Suzanne M. Appleyard - US grants

Obesity is a major health problem in the United States affecting a large portion of the population. It can lead to serious diseases such as heart failure, stroke and diabetes. A more detailed understanding of the homeostatic regulation of weight is required to find the best method of both preventing and treating this serious disease. POMC neurons in the hypothalamus have been demonstrated to play a critical role m weight homeostasis. Melanocortins inhibit feeding and are essential for appropriate weight homeostasis as mice deficient in melanocortin signaling are obese. The effects of beta-endorphin on weight homeostasis are less clear. Surprisingly, our preliminary results indicate that mice lacking beta-endorphin are also obese. The first goal of this grant is to establish the mechanism of the obesity in beta-endorphin deficient mice and to determine whether this obesity is additive with MC4 receptor deficient mice. NPY modulates the release of alpha-MSH and beta-endorphin from these neurons, and opioid antagonists have been shown to block NPY stimulated food intake. We therefore plan to test the hypothesis that beta-endorphin is required for the action of NPY and predict that mice lacking beta-endorphin will be deficient in NPY- stimulated feeding. The ventrobasal hypothalamus has an inhibitory tone on weight homeostasis and high levels of expression of leptin receptors. Both POMC and NPY neurons have cell bodies in this region and express leptin receptors. In addition to the melanocortins, POMC neurons also express CART, a transmitter shown to potently inhibit feeding and predicted to play an important role in weight homeostasis, as well as potentially unidentified transmitters. We hypothesize that other transmitters in the POMC neurons, aside from the melanocortins, are important for weight homeostasis and leptin action. To test this, we propose to remove all signaling through these POMC neurons by specifically ablating the POMC neurons in adult mice. Weight homeostasis and leptin function in these mice will then be compared to mice deficient solely in POMC signaling.

Obesity is a major health problem in the Western wodd and is a leading contributor to cardiovascular disease and diabetes mellitus. The causes of obesity are multifactorial. However, its severity and early onset in children with monogenic mutations in the proopiomelanocortin (POMC) and metanocortin-4 receptor genes highlight the importance of the POMC system in the regulation of energy balance. Remarkably, the cell bodies of POMC neurons are located in only two regions of the brain: the arcuate nucleus of the hypothalamus (ARC) and the nucleus of the solitary tract (NTS) in the medulla. As any pharmacological intervention using either melanocortin or opioid drugs will affect the signaling from both regions, it is critical to determine the function of the NTS-POMC neurons for rational predictions concerning the long-term consequences of drug therapy aimed at these targets. However, although the NTS receives visceral sensory inputs and is interconnected with regions proposed to act as feeding centers, the role of the POMC neurons in the NTS in energy homeostasis remains unknown. I hypothesize that these NTS-POMC neurons are a vital component of neural pathways that control energy homeostasis and are capable of integrating the sensory afferent and hormonal input into the NTS. This research plan uses a multi-disciplinary approach to address key aspects of this hypothesis. The Low lab has recently developed transgenic mice in which the POMC neurons are identified by EGFP, allowing the examination of the rote of these POMC neurons in the NTS pathways, using both electrophysiologicat and immunocytochemical techniques The proposed specific aims wilt identify neuroanatomic, neurochemical, regulatory, and molecular properties of NTS-POMC neurons, tn addition, I plan to ablate the NTS-POMC neurons to directly determine if they are important for energy balance. An important aspect of this proposal are the training goals. Dr. Michael Andresen's knowledge and experience with his unique set up for studying NTS neurons and their regulation by afferent inputs are critical for this proposal to characterize the NTS POMC neurons using electrophysiological approaches. Additionally, 1plan to expand my training in immunocytochemicat and retrotabeting studies by utilizing the extensive experience and knowledge in this area available at OHSU, as these techniques will be invaluable for my long-term career goal of becoming an independent investigator.

