A New Method for Imaging Neuropeptide Release in the Brain

大脑中神经肽释放成像的新方法

• 批准号: 10016856
• 负责人: EDWIN S LEVITAN
• 金额: $ 19.67万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10016856
• 关键词: Address; Adult; Anatomy; Behavior; Behavior Control; Behavioral; Binding; Blood Circulation; Brain; Bypass; Circadian Rhythms; Clustered Regularly Interspaced Short Palindromic Repeats; Dense Core Vesicle; Desire for food; Detection; Drosophila genus; Dyes; Engineering; Exocytosis; Fluorescence; Genes; Genetic; Genomics; Image; Individual; Knock-in; Label; Mammals; Measurement; Measures; Membrane; Methodology; Methods; Monitor; Moods; Morphology; Neuraxis; Neuromuscular Junction; Neurons; Neuropeptides; Noise; Optics; PHluorin; Pain; Physiological; Pigments; Proteins; Resistance; Running; Signal Transduction; Site; Sleep; Synapses; System; Testing; Tetanus Toxin; Tetrodotoxin; Time; Tissues; Transcriptional Regulation; Transgenic Organisms; Variant; Vesicle; aqueous; base; circadian; experimental study; extracellular; fly; imaging approach; imaging modality; imaging study; insight; mind control; neuronal cell body; neuropeptide F; novel; pain perception; protein complex; targeted treatment; thiazole orange; tool; trafficking; transmission process; vesicle transport; vesicular release

Neuropeptides modulate synapses and circuits to control mood and a wide variety of behaviors including appetite, pain perception and circadian rhythms. Despite the importance of neuropeptides, it is not possible to detect synaptic neuropeptide release in the intact living brain with current methods. However, we recently developed a new optical approach for imaging exocytosis of neuropeptide-containing dense-core vesicles (DCVs) at intact living Drosophila synapses. This approach is based on inserting a fluorogen activating protein (FAP), which confers fluorescence on the normally nonfluorescent dye malachite green (MG), into a proneuropeptide, thus targeting the FAP to the DCV lumen. Following extracellular application of membrane impermeant MG derivatives that are small enough to pass through fusion pores, activity-evoked fusions of individual DCVs can readily be resolved at the Drosophila neuromuscular junction. Furthermore, we detected a novel mode of DCV spontaneous exocytosis that is distinguished by its sensitivity to perturbations of the secretory apparatus (e.g. resistance to tetanus toxin). Finally, preliminary studies show that the neuropeptide-FAP approach is applicable to studying circadian peptidergic neurons in the intact adult Drosophila brain. Therefore, to demonstrate the utility of FAP imaging, this approach will be used to answer fundamental questions regarding the function of the circadian circuit in the Drosophila. For example, we will determine the timing of neuropeptide release by multiple neurons and address whether a newly discovered mode of spontaneous release accounts for tetanus toxin resistant behavioral effects of a fly neuropeptide. Furthermore, we will use new dyes, a second spectrally distinct FAP variant and genomic engineering to enable simultaneous imaging of synaptic release of two neuropeptides under native transcriptional control. In addition to testing specific hypotheses about peptidergic transmission in the circadian circuit of the adult fly brain, these studies will serve as proof of principle examples for applying real time neuropeptide-FAP imaging to other systems including the mammalian central nervous system.

神经肽调节突触和电路来控制情绪和各种行为 包括食欲、疼痛感知和昼夜节律。尽管神经肽很重要，但它 用目前的方法不可能检测完整的活体大脑中突触神经肽的释放。 然而，我们最近开发了一种新的光学方法，用于对完整的活果蝇突触中含有神经肽的致密核心囊泡（DCV）的胞吐作用进行成像。该方法基于 插入荧光激活蛋白（FAP），该蛋白赋予正常细胞荧光 非荧光染料孔雀石绿 (MG) 转化为原神经肽，从而将 FAP 靶向 DCV 流明。在细胞外应用小的膜非渗透性 MG 衍生物后 足以通过融合孔，单个 DCV 的活动诱发融合可以很容易地被 在果蝇神经肌肉接头处解决。此外，我们还发现了一种新的 DCV 模式 自发胞吐作用，其特征在于其对分泌扰动的敏感性 设备（例如对破伤风毒素的抵抗力）。最后，初步研究表明神经肽-FAP方法适用于研究完整成年果蝇的昼夜节律肽能神经元 脑。因此，为了证明 FAP 成像的实用性，该方法将用于回答 有关果蝇昼夜节律回路功能的基本问题。例如， 我们将确定多个神经元释放神经肽的时间，并解决是否 新发现的自发释放模式解释了破伤风毒素抗性行为效应 果蝇神经肽。此外，我们将使用新的染料、第二种光谱上不同的 FAP 变体和 基因组工程能够同时对两种神经肽的突触释放进行成像 天然转录控制。除了测试有关肽能传递的具体假设 在成年果蝇大脑的昼夜节律回路中，这些研究将作为原理示例的证明 将实时神经肽-FAP 成像应用于其他系统，包括哺乳动物中枢神经系统 神经系统。