A New Method for Imaging Neuropeptide Release in the Brain

神经肽调节突触和回路来控制情绪和各种行为 包括食欲、痛觉和昼夜节律。尽管神经肽很重要，但它 用目前的方法无法在完整的活脑中检测到突触神经肽的释放。 然而，我们最近开发了一种新的光学方法来成像完整的果蝇突触上含有神经肽的致密核小泡(DCV)的胞吐。这种方法是基于 关于插入荧光素激活蛋白(FAP)，使其在正常情况下发出荧光 非荧光染料孔雀石绿(MG)，转化为前神经肽，从而将FAP靶向DCV 流明。在细胞外应用膜无意义的MG小的衍生物 足以穿过融合毛孔，单个DCV的活动诱发融合很容易被 在果蝇神经肌肉交界处分解。此外，我们还检测到了一种新的DCV模式 自发性胞吐，以其对分泌扰动的敏感性为特征 仪器(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性多肽能神经元。 大脑。因此，为了演示FAP成像的实用性，将使用此方法来回答 关于果蝇昼夜节律功能的基本问题。例如, 我们将确定多个神经元释放神经肽的时间，并解决 新发现的自发释放模式解释了破伤风毒素抵抗行为的影响 一种苍蝇神经肽。此外，我们将使用新的染料，第二个光谱不同的FAP变体和 基因组工程使两种神经肽突触释放的同时成像成为可能 原生转录控制。除了测试关于肽能传递的具体假设之外 在成年苍蝇大脑的昼夜节律回路中，这些研究将作为主要例子的证据 将神经肽-FAP实时成像应用于其他系统，包括哺乳动物的中枢 神经系统。

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.