells and on the release of LHRH. Slices will be prepared from physically dependent and tolerant guinea pigs, and the changes in the opioid dose response and in their coupling to specific membrane conductances determined in ARC-CPZ neurons. It is envisioned that these studies will elucidate the physiological role of the opioids in the control of the mammalian reproductive cycle and the basis for the inhibition of the cycle with chronic abuse.

DESCRIPTION: The long-term goal of the proposal is to understand the cellular actions of estrogen and morphine on hypothalamic neurosecretory neurons which control anterior pituitary secretions. The application is based upon an ongoing NIDA- funded research program regarding the electrophysiology of hypothalamic neurosecretory neurons. The Principal Investigator is now seeking to increase the power of his techniques by including whole-cell patch recordings. This will greatly expand the candidate's research capabilities and is in keeping with his career development plans. The proposal has a training and research component. The award would allow the applicant to increase his time in the laboratory and to receive training in vital new biological techniques. The research component of the application seeks to further our understanding of the role of estrogens in controlling neurosecretory neurons of the hypothalamus. Since estrogen appears to decrease the number of functional mu opioid receptors in a manner similar to chronic morphine treatment, the research focuses on the interaction of and the common cellular components affected by both agents. Hypothalamic slices will be prepared from ovariectomized female guinea pigs which have been treated with morphine or placebo. The first series of experiments will study the interaction of chronic morphine in vivo and acute estrogen in vitro in altering the mu opioid mediated membrane hyperpolarization of identified arcuate (ARC) neurons by measuring the shift in the mu opioid dose-response curve in the two groups of animals.Cells from morphine- treated or placebo- treated, ovariectomized (oil-treated) guinea pigs will be tested with mu opioid agonists before and after perfusion with 17 Beta-estradiol. The time- course and site of estrogen's rapid actions to decrease the potency of the mu opioid response will also be determined. Finally, the effects of chronic morphine on a heterologous receptor response (GABA-B) which is coupled to the inwardly rectifying K channel and affected by estrogen will be measured. Other experiments will study the effects of mu opioid agonists on supraoptic (SON) vasopressin and oxytocin magnocellular neurons by ascertaining the specific K and Ca conductances affected by mu opioid agonists and characterize the interaction of estrogen and chronic morphine on SON neurons.A third series of experiments will investigate the cellular mechanism by which estrogen and morphine decrease the potency of mu opioid agonists to hyperpolarize ARC (SON) neurons by measuring the expression of G-alpha-i and G-alpha-o mRNA using in situ hybridization and immunocytochemistry to identify the cells. These experiments will be followed by whole cell patch recording from ARC (SON) neurons to study the role of G-proteins in the estrogen- mediated attenuation of the mu opioid response.

DESCRIPTION (provided by applicant): The long range goal of the proposed research is to elucidate the mechanisms by which 17¿-estradiol (E2) signals in hypothalamic neurons to control energy homeostasis. At the core of the regulation of energy homeostasis, and hence the central feedback of insulin (and leptin), are the hypothalamic arcuate proopiomelanocortin (POMC) and neuropeptide Y/agouti related peptide (NPY/AgRP) neurons. These neurons form a reciprocal circuit that controls energy homeostasis. We have recently discovered that both leptin and insulin depolarize POMC neurons via activation of canonical transient receptor potential (TRPC) channels, and hyperpolarize NPY/AgRP neurons via activation of KATP channels. Moreover, E2 exerts rapid effects through a putative Gq coupled membrane estrogen receptor (GqmER) that either attenuates or augments GABAB mediated inhibition to increase POMC or decrease NPY/AgRP neuronal excitability, respectively. Given the functional convergence of E2 and insulin in the hypothalamus, we propose the novel hypothesis that E2 signaling pathways act in concert with the insulin signaling pathway to upregulate POMC and downregulate NPY/AgRP neuronal excitability and gene expression in a cell specific manner. These estrogenic actions protect against the development of diet induced insulin resistance in POMC neurons. Elucidating the cell specific signaling pathways and gene expression at the single cell level will help in developing new therapies for targeting hormone actions in CNS neurons and also for countering insulin resistance in CNS neurons that leads to diabetic neuropathy and stroke. Our multidisciplinary approach incorporates a unique array of cellular, molecular and optogenetic tools and our combined expertise in electrophysiology, chemical genetics, molecular biology, histochemistry and whole animal physiology. Our specific aims are the following: (1) To elucidate the cellular/molecular mechanisms by which insulin activates TRPC channels in POMC neurons; (2) to elucidate the mechanism by which E2 augments insulin signaling in POMC neurons; (3) to elucidate the direct inhibitory effects of POMC synaptic input to NPY/AgRP neurons using optogenetic stimulation of POMC neurons and recording of postsynaptic inhibitory responses in NPY/AgRP neurons; and (4) to elucidate the changes in insulin signaling in POMC neurons associated with diet induced insulin resistance. Women show increased risk of insulin resistance in hypoestrogenic states (e.g., with the onset of menopause), which in turn can lead to severe injury to the nervous system as seen in diabetic neuropathies and stroke. Therefore, a greater understanding of how E2 protects against insulin resistance and specifically how E2 signaling cross talks with insulin signaling wil provide unique insights and novel approaches to prevent insulin resistance in the CNS.

