Anatomical and molecular characterization of appetite input to dopamine neurons

描述（由申请人提供）：中脑中的多巴胺神经元在药物滥用的奖励和成瘾效应中起着核心作用。它们对食欲也很重要。食欲是由下丘脑大脑中心的神经元亚群调节的。然而，尚不清楚这些神经元是否向涉及奖励的中脑多巴胺神经元传递信号。在这个提议中，我将研究与食欲有关的神经元如何将信号传递给多巴胺神经元。使用一种可以追踪神经回路的伪狂犬病毒，我将在与食欲行为相关的大脑区域中识别中脑多巴胺神经元上游的神经元亚群。为了鉴定食欲神经元突触前与多巴胺神经元表达的神经递质或神经调节剂，我将从下丘脑分离荧光标记的食欲神经元亚群，在第一链合成后分离RNA并扩增cDNA，并进行微阵列分析。在确定候选神经递质和神经调节剂基因后，我将进行双免疫荧光原位杂交，以确定DA神经元上游的食欲神经元是否表达任何候选神经递质、神经调节剂和生物合成酶基因。为了鉴定中脑多巴胺神经元中的受体，我将解离荧光标记的活多巴胺神经元，分离RNA并在第一链合成后扩增cDNA，并进行定量实时pcr筛选。根据这些结果，我将构建一个上游食欲中心和多巴胺神经元之间的电路解剖图，然后将其叠加到细胞和分子图上。这种组织将为理解多巴胺奖励系统和食欲之间的联系提供一个结构，可能暗示多巴胺在饮食成瘾中的作用，并可能为成瘾和疾病的新药物靶点奠定基础。

DESCRIPTION (provided by applicant): Dopamine neurons in the midbrain play a central role in the rewarding and addictive effects of drugs of abuse. They are also important for appetite. Appetite is regulated by subsets of neurons in brain centers in the hypothalamus. However, it is not known whether these neurons transmit signals to midbrain dopamine neurons implicated in reward. In this proposal, I will examine how neurons linked to appetite transmit signals to dopamine neurons. Using a pseudorabies virus that can trace neural circuits, I will identify neuronal subsets upstream of midbrain dopamine neurons in brain regions linked to appetite behaviors. To identify neurotransmitters or neuromodulators expressed in appetite neurons presynaptic to dopamine neurons, I will dissociate fluorescently labeled appetite neuron subsets from the hypothalamus, isolate RNA and amplify cDNA after first-strand synthesis, and perform microarray analysis. After identifying candidate neurotransmitter and neuromodulator genes, I will perform dual immunofluorescent in situ hybridization to determine whether appetite neurons that are upstream of DA neurons express any of the candidate neurotransmitters, neuromodulators and biosynthetic enzymes genes. To identify receptors in midbrain dopamine neurons, I will dissociate live fluorescently labeled dopamine neurons, isolate RNA and amplify cDNA after first-strand synthesis, and perform a quantitative real-time PCR-based screen. From these results, I will construct an anatomical map of circuitry between upstream appetite centers and the dopamine neurons they convey signals to, and then superimpose onto that a cellular and molecular map. This kind of organization will provide a structure to understand the connections between the dopamine reward system and appetite, may implicate roles for dopamine in eating addictions, and could lay groundwork for novel drug targets for addictions and disorders. PUBLIC HEALTH RELEVANCE: Ventral midbrain dopamine neurons are responsible for the rewarding and addictive effects of drugs of abuse. They also are important for innate behaviors such as appetite behavior. The studies described here will clarify the relationship between appetite and rewards and addictions, and provide a platform for further studies in food addictions.