DESCRIPTION (provided by applicant): Obesity is a major health problem in the United States and is a leading contributor to cardiovascular disease, diabetes mellitus and stroke. One of the less well understood areas of obesity research are the mechanisms by which neurons respond to, integrate and pass on central and peripheral signals about energy state and how these signaling pathways are altered in obesity. Our long-range goal is to understand the molecular and cellular mechanisms by which neurons in the nucleus of the solitary tract (NTS) of the brainstem control body- weight and how these mechanisms are altered in obesity. Visceral afferents, including gastric afferents, carrying information about satiety terminate in the NTS. As such, NTS neurons act as gates, determining what information contained in afferents is passed onto other brain regions. Ablation of NTS neurons expressing catecholamines (NTS-CA neurons) disrupts control of food intake by several hormones, including ghrelin and cholecystokinin (CCK). The overall objective of this proposal is to identify the mechanisms by which NTS-CA neurons respond to appetite-regulating inputs under normal and obese conditions. Our central hypothesis is that the output of NTS-CA neurons is an integration of afferent and hormonal inputs and that it is altered by different energy states, such as fasting or obesity. We will test this hypothesis with the following three specific aims. (1) Determine how afferent inputs regulate NTS-CA neurons. Our working hypothesis for this aim is that the firing rate of NTS-CA neurons is controlled by visceral afferents, including gastric afferents, both through direct and indirect inputs. (2) Identify mechanism(s) by which key hormonal appetite modulators regulate NTS- CA neurons. Our working hypothesis for this aim is that CCK, which inhibits food intake and ghrelin, which stimulates food intake, regulate the output of NTS-CA neurons. (3) Determine how NTS-CA neurons adapt to different energy states. Our working hypothesis for this aim is that NTS-CA neurons adapt to prolonged exposure to hormones or conditions of altered energy states. We are well prepared to undertake the proposed research because we have developed a mouse horizontal brain slice that allows us to selectively stimulate visceral afferents while recording from identified NTS-CA neurons using electrophysiological patch clamp techniques. This is an extremely powerful system to address the cellular and molecular mechanisms by which NTS-CA neurons integrate neuronal and hormonal responses and how these mechanisms are altered in obesity. In addition, we will use labeling techniques to specifically identify NTS-CA neurons receiving gastric afferent inputs, pharmacology to dissect out molecular signaling pathways and behavioral paradigms to examine how these pathways/mechanisms are altered in different energy states. The contribution of this work is expected to be significant, because increasing our understanding of the molecular mechanisms underlying appetite control and how these mechanisms are altered in different energy states, will lead to more precisely targeted approaches for the prevention treatment of obesity. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because obesity is a major health problem in the United States and is a leading contributor to cardiovascular disease, diabetes mellitus and stroke. The expected contribution of these studies will be the identification of molecular mechanisms by which one group of critical neurons control weight regulation and how these are altered in obesity. The belief is that increasing our understanding of these mechanisms will lead to more precisely targeted approaches for the prevention and treatment of obesity.

ABSTRACT This application for renewal continues an enduring interest in neural mechanisms by which gastrointestinal (GI) satiation signals are communicated to the hindbrain nucleus of the solitary tract (NTS) by vagal afferents and are integrated with other controls of food intake. Glutamate is the principal neurotransmitter released by vagal afferent terminals in the nucleus tractus solitarius (NTS). As such, glutamate receptors in the NTS are pivotal to the transmission and processing of satiation signals. Our published results, demonstrating that hindbrain injections of N-methyl-D-aspartate-type glutamate receptor (NMDAR) antagonists increase meal size and prevent reduction of food intake by cholecystokinin (CCK) support this assertion. Although vagal afferent activation by GI stimuli reduce meal size, central neuropeptides and circulating hormones also control food intake by controlling meal size, suggesting that they may modulate or imitate the effects of GI stimuli. We postulate that brain peptides interact with NMDAR to control meal size by modulating the strength of glutamatergic vagal afferent synapses in the NTS. Our preliminary results suggest that interaction of vagal afferent NMDAR and melanocortin 4 receptors (MC4R) triggers long-lasting changes in vagal afferent synapsin phosphorylation that are consistent with strengthened vagal afferent synaptic function. Experiments of Aim 1 utilize pharmacological, immunochemical, chemogenetic and electrophysiological approaches to determine the nature of NMDAR participation in MC4R effects on vagal afferent synaptic function and control of meal size. Vagal afferents express type 1 and type 2 NPY receptors (Y1R and Y2R) as well as MC4R and NMDAR. We find that NTS injection of NPY or the Y2R agonist, PYY 3-36, increases food intake, an effect that is attenuated by NTS co-injection of SP-cAMP. In Aim 2 of the application we test the hypothesis that vagal afferent Y2R control food intake by antagonizing MC4R effects on PKA activation, synapsin phosphorylation and vagal afferent synaptic strength. Our long- term goal is to determine how NMDAR participate in modulation of vagal afferent synaptic strength to reduce food intake. A detailed appreciation of the mechanisms by which peptides and hormone interact with NMDAR to control of food intake is of significance to human health because it may provide avenues for therapeutic intervention in eating disorders and obesity.