Xiao-Bing Gao - US grants

DESCRIPTION (provided by applicant): In this proposal, the role and mechanism of melanin-concentrating hormone (MCH) in the regulation and function of the lateral hypothalamus (LH) at the cellular level will be addressed. In feeding regulation LH plays a key role and has been considered as a feeding center. The regulation of food intake is crucial in energy- balance homeostasis. Unbalanced energy accumulation leads to obesity. MCH is a cyclic 19-amino-acid peptide. A substantial body of evidence from systemic, morphological and molecular biological studies suggests that MCH is involved in feeding regulation. However, there is little evidence as to the physiological role of MCH in LH neurons at the cellular level. Also there is little evidence as to how MCH and other neuropeptides interact in LH neurons. Furthermore, there is little data about the cellular physiology as well as properties of neurons and neuronal organization in the LH area. In this proposal, electrophysiological methods (including extracellular recording, whole-cell voltage and current clamp recording), immunocytochemistry and digital calcium imaging will be employed in cultured neurons and acute hypothalamic slices from rats. The chief hypothesis to be tested is that MCH acts as an inhibitory neuropeptide to modulate neuronal activity in the LH. The following hypotheses will be examined: 1. MCH depresses the activity-dependent gene transcription. 2. MCH inhibits the function of voltage-dependent calcium channel subtypes via distinct signaling pathways. 3. MCH depresses glutamatergic and GABAergic synaptic transmission in the LH slices. 4. Characterization and organization of MCH responding neurons in LH slices will be documented. This proposal will begin to bridge the gap between molecular research and systemic studies on the regulation of feeding to benefit those suffering from obesity and obesity-related diseases. The applicant's previous work in characterizing the cellular physiology of the newly discovered neuropeptide hypocretin/orexin is the first step in his journey to pursue his long-term goal of understanding neurotransmission in the LH. The funding of this research proposal is important for the applicant to continue this course. A career development plan is also documented in this proposal.

DESCRIPTION (provided by applicant): The lateral hypothalamus (LH) serves as a central hub, integrating a wide range of inputs from various brain regions and projecting neuropeptide-containing nerve fibers all over the brain. The LH has long been recognized as a feeding center. The regulation of food intake plays an important role in energy-balance homeostasis. Unbalanced energy accumulation leads to obesity, which has been recognized as a risk factor in many diseases and disorders. Despite the overwhelming importance of this structure, it remains poorly understood how the LH integrates different sources of signals and exerts its functions via hypocretin/orexin and melanin-concentrating hormone (MCH) systems in the LH. Both hypocretin and MCH have been implicated as orexigenic: Previous studies have suggested that hypocretin/orexin is an excitatory peptide while MCH is inhibitory. Given their opposite actions at the cellular level, it is intriguing to understand how these two systems interact dynamically to determine the final output and hence to determine the behavioral states of animals. The long-term goal of this project is to understand how these neurons interact with each other to form specific functional organizations for regulating different behaviors. In this proposal, we will use molecular biological, electrophysiological, immunocytochemical and electron microscopic methods as well as digital calcium imaging to address the interaction or hypocretin/orexin and MCH at the cellular level in both normal animals and transgenic mouse models. The chief hypothesis is that the excitatory role of hypocretin/orexin in the LH may serve as the engine to propel this brain region, while the inhibitory modulation induced by MCH may substantially adjust the tone of neuronal activity in the LH area and thus may shape the decision making of the LH and the arcuate nucleus. The following hypotheses will be examined: 1. Hypocretin/orexin serves as a major excitatory force to both hypocretin/orexin and MCH systems. 2. MCH modulates excitability in both hypocretin/orexin and MCH containing neurons. 3. Crosstalk of hypocretin and MCH underlies appropriate regulation of the melanocortin system in the arcuate nucleus. This proposal will begin to bridge the gap between studies at the molecular level and the systemic level on the role of the LH in regulation of food intake. We hope that results generated from this proposal will complement to the current framework in understanding energy homeostasis in mammals. Ultimately, we hope that our research will benefit those suffering from obesity and obesity-related diseases.