DESCRIPTION (provided by applicant): Hypothalamic proopiomelanocortin (POMC) neurons have been shown to play a critical role in energy homeostasis. The long-range goal of the proposed research is to define the differences in signaling mechanism(s) by which estrogen (E2) modulates opiomelanocortin tone and subsequently homeostatic functions in a sex-specific manner. Understanding the actions of E2 on POMC neurons will provide insight into fundamental differences between females and males in the control of feeding, and as a consequence eating disorders, which are more common in females after puberty. The biological bases for these discrepancies are unknown, but likely involve hypothalamic POMC neurons and their response to E2 and serotonin (5HT) in a sex-specific manner. We have found that E2 can rapidly disinhibit female POMC neurons via uncoupling mu-opioid and GABAB receptors, and have identified a putative membrane-associated E2 receptor (mER) that is Gq-coupled to a phospholipase C-protein kinase C-protein kinase A pathway, which is very similar to what has been described for serotonin 5TH2A/C receptors. In addition, we have synthesized a new SERM, STX that specifically targets this novel E2 signaling pathway. Rapid signaling by E2 (STX) has a number of downstream targets including uncoupling G i,o coupled neurotransmitter receptors from K+ (GIRK) channels in POMC neurons, which enhances neuronal activity. Our hypothesis is that sex differences in the control of feeding and energy homeostasis are due, in part, to the greater efficacy of E2 in females versus males to dis-inhibit POMC neurons and that the mER and 5HT2c intracellular signaling pathways converge in POMC neurons in a sex specific manner. In this proposal, we seek to elucidate the cellular cascades activated by E2 and serotonin in both sexes. We will use a unique range of cellular, molecular and chemical tools to characterize the signaling pathways in POMC neurons and its functional consequences in both male and females. The specific aims are: (1) To test the efficacy of STX in gonadectomized animals to uncouple GABAB, f-opioid and 5HTiA receptors from K+ channels in POMC neurons. (2) To delineate the 5HT2A/c signaling pathway and determine its convergence with the mER signaling pathway in POMC neurons. (3) To measure the effects of STX on food intake and energy metabolism in gonadectomized male and female guinea pigs. (4) To measure the effects of STX on POMC neurons in wild type and ER? deficient mice. The results from these studies will not only help to elucidate sex differences in estrogen and serotonin signaling pathways in hypothalamic POMC neurons that control feeding and energy homeostasis, but potentially will allow the development of SERMs for treatment of eating disorders.

The synthesis, release and actions of steroid hormones such as estrogens, progesterone, testosterone, and cortisol are vital for normal daily living. For example, the absence of cortisol production is incompatible with life, and insufficient estrogen production in women leads to hot flashes, osteoporosis and infertility. In addition, steroids promote the growth of some cancers. The purpose of this conference is to bring together world-renowned scientists studying the effects of steroid hormones in the brain and other organ systems (heart, pancreas, gonads, colon, etc.). The overarching goal is to develop broad new concepts about the way in which steroids have immediate impact on individual cells in various organ systems as well as long term effects on growth and development of organ systems. This meeting will not only foster new collaborations among the experts in the field but will also provide a venue for junior scientists of diverse background to interact and collaborate with these expert leaders. Moreover, the FASEB conference will foster the development of novel systems level biological approaches toward understanding the communication networks (via blood, nerves, etc.) involved in steroid actions throughout the body. The cross-talk between this distinguished group of scientists, which include marine biologists that work on simple marine organisms and physicians that treat patients, will allow the development of common research approaches to better understand endocrine systems.

