Neurochemistry/Physiology of Proopiomelanocortin Neurons

描述（由申请人提供）：肥胖及其相关代谢疾病是世界范围内的主要健康问题。尽管肥胖率快速增长的原因是多方面的，但很明显，大脑中的神经回路在感知能量储存和调节能量平衡方面起着重要作用。这些神经通路是开发旨在逆转肥胖流行的新药物治疗的候选靶点，因此我们有必要详细了解它们的功能，包括与其他神经系统的相互联系的完整图谱，以及它们对多种神经递质和细胞内信号通路的利用。该项目聚焦于大脑能量平衡回路的一个关键组成部分，即位于下丘脑和脑干的proopiomelanocortin （POMC）神经元。大脑中POMC功能的基因缺失会导致重度肥胖和代谢综合征，其特征是极度嗜食和基础代谢率降低。然而，POMC神经元在许多方面是异质的，越来越多的证据表明，不同的神经元亚群调节不同的神经过程，这些神经过程共同导致热量平衡的正常或病理控制。该项目的总体目标是确定这些神经元亚群的特定功能以及它们利用的神经解剖学和分子途径。具体目标1包括一系列行为学和药理学研究，以探索pomc缺陷小鼠模型中导致贪食的潜在成分过程和神经基质。这些实验利用一种新开发的方法进行进食模式分析，并将测试黑素皮质素信号协调调节进食行为的刻板运动，奖励和享乐方面的假设；在肥胖的临床发病机制中，与食物选择和膳食大小等人类问题特别相关的过程。具体目标2将进一步定义连接弓形POMC神经元到边缘前脑核的神经回路。一种新的逆行追踪方法，将表达Cre重组酶的犬腺病毒载体显微注射到具有可逆沉默POMC基因等位基因的突变小鼠中，将用于将轴突侧枝定位到特定靶点的组合。小麦胚芽凝集素在POMC细胞体中表达，并从POMC细胞体顺行转运，通过反突触标记可以识别由POMC末梢支配的边缘区二级神经元。我们还将使用遗传技术和多标记免疫组织化学来研究POMC神经元出人意料的复杂树突，这些树突接收来自远端位点的突触输入。最后，在具体目标3中，我们将使用互补的遗传学方法来研究空间不同的POMC神经元亚群或发育改变的POMC基因表达在预防或减轻肥胖中的独特功能作用。所涉及的技术是聚集嵌合体的形成和Cre重组酶介导的从神经元特异性和可逆沉默的POMC等位基因的POMC表达的再激活。公共卫生相关性：目前对公共卫生的最大威胁之一是超重、肥胖、糖尿病和代谢综合征的发病率不断上升。一组复杂的神经回路将热量需求和利用与进食相关的行为和心理过程之间的平衡整合在一起。该项目以大脑进食回路的一个关键组成部分——丙黑素皮质素神经元为中心，解释这些神经元如何协调调节食欲、进餐开始和结束以及代谢率，从而将体重维持在严格控制的范围内。

DESCRIPTION (provided by applicant): Obesity and its associated metabolic diseases are major health problems world-wide. Although the reasons for the rapid increase in rates of obesity are multifactorial, it is clear that neural circuits in the brain play a major role in sensing energy stores and regulating energy balance. These neural pathways are candidate targets for the development of new pharmacotherapies aimed at reversing the obesity epidemic and therefore it is essential that we understand their function in detail, including the complete map of interconnections with other neural systems and their utilization of multiple neurotransmitters and intracellular signaling pathways. This project focuses on a key component of the brain's energy balance circuitry, the proopiomelanocortin (POMC) neurons located in the hypothalamus and brainstem. Genetic deletion of POMC function from the brain results in profound obesity and a metabolic syndrome characterized by extreme hyperphagia and reduced basal metabolic rate. However, POMC neurons are heterogeneous in many aspects and accumulating evidence suggests that different subpopulations of the neurons regulate separate neurological processes that together result in normal or pathological control of caloric balance. The overall goals of this project are to identify specific functions of these neuronal subpopulations and the neuroanatomic and molecular pathways that they utilize. Specific aim 1 includes a series of behavioral and pharmacological studies to probe the underlying component processes and neural substrates contributing to hyperphagia in POMC-deficient mouse models. These experiments utilize a newly developed method for meal pattern analysis and will test the hypothesis that melanocortin signaling coordinately modulates stereotyped motor, reward, and hedonic aspects of feeding behavior; processes which are particularly relevant to human issues surrounding food choice and meal size in the clinical pathogenesis of obesity. Specific aim 2 will further define the neurocircuitry connecting arcuate POMC neurons to limbic forebrain nuclei. A novel retrograde tracing method involving site-specific microinjections of a canine adenoviral vector expressing Cre recombinase into mutant mice with a reversibly silenced POMC gene allele will be used to map axon collaterals to specific combinations of target sites. Second order neurons in limbic areas innervated by POMC terminals will be identified by trans-synaptic labeling with wheat germ agglutinin expressed in, and transported anterogradely from, POMC cell bodies. We will also use genetic techniques and multilabel immunohistochemistry to study the unexpectedly complex dendrites of POMC neurons that receive synaptic inputs from distal sites. Finally, in Specific aim 3 we will use complementary genetic approaches to study the unique functional role of spatially distinct POMC neuron subpopulations or developmentally altered POMC gene expression in the prevention or mitigation of obesity. The techniques involved are aggregation chimera formation and Cre recombinase-mediated reactivation of POMC expression from a neuron-specific and reversibly silenced POMC allele. PUBLIC HEALTH RELEVANCE: Among the greatest current threats to public health are the continually increasing rates of overweight, obesity, diabetes, and the metabolic syndrome. A complex set of neural circuits integrates the balance between caloric demand and utilization with the behavioral and psychological processes related to feeding. This project centers on a key component of the brain's feeding circuits, propiomelanocortin neurons, to explain how these neurons coordinately regulate appetite, meal initiation and termination, and metabolic rate to normally maintain body weight within tightly controlled limits.