Zachary A. Knight - US grants

DESCRIPTION (provided by applicant): Leptin is a hormone secreted by adipocytes that acts as the major signal in a negative feedback loop controlling bodyweight. Leptin treatment of leptin deficient (ob/ob) mice and humans results in profound weight loss, but more common diet-induced obesity is associated with high plasma leptin levels and resistance to leptin's weight-reducing effects. The molecules and signaling pathways that are responsible for the development of leptin resistance are largely unknown, but such molecules would be attractive targets for obesity therapy. This application describes experiments that will clarify the physiological events that lead to leptin resistance and identify candidates that represent novel cellular regulators of leptin sensitivity. These experiments make use of new approaches and models that overcome one of the key technical challenges that has frustrated efforts to study leptin resistance: the difficulty of accessing leptin's direct target cells, a small subset of neurons dispersed throughout the hypothalamus. Finally, the physiological function of candidate leptin regulators will be explored in rodent models of obesity, using a combination of genetic, anatomical, and pharmacological approaches. Special emphasis will be placed on the use of small molecule drugs, accessed through synthetic chemistry, to rapidly validate candidates in vivo. This research plan will help advance my career goal to lead an interdisplinary research laboratory that applies my background in synthetic chemistry and small molecule discovery to address questions in obesity and metabolic disease. The mentored phase of this research will be conducted in the laboratory of Jeffrey Friedman at Rockefeller University, who has been a leader in field of molecular obesity research. PUBLIC HEALTH RELEVANCE: Obesity is a major public heath problem. This research seeks to understand basic mechanisms that control body weight and identify novel drug targets for obesity therapy.

? DESCRIPTION (provided by applicant): A deeper understanding of the biologic origins of obesity will require mapping the neural networks that control feeding. Two key neural populations that control feeding are AgRP and POMC neurons in the hypothalamus. Despite extensive study of these cells over the past 20 years little is known about their natural dynamics in vivo. We have used fiber photometry to record the natural activity of AgRP and POMC neurons in awake behaving mice. Using this approach we have discovered that AgRP and POMC neurons are rapidly (seconds) and dramatically modulated by sensory cues associated with food. This regulation is cell-type-specific, is sensitive to food palatability and nutritional state, and occurs before any food is consumed. These data indicate the existence of an unanticipated neural pathway by which sensory detection of food generates rapid anticipatory changes in the activity of AgRP and POMC neurons. Importantly, this rapid regulation provides a mechanism for AgRP and POMC neurons to integrate sensory and hedonic cues with homeostatic information about nutritional state, suggesting a more complex role for these first order neurons than is currently appreciated. We propose here to delineate the neural mechanisms underlying the remarkable rapid regulation of these cells by food detection. We propose further to explore the downstream pathways to which these sensory cues are communicated in order to understand their role within a distributed feeding network.

? DESCRIPTION (provided by applicant): One the most remarkable features of animals is their ability to sense changes in their internal physiologic state and then generate flexible behaviors that restore homeostasis. These survival processes are mediated by neural circuits in the brain that sense the state of the body and then convert this information into specific behavioral and autonomic responses. However our understanding of these homeostatic circuits has remained limited due to their structural complexity, as they are embedded within brain regions that contain a vast diversity of intermingled neural cell types. Understanding how this vast cellular diversity s organized into circuits that give rise to behavior is one of the central challenges in neuroscience I describe in this proposal how we have used a technology for activity-dependent RNA sequencing to generate a map of the key thermoregulatory neurons in the mouse brain. I then describe how we will develop two new technologies that will allow us to delineate the complete neural circuit that is downstream of these thermosensitive cells. These transformative new technologies enable the molecular identification of neurons that are anatomically or functionally connected and thereby allow for the first time the systematic application of RNA sequencing to deconstruct the cellular organization of neural circuits in the brain. I further outline how we wil use these new approaches to investigate the logic underlying the distributed and overlapping structure of neural circuits that mediate various aspects of physiologic homeostasis. This analysis takes advantage of information about the cellular organization of these circuits that could not be generated without the new technologies described in this proposal. Thus the experiments in this proposal will generate new tools for the neuroscience community and further apply these tools to address fundamental questions about the neural mechanisms that control homeostatic processes such as thermoregulation and feeding.

? DESCRIPTION (provided by applicant): A better understanding of the mammalian thermoregulatory system may lead to new therapeutic strategies for the treatment of conditions associated with hyperthermia such as infection, drug abuse, and stroke. Classical studies identified the preoptic area (POA) of the anterior hypothalamus as the principal site of mammalian thermoregulation. This region is postulated to contain warm-sensitive neurons that are activated by environmental heat and in response trigger an array of autonomic and behavioral responses that restore temperature homeostasis. An extensive scientific literature accumulated over the past 75 years has supported the existence and importance of these warm-sensitive cells, yet their neurochemical identity remains unknown. For this reason key components of the mammalian thermoregulatory circuit have remained inaccessible to modern genetically-targeted approaches in neuroscience. Recently we have used an approach for activity- based RNA sequencing to identify molecular markers that selectively label these long-sought warm-sensitive cells. We have shown using cell-type-specific optogenetic manipulations that activation of these neurons inhibits brown adipose tissue thermogenesis and lowers body temperature. We have also recorded the activity of these neurons in awake behaving mice and shown that they are activated by ambient heat and inhibited by ambient cold. Thus we have identified a novel, molecularly-defined neural population that functionally resembles the long-sought warm-sensitive cells of the POA. We propose here to apply approaches for genetic and projection-targeted neural manipulation and recording to elucidate the downstream circuit by which these cells control behavioral and autonomic thermoregulation.

Abstract Behavior is motivated by reward, and the most powerful rewards are those that satisfy a physiologic need. For decades, neuroscientists have studied the midbrain dopamine system to understand reward and hypothalamic circuits to understand sensing of internal needs. But how these two neural systems are interact to give rise to behaviors like eating and drinking remains poorly understood. Recently, we have used approaches for simultaneous neural recording and manipulation to observe directly the communication between these two systems. We have also mapped the signals they each receive from the gut in response to ingestion of food and fluids. This has revealed that hunger and thirst powerfully modulate the dopamine system, but do so in different ways and likely involve distinct circuit mechanisms. We propose here to build on these findings to systematically delineate how these neural circuits for need and reward interact in the brain. In Aim 1, we investigate how these circuits represent internal needs, by recording their dynamics at multiple levels of analysis under different physiologic states, and further measuring how those dynamics are influenced by targeted circuit manipulations. In Aim 2, we investigate how these circuits use information about bodily needs to drive learning about food, by monitoring and manipulating their activity during the learning process. In Aim 3, we investigate how these circuits use information about internal state to drive motivation, by monitoring and manipulating their activity during tasks where animals must evaluate competing needs and rewards. These studies will provide fundamental insight into the mechanisms by which information about body needs is utilized by the brain to generate learning and motivation.