Amber L. Alhadeff - US grants

DESCRIPTION (provided by applicant): Obesity is a major public health concern, especially in the United States, as currently over two-thirds of the US population is considered overweight or obese. The lack of safe and effective obesity treatments highlights the urgency of developing effective anti-obesity drug therapies. Since obesity is driven, in part, by excess caloric intake o palatable foods, research must focus on defining the neural circuits involved in the control of such food intake. Leptin is among the most critical anorectic signals involved in the control of energy balance, and signaling through its receptor (LepRb) in certain hindbrain, midbrain, and forebrain nuclei reduces food intake and food-motivated behaviors. Leptin receptor signaling in the nucleus tractus solitarius (NTS) of the caudal brainstem is necessary for the control of food intake, body weight, and processing of vagally-derived GI satiation signals, and recently it has also been implicated in the regulation of food-motivated and appetitive behaviors. The main goal of this proposal is to test the novel hypothesis that a population of neurons in the NTS integrates leptin and GI-derived satiation signals and projects directly to midbrain and forebrain reward- related structures that contribute to the control of food-motivated and appetitive (food-seeking) behaviors. Specific Aim I investigates the anatomical connectivity of NTS neurons that respond to both leptin and gastric stimulation with three reward-related nuclei [ventral tegmental area (VTA), nucleus accumbens (NAc) shell, and lateral hypothalamus (LH)]. Triple immunohistochemistry for leptin-induced pSTAT3 (a well-established marker of LepRb signaling), GI-stimulation-induced cFos, and neuronal tracers (Fluorogold and Retrobeads) will be used to identify neurons that integrate these anorectic signals and project to one or more reward-related nuclei. The experiment proposed in Specific Aim II examines the hypothesis that effects of NTS leptin signaling on food intake and reward-related feeding behavior may involve changes in the transcription of energy-balance relevant genes in the VTA, NAc shell, and/or LH, and that these changes may be potentiated by GI-derived satiation signals. This experiment will utilize qPCR to examine a deductively-chosen set of genes whose protein products influence reward-related feeding and food-motivated behaviors. Research proposed in this application has the potential to deepen the understanding of the anatomical and molecular mechanisms that mediate the control of palatable food intake.

PROJECT SUMMARY The recent increase in obesity prevalence is a major public health concern in the United States. Since energy balance regulation is rooted in the brain, understanding the neural feeding circuits that impede weight loss and maintenance is necessary for the development of novel obesity treatments. Agouti-related protein-expressing neurons in the hypothalamic arcuate nucleus (ARCAGRP) are both necessary and sufficient for food intake. Indeed, acute stimulation of ARCAGRP neurons drives food intake, and chronic stimulation leads to dramatic hyperphagia and weight gain. ARCAGRP neurons can be divided into distinct subpopulations that project to one of several target regions. Independent stimulation of four of these distinct projection subpopulations [ARCAGRP- bed nucleus of the stria terminalis (BNST), -paraventricular hypothalamic nucleus (PVH), -lateral hypothalamic area (LHA) and paraventricular thalamic nucleus (PVT)] is sufficient to drive food intake. We have also shown that ARCAGRP neurons transmit a negative valence that may be likened to the negative affect that is associated with hunger. Here, we take advantage of the one-to-one neuron connectivity to test the affective, functional and physiological relevance of ARCAGRP neuron subpopulations. The main goal of this proposal is to test the hypothesis that ARCAGRP neurons contribute to diet-induced obesity by driving food intake and negative valence through distinct neuron subpopulations. Specific Aim I experiments will examine the valence of the four ARCAGRP neuron subpopulations that increase feeding upon stimulation to provide insight into the specific neural projections that mediate negative affect associated with dieting for weight loss. Specific Aim II examines the neural activity dynamics of feeding-sufficient ARCAGRP neuron subpopulations during the gradual onset of hunger and feeding in lean and obese mice to better understand the endogenous role of feeding-sufficient ARCAGRP subpopulations in hunger and weight gain. Finally, given that persistent ARCAGRP neuron firing is associated with obesity, Specific Aim III experiments will directly examine the role of ARCAGRP neurons in the development and treatment of diet-induced obesity. Overall, results from these experiments will identify ARCAGRP neuron subpopulations, target regions, and mechanisms that can be leveraged to develop novel treatments for obesity.

PROJECT SUMMARY The recent increase in obesity is a major public health concern. Since energy balance regulation is coordinated by communication between the gastrointestinal (GI) tract and the brain, understanding these gut-brain interactions will enable the development of novel obesity treatments. The hypothalamus and the hindbrain are critical brain regions that integrate information from the gut to control food intake. Here, I will leverage recent technological advances to explore the regulation of both these brain regions in awake, behaving animals. Within the hypothalamus, agouti-related protein (AgRP)-expressing neurons are essential for food intake control. Activity in AgRP neurons is high during hunger and is rapidly inhibited by food. My recent work demonstrates that AgRP neurons are primarily regulated by calorie intake, rather than sensory detection of food, since direct gastric infusion of macronutrients into the stomach rapidly suppresses AgRP neuron activity in vivo. Further, this effect is recapitulated by administration of GI satiation signals normally released following food consumption. However, the mechanisms through which the gut transmits signals to AgRP neurons remain unknown. The mentored phase (Aims I and II) of this grant will build upon my previous work by elucidating the mechanisms through which nutrient detection in the gut leads to AgRP neuron activity reductions. Specifically, these aims will determine whether GI signals are transmitted vagally or through direct action on the brain, and will uncover the AgRP axon projections that transmit nutritive signals throughout the brain. Importantly, the mentored experiments will afford me training in peripheral manipulation of the GI tract, as well as in vivo calcium imaging of individual neurons using microendoscopy and 2-photon microscopy, expanding my technical expertise and enabling the proposed R00 experiments. The hindbrain nucleus tractus solitarius (NTS) is the first central site of integration of GI-derived signals from vagal afferents, and is a key signaling node that transmits signals from the gut to higher-order brain structures such as the hypothalamus. For the independent phase (Aims III and IV) of my grant, I have designed experiments that build upon both my graduate and postdoctoral training to determine how different hindbrain NTS neuron populations receive signals from the GI tract, at unprecedented levels of temporal and cellular detail. These complementary research aims combined with the proposed career development activities will provide me with the training necessary to successfully transition to independence, under the guidance of my mentorship team who have extensive collective experience with neuroscience techniques and mentorship. Overall, this award will facilitate my career as an independent investigator characterizing the role of gut-brain signaling on the in vivo activity dynamics of feeding-relevant neurons.