DESCRIPTION (provided by applicant): The primary goal of this project is to elucidate the cross-talk between leptin and 17-estradiol signaling in kisspeptin neurons. Congenital leptin deficiency and/or loss of leptin function due to mutations in leptin can cause obesity and hypogonadotropic hypogonadism. Hypothalamic hypogonadism and its associated disturbances can be reversed by administration of leptin. Leptin signals via its cognate receptors, leptin receptors (LRs). The long isoform (LRb) is the predominant signaling form of the receptor and is abundantly expressed in hypothalamic neurons, including arcuate proopiomelanocortin (POMC) and kisspeptin neurons, but not in gonadotropin releasing-hormone (GnRH) neurons. Therefore, the effects of leptin on GnRH neurons are thought to be mediated indirectly via neurons synapsing on GnRH neurons. Hypothalamic kisspeptin neurons play a critical role in modulating GnRH release and hence the control of reproduction. Moreover, KiSS1 mRNA is reduced in obese and infertile ob/ob mice, and the levels of Kiss1 mRNA increase after administration of leptin. Furthermore, KiSS1 mRNA and kisspeptin protein are highly regulated by 17- estradiol (E2), and recently, we have found that E2 differentially regulates arcuate kisspeptin neurons in the female guinea pig, inhibiting expression during negative feedback and augmenting expression during positive feedback. In addition, we have discovered that leptin depolarizes POMC neurons via a novel signaling pathway that is coupled to activation of canonical transient receptor potential (TRPC) channels, and kisspeptin neurons may be similarly regulated. Therefore, our current work focuses on hypothalamic arcuate kisspeptin neurons and the interaction between E2 and leptin acting through multiple signaling cascades to affect kisspeptin neuronal excitability and ultimately the reproductive cycle. Our multidisciplinary approach incorporates a unique array of cellular and molecular tools and our combined expertise (electrophysiology, molecular biology, histochemistry and whole animal physiology). Our working hypothesis is that the kisspeptin neurons are the gate-keeper of excitatory drive to GnRH neurons in the female, and it is the complex interaction of E2 and leptin in these neurons that control the ovulatory cycle in fed and fasted states. Therefore, our specific aims are the following: (1) To characterize the leptin signaling pathway in arcuate kisspeptin neurons in ovariectomized female guinea pigs. (2) To characterize the effects of E2 on arcuate kisspeptin neurons during positive feedback versus negative feedback. (3) To elucidate the actions of E2 on arcuate kisspeptin neurons during positive feedback in fasted versus fed guinea pigs. (4) To elucidate the effects of E2 and fasting on the expression of K-ATP channels in kisspeptin neurons. Understanding the convergence of leptin and E2 signaling in arcuate kisspeptin neurons will provide insight into the fundamental role of these hormones in conveying metabolic cues to the reproductive axis.

Estrogens are steroid hormones that affect virtually every tissue in the body including the brain. All of the actions of estrogen have been ascribed to its binding to the ?classical? intracellular estrogen receptors ERá and ERâ and subsequent gene activation, which is a slow event. However, there is overwhelming physiological evidence that estrogens have rapid effects in the brain and other non-neural tissues that do not involve these classical receptors but rather is thought to involve a plasma membrane localized estrogen receptor. Indeed, a novel compound called STX has been synthesized that selectively activates this membrane estrogen receptor and mediates many of the physiological processes ascribed to estrogen without the growth promoting effects. The structure of the membrane estrogen receptor is not yet known although aspects of its cell signaling properties have been elucidated. Therefore, this project aims to identify and clone the membrane estrogen receptor using well-established functional cloning strategies. The specific sites expressing the membrane estrogen receptor will be identified in the brain and non-neural tissues by our two neuroscience graduate students. Identification of the membrane estrogen receptor will break new ground and generate new approaches for studying estrogen biology in particular physiological functions (energy and bone homeostasis, temperature regulation, stress responses, circadian rhythms, sleep cycles, learning/memory and mood) that become dis-regulated in hypo-estrogenic states in females (e.g., menopause). In addition, students and fellows world-wide have utilized STX to probe the function of the membrane estrogen receptor in multiple tissues (brain, bone, liver, and pancreas) and will benefit tremendously from having the membrane estrogen receptor clone since the gene sequence of the cloned estrogen receptor will be deposited immediately in GeneBank for the broader research community. Therefore, cloning the membrane estrogen receptor will have an enormous benefit not only for neuroscientists but for students of life sciences worldwide.