DESCRIPTION (provided by applicant): Candidate: My career goal is to be a research scientist addressing mechanisms underlying complicated behaviors in humans and animals under physiological and pathological conditions. I have a substantial record of accomplishment to demonstrate my pursuit of this goal. During my postdoctoral training at Yale, my work on the role of neurotransmitters, neuropeptides and neurotrophic factors in the modulation of neuronal activity in developing and mature hypothalamic neurons, led to peer-reviewed publications (including seven first-author papers and nine secondary author papers) in high quality journals. I was awarded career development grants/awards from the American Heart Association and the NIH. During the last seven years, I established my own independent research program and was awarded a R01 grant from the NIH to address cellular mechanisms underlying feeding regulation in the hypothalamus. We have demonstrated that neuronal plasticity in hypocretin neurons underlies energy homeostasis and sleep regulation, which has been published in high- impact peer-reviewed journals. In this application, I propose that through a period of rigorous research training outlined here, my research ability in basic behavioral science will be substantially enhanced and I will be capable to pursue a new direction (drug addiction) in my research. Environment: I am an Associate Professor in the Section of Comparative Medicine at Yale University School of Medicine. Yale University has provided an outstanding environment for career development during my pursuit to be an independent investigator. I feel that I will be able to take full advantage of the environment and resources here to achieve my goals outlined in this application. Research: Drug addiction has been considered a chronic disease and a risk factor for many other diseases and disorders. There is compelling evidence that drug addiction is a learned behavior. Mechanisms underlying learning and memory (experience-dependent neuroplasticity) in the normal brain are "hijacked" by addictive substances to produce an enduring and compelling urge to consume illicit drugs. The lateral hypothalamus (LH) has long been recognized to participate in the regulation of reward-related behaviors in animals. It is now clear that the hypocretin system participates in behaviors related to drug reward. It is not clear whether the experience-dependent neuroplasticity in hypocretin neurons is essential for the development of drug abuse. In this proposal, our chief hypothesis is that the experience-dependent plasticity in hypocretin neurons is essential for the expression of behaviors related to cocaine reinforcement. The specific aim is to determine whether cocaine self-administration results in enduring changes in synaptic strength of hypocretin glutamatergic inputs and to determine whether manipulations that abolish synaptic plasticity in hypocretin neurons alter cocaine reinforcement in animals. Relevance: Our long-term goal is to bridge the gap between clinical studies and basic research on the role of the CNS in cocaine addiction. The research training proposed in this application is highly relevant to the mission of the NIH and the OppNet. PUBLIC HEALTH RELEVANCE: Drug addiction has been considered a chronic disease and a risk factor for many other diseases and disorders. About 19.7 million Americans are current users of illicit drugs, which has posed a significant burden on the health care system, economy and society in the country. The long-term goal of this proposal is to bridge the gap between clinical studies and basic research on the role of the hypocretin system in cocaine addiction-related behaviors.

Drug addiction has been considered a chronic disease and a risk factor for many other diseases and disorders. To better treat addiction and prevent future abuse of illicit drugs, it is essential to understand the mechanisms underlying addictive behaviors. Clinical and animal studies have established that the metabolic status contributes to the determination of reward threshold in humans and animals. Food restriction increases the sensitivity to drugs of abuse, while over-nutrition decreases the sensitivity to drugs. However, it is still elusive how the brain circuitry regulating the metabolic status interacts with the reward circuitry. The lateral hypothalamus (LH), a central hub integrating a wide range of inputs from various brain regions encoding metabolic, behavioral and environmental cues, is a critical brain area to regulate both energy homeostasis and food/drug reward. Particularly, a selective group of neurons exclusively synthesizing the neuropeptide hypocretin (Hcrt, also called orexin) affect food intake and play a prominent role in food award and drug addiction. Currently it is not entirely clear what role the Hcrt system plays in the hierarchy of circuitry responsible for food reward and drug addiction. Recent studies by others and us indicate that the Hcrt system undergoes experience-dependent synaptic plasticity in animals exposed to cocaine, which leads to our overall hypothesis that the expression of experience-dependent synaptic plasticity in Hcrt cells contributes to the development of addictive behaviors in animals. If this is true, the ability to establish synaptic plasticity in Hcrt neurons may contribute to the susceptibility of animals to addictive behaviors. Based on our previous studies, we hypothesize that the metabolic status of animals may contribute to determination of sensitivity to cocaine through modulating synaptic plasticity in Hcrt neurons. In this R21 application we will begin to address this hypothesis by determining whether either diet-induced obesity (DIO) or chronic calorie restriction (CR) alters the ability of cocaine to trigger plasticity in the Hcrt system with molecular (Hcrt-IRES-Cre mice and DREADDs), cellular (electrophysiological and EM studies) and behavioral (cocaine conditioned place preference) approaches. Two specific aims are: 1) To determine whether DIO induces adaptation in Hcrt neurons, which impedes the expression of activity (or experience)-dependent synaptic plasticity. 2) To determine whether chronic CR induces adaptation in Hcrt neurons, which facilitates the expression of activity (or experience)-dependent synaptic plasticity. Following completion of these important proof-of-concept studies, we will conduct a more comprehensive study to determine how molecular and cellular signaling pathways in Hcrt neurons contribute to the development of addictive behaviors in animals under different metabolic status. Our long-term goal is to bridge the knowledge gap in our current understanding of addiction and to bridge the gap between clinical studies and basic research on the role of the Hcrt system in addictive behaviors, an area of study that has not been well explored thus far.

