Diet-Induced Plasticity of Proopiomelanocortin Neurons

描述（由申请人提供）：中枢阿黑皮素原（POMC）神经元是一种促进厌食进食行为和负能量平衡的回路，是调节代谢功能的最佳定义的神经网络之一。然而，POMC神经元在整个CNS中整合这些代谢信号的程度仍不清楚。POMC神经元位于下丘脑内侧基底部，但明显缺乏细胞学组织，使其相对难以解剖分析。同样，已经确定生物体进食状态的变化可以重新连接下丘脑回路，但饮食对POMC神经可塑性的影响尚未直接研究。为了完成中央POMC整合的解剖图，并检查饮食诱导的下丘脑回路重新布线的影响，我们将利用转基因动物模型，跨突触病毒示踪，和单细胞电生理技术。在第一组实验中，复制缺陷型表达Cr的犬腺病毒（CAV-Cre）载体将用于在Pomc的神经增强子模块中用LoxP侧翼的neo盒功能性地再激活神经特异性Pomc缺陷型小鼠。由于CAV-Cre特异性感染突触末端的受体，因此重新激活的POMC神经元的数量将取决于远端靶位点处POMC神经支配的丰度。为了在细胞水平上直接检查POMC的形态学和生理学，我们将使用单细胞膜片钳技术来测量基础神经活性，同时用神经生物素标记细胞。这些实验将遵循不同的饮食模式，这将使我们能够检查急性与慢性变化的影响，在POMC神经元的形态和突触活动的喂养。最后，我们将通过将我们的喂养模式应用于表达Cre依赖性荧光树突和轴突标志物的转基因Pomc-Cre小鼠来合成饮食对POMC网络的影响。这些小鼠将使我们能够研究POMC神经支配的回路水平变化，并将使我们能够将中央POMC网络重建成一个完整的图谱。总之，破译中央POMC神经元的结构和功能将有助于深入了解能量稳态的神经控制，这对于对抗全球肥胖流行病至关重要。 公共卫生相关性：肥胖和相关的代谢并发症每年在医疗保健支出中占超过1500亿美元;因此，美国的健康和经济都必须开发治疗方法来对抗这种流行病。中枢阿黑皮素原神经元在厌食性摄食行为和能量消耗中的作用是众所周知的，但该回路的解剖和可塑性仍不清楚。为了促进对神经系统如何调节代谢的理解，并加快肥胖相关疗法的发展，本提案的目的是阐明阿黑皮素原网络的完整解剖图，并确定饮食变化如何影响阿黑皮素原的生理学。

DESCRIPTION (provided by applicant): Central proopiomelanocortin (POMC) neuron, a circuit that promotes anorectic feeding behavior and negative energy balance, is one of the best-defined neural networks regulating metabolic function. Nevertheless, the extent to which POMC neurons integrate these metabolic signals throughout the CNS remains unclear. POMC neurons reside in the mediobasal hypothalamus, but a distinct lack of cytological organization has made them relatively intractable to anatomical analysis. Likewise, it is well-established that changes in the feeding state of an organism can rewire hypothalamic circuits, but the effects of diet on POMC neural plasticity has not been directly examined. To complete the anatomical map of central POMC integration and examine the effects of diet-induced rewiring of hypothalamic circuits, we will utilize transgenic animal models, trans-synaptic viral tracing, and single cell electrophysiology techniques. In the first set of experiments, replication-deficient Cr-expressing canine adenovirus (CAV-Cre) vectors will be used to functionally re- activate neural-specific Pomc deficient mice with a LoxP-flanked neo cassette in the neural enhancer module of Pomc. Because CAV-Cre specifically infects receptors at the synaptic terminal, the number of re-activated POMC neurons will be dependent on the abundance of POMC innervations at a distal target site. To directly examine POMC morphology and physiology at the cellular level, we will use single cell patch clamp techniques to measure basal neural activity and simultaneously label cells with neurobiotin. These experiments will be followed by different diet paradigms, which will allow us to examine the effects of acute versus chronic changes in feeding on the morphology and synaptic activity of POMC neurons. Finally, we will synthesize the effects of diet on the POMC network by applying our feeding paradigms to transgenic Pomc-Cre mice expressing Cre- dependent fluorescent dendritic and axonal markers. These mice will allow us to study circuit-level changes in POMC innervations, and will allow us to reconstruct the central POMC network into a complete atlas. Taken together, deciphering the structure and function of central POMC neurons will lend insight into neural control of energy homeostasis, critical for combating the worldwide obesity epidemic. PUBLIC HEALTH RELEVANCE: Obesity and related metabolic complications constitute over $150 billion in healthcare spending annually; therefore it is imperative for both the health and economy of the United States to develop therapeutics to combat this epidemic. Central proopiomelanocortin neurons are well known for their role in anorectic feeding behavior and energy expenditure, but the anatomy and plasticity of this circuit remains unclear. To facilitate an understanding of how the nervous system regulates metabolism and expedite the development of obesity-related therapeutics, the objective of this proposal is to elucidate the ful anatomical map of the proopiomelanocortin network and to determine how changes in diet can affect proopiomelanocortin physiology.