Project Summary The long range goals of the proposed research are to elucidate the mechanism(s) by which metabolic states and 17?-estradiol (E2) regulate arcuate nucleus (ARC) kisspeptin (Kiss1) neuronal circuits that are critical for coordinating energy homeostasis and reproduction in females. It is well known that E2 is anorexigenic, and that Kiss1 neurons which are directly regulated by E2, are essential for pubertal development and adult reproductive success. However, their role in the control of energy homeostasis is less understood. We have shown that the ARC Kiss1 neurons are directly excited by leptin and insulin indicating that they may serve an important role in the control of energy homeostasis. Also, we have evidence that glutamate is released from ARC Kiss1 neurons and targets anorexigenic proopiomelanocortin (POMC) neurons and orexigenic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons. In addition, we have found that glutamate can differentially regulate POMC and NPY/AgRP neurons by acting on separate groups of metabotropic glutamate receptors (mGluRs). Moreover, we have discovered that E2 increases vesicular glutamate transporter 2 (vGluT2) mRNA in female ARC Kiss1 neurons, an indication of heightened vesicular glutamate packaging and release. We also have evidence that ARC Kiss1 neurons project to and excite AVPV/PeN Kiss1 neurons, which are important for the induction of the GnRH/LH surge. Thus, we believe that ARC Kiss1 neurons integrate metabolic hormone and steroid cues to regulate both energy homeostasis and reproduction. Therefore, we propose the novel hypothesis that the excitability of ARC Kiss1 neurons is increased in high estrogenic states thereby releasing glutamate to excite POMC neurons and inhibit NPY/AgRP neurons via group I and group II/III mGluRs, respectively, which decreases food intake. In addition, excitatory glutamatergic input to AVPV/PeN Kiss1 neurons from ARC Kiss1 neurons constitutes a critical stimulatory drive to GnRH neurons at the time of GnRH/LH surge. Our multidisciplinary approach incorporates a powerful set of cellular, molecular and optogenetic tools to address the following aims: 1) To elucidate in ARC Kiss1 neurons the effects of E2 on the mRNA expression and function of Cav3 and HCN ion channels and the expression of vGluT2 mRNA; 2) to elucidate the direct synaptic input to ARC POMC and NPY/AgRP neurons from ARC Kiss1 neurons using optogenetic stimulation in combination with whole-cell recording in E2-treated females; 3) to elucidate the direct synaptic input to AVPV/PeN Kiss1 neurons from ARC Kiss1 neurons using optogenetic stimulation and whole-cell recording in E2-treated females; 4) to elucidate the effects of high frequency optogenetic stimulation of ARC Kiss1 neurons on GnRH release and on food intake in E2-treated females. Therefore, elucidating the circuits and signaling cascades underlying the actions of E2 in the hypothalamus will provide a neurophysiological framework whereby Kiss1 neurons could coordinate reproduction with changes in energy status.

Project Summary The long range goals of our research program has been to elucidate the mechanism(s) by which metabolic states and 17?-estradiol (E2) regulate arcuate nucleus kisspeptin (Kiss1ARH) neuronal circuits that are critical for coordinating energy homeostasis and reproduction in females. It is well known that E2 is anorexigenic, and that Kiss1 neurons, which are directly regulated by E2, are essential for pubertal development and adult reproductive success. However, their role in the control of other homeostatic functions is just emerging. Earlier, we found that Kiss1ARH neurons are excited by leptin and insulin via canonical transient receptor potential (TRPC) 5 channel signaling and proposed that they may serve as an important hub in the control of energy homeostasis. Recently, we found that high frequency optogenetic stimulation of Kiss1ARH neurons releases glutamate to excite the anorexigenic proopiomelanocortin (POMC) neurons but inhibit the orexigenic neuropeptide Y/agouti-related peptide (AgRP) neurons in both females and males. E2 increases vesicular glutamate transporter 2 (Vglut2) mRNA expression and glutamate release from female Kiss1ARH neurons to augment the POMC excitation and AgRP inhibition. In contrast, Vglut2 mRNA expression and glutamate release are increased in castrates as compared to intact males, illustrating an important sex difference in the synthesis and release of glutamate. Key excitatory cationic channels are upregulated by E2 leading to increased excitability and glutamatergic synaptic transmission. Recently, we have found that the selective membrane estrogen receptor (GqmER) agonist STX increases the excitability of Kiss1ARH neurons without downregulating the peptide expression. It also decreases food-intake in both females and males. Therefore, we hypothesize that estrogenic signaling in Kiss1ARH neurons is important for increasing Kiss1ARH neuronal excitability and maintenance of homeostatic functions critical for reproductive success. Our multidisciplinary approach incorporates a powerful set of cellular, molecular, genetic and optogenetic tools, and our combined expertise in molecular biology, electrophysiology, and whole animal physiology to address the following aims: (1) to measure the estrogenic-mediated increase in excitability of Kiss1ARH neurons using GCaMP6 and Voltron recordings; (2) to elucidate the estrogenic modulation of the synaptic input from Kiss1ARH to hypothalamic paraventricular nucleus neurons using optogenetic stimulation and its effects on food intake in E2 (STX)-treated females and STX-treated males; and (3) to elucidate the estrogenic modulation of synaptic input from Kiss1ARH neurons to hypothalamic dorsomedial nucleus neurons and its effects on energy expenditure in E2 (STX)-treated females and STX-treated males. Elucidating the circuits and signaling cascades underlying the actions of E2 and STX will provide a neurophysiological/neuropharmacological framework for a more thorough understanding of the cellular mechanisms by which Kiss1ARH neurons coordinate homeostatic functions with reproduction.