Drug addiction has been considered a chronic disease and a risk factor for many other diseases and disorders. To better treat addiction and prevent future abuse of illicit drugs, it is essential to understand the mechanisms underlying addictive behaviors. Clinical and animal studies have established that the metabolic status contributes to the determination of reward threshold in humans and animals. Food restriction increases the sensitivity to drugs of abuse, while over-nutrition decreases the sensitivity to drugs. However, it is still elusive how the brain circuitry regulating the metabolic status interacts with the reward circuitry. The lateral hypothalamus (LH), a central hub integrating a wide range of inputs from various brain regions encoding metabolic, behavioral and environmental cues, is a critical brain area to regulate both energy homeostasis and food/drug reward. Specifically, a selective group of neurons exclusively synthesizing the neuropeptide hypocretin (Hcrt, also called orexin) affect food intake and play a prominent role in food award and drug addiction. It is not entirely clear what role the Hcrt system plays in the hierarchy of circuitry responsible for food reward and drug addiction. Recent studies by others and us indicate that the Hcrt system undergoes experience-dependent synaptic plasticity in animals exposed to cocaine, which leads to our overall hypothesis that the expression of experience-dependent synaptic plasticity in Hcrt cells contributes to the development of addictive behaviors in animals. If this is true, the ability to establish synaptic plasticity in Hcrt neurons may contribute to the susceptibility of animals to addictive behaviors. Based on our previous studies, we hypothesize that metabolic/energy status may determine the sensitivity to reward reinforcers through modulating activity and plasticity in Hcrt neurons in animals. In this application stemmed from an R21 grant, we will begin to address this hypothesis by determining whether either over- nutrition or chronic energy deficiency alters the ability of cocaine to trigger plasticity in the Hcrt system with molecular (Hcrt-IRES-Cre mice and DREADDs), cellular (electrophysiological and EM studies) and behavioral (cocaine conditioned place preference) approaches. Three specific aims are: 1) To determine whether over-nutrition causes adaptive changes in Hcrt neurons, which is required in the impairment of drug- seeking behaviors in animals. 2) To test whether chronic energy deficiency leads to adaptation in Hcrt neurons, which facilitates the expression of drug reward in animals. 3) To interrogate whether over-nutrition and energy deficiency-induced adaptations in Hcrt neurons lead to altered responses of target areas of the Hcrt system when animals exposed to cocaine. Our long-term goal is to bridge the knowledge gap in our current understanding of addiction and to bridge the gap between clinical studies and basic research on the role of the Hcrt system in addictive behaviors, an area of study that has not been well explored thus far.

Obesity, referring to having an abnormally high proportion of body fat, has become a health and economic problem in wealthy countries such as the United States. Therefore, intensive investigations have been conducted to determine the factors leading to the positive energy balance between energy intake and energy expenditure. The hypothalamus plays a substantial role in the regulation of energy intake and expenditure. However, it is still not clear how various neuronal systems in the hypothalamus interact to maintain normal body weight and how the dys-regulated interactions may compromise the balance between energy intake and expenditure. In this application we propose to identify interactions between melanin-concentrating hormone- synthesizing (MCH) neurons and hypocretin (Hcrt) neurons in the lateral hypothalamus in the regulation of energy balance and body weight at molecular, cellular and whole animal levels. Both MCH and Hcrt systems are critical players in the regulation of energy balance in animals and humans. The MCH system promotes energy intake and decreases energy expenditure while the Hcrt system promotes both energy intake and expenditure. There has been morphological and functional evidence on the possible interactions between these two systems. However, it is still not entirely clear whether the interaction between these two systems is required in the regulation of energy balance in animals and humans and whether changes in the interaction may lead to altered responses of the brain to the energy status of organisms and compromised energy metabolism. Based on current data we hypothesize that a crosstalk between MCH and Hcrt systems is required to maintain normal body weight. Specifically, we propose that the MCH system requires activation from Hcrt cells to promote energy intake, while MCH neurons may provide a feedback pathway to limit the activity in the Hcrt system to curb energy expenditure and maintain the normal body weight in animals. Since neuronal plasticity plays a critical role in the formation and modification of homeostatic and behavioral functions in animals, it is very likely that the modulation of synaptic function in MCH and Hcrt neurons could be one of the mechanisms underlying the interaction between these two systems. Therefore, three goals of this application are: 1) To test the hypothesis that MCH-mediated food intake requires activation from the Hcrt system in animals.; 2) To interrogate the hypothesis that the MCH system is required to limit activity in the Hcrt system to curb energy expenditure; and 3) To examine whether the imbalance between the activities in MCH and Hcrt cells underlie dys-regulated metabolic status (DIO and anorexia) in animals. The answers to these questions will help address the crosstalk between homeostatic centers in the hypothalamus and the role of the crosstalk in the determination of energy metabolism. We hope that this project will benefit people suffering from diseases/conditions (such as obesity and anorexia) resulting from impaired regulation of energy metabolism.