Development of a scalable methodology for imaging neuropeptide release in the brain

 描述(申请人提供)：描述大脑的一个常见比喻是，它就像一台超级计算机。因此，目前改进大规模记录大脑功能的技术的努力主要集中在测量其电活动上。然而，与超级计算机不同的是，大脑是一台电化学机器。在它的突触连接网络上叠加了一个“化学连接体”，这是一个很大程度上看不见的神经调节剂网络，如5-羟色胺和神经肽(NP)，对大脑功能产生深远影响。神经调节剂影响大脑状态，从而改变神经回路执行的计算，并且是情绪、情绪和情感的核心。因此，对神经调节影响的理解与人类的精神障碍有关。如果没有能力测量和操纵特定神经调节剂的释放，我们对神经元回路功能的理解将从根本上不完整。然而，在我们监测大脑电活动的能力和我们以相应的时空分辨率监测化学活动的能力之间仍然存在差距。具体地说，没有方法可以可视化特定NPs在大脑中单个突触的释放。为了填补这一空白，我们建议开发一种在体内神经末梢水平上对特定NPs释放进行成像的方法。长期的目标是开发新的方法来可视化、检测和抑制体内NP的释放，并将这些方法应用于了解特定的、与行为相关的神经回路的神经调节动力学。这项提议的总体目标是开发一种新的方法，用于神经末梢释放NP的时间分辨成像。这项提议的中心目标是用对pH敏感的荧光蛋白标记大致密核心囊泡(LDCV)的成分和特定的NPs，并确定这些报告是否可以用于成像神经分泌颗粒释放。果蝇为这项技术提供了一个有用的试验台，因为它的基因可操纵性和复杂的成像方法。为了实现我们的目标，在目标1中，我们将不同的pH敏感荧光报告融合到几个LDCV特异性蛋白和NPs的蛋白质编码序列中，并产生转基因果蝇。在目标2中，我们将利用体内含有这些报告的特定神经肽能神经元的光遗传激活来确定是否可以检测到激活依赖的荧光增加，并将其与突触小泡(SV)释放区分开来。其贡献将是确定拟议方法的可行性，并实现该方法的原则证明应用。这一贡献意义重大，因为它有可能创造一种具有广泛普遍应用的变革性新技术。这项贡献是创新的，因为它结合了细胞生物学、分子遗传学和神经电路分析的专业知识，开发了一种新的方法学。因此，申请中提出的工作将使神经科学领域作为一个整体受益，并使神经化学和电路的研究成为可能，这些神经化学和电路的功能障碍可能是精神疾病的基础。

 DESCRIPTION (provided by applicant): A common metaphor to describe the brain is that it is like a supercomputer. Consequently, current efforts at improving technologies for large-scale recording of brain function are primarily focused on measuring its electrical activity. However, unlike a supercomputer, the brain is an electrochemical machine. Superimposed upon its network of synaptic connections is a "chemical connectome," a largely invisible network of neuromodulators, such as serotonin and neuropeptides (NPs), which exert a profound influence on brain function. Neuromodulators influence brain states that alter the computations performed by neural circuits, and are central to emotion, mood and affect. An understanding of neuromodulatory influences is therefore relevant to psychiatric disorders in humans. Without the ability to measure and manipulate the release of specific neuromodulators, our understanding of neuronal circuit function will be fundamentally incomplete. Nevertheless, a gap remains between our ability to monitor brain electrical activity, and our ability to monitor chemical activty with commensurate spatio-temporal resolution. Specifically, there is no method to visualize the release of specific NPs in the brain, at individual synapses. To fill this gap, we propose to develop a method for imaging the release of specific NPs at the level of nerve terminals, in vivo. The long-term goal is to develop new methods for visualizing, detecting and inhibiting NP release in vivo, and to apply these methods to understanding the dynamics of neuromodulation of specific, behaviorally relevant neural circuits. The overall goal of this proposal is to developa novel approach for time-resolved imaging of NP release from nerve terminals. The central objective of this proposal is to tag components of large dense core vesicles (LDCVs), and specific NPs, with pH-sensitive fluorescent proteins and to determine whether these reporters can be used to image neurosecretory granule release. Drosophila melanogaster provides a useful test-bed for this technology because of its genetic manipulability and sophisticated imaging methodologies. To achieve our objective, in Aim 1 we will fuse different pH-sensitive fluorescent reporters to the protein coding sequences of several LDCV-specific proteins and NPs, and generate transgenic flies. In Aim 2, we will use optogenetic activation of specific neuropeptidergic neurons containing these reporters in vivo, to determine whether activation-dependent increases in fluorescence can be detected, and distinguished from synaptic vesicle (SV) release. The contribution will be to determine the feasibility of the proposed approach, and to achieve a proof- of-principle application of the method. This contribution is significant, because it has the potential to create a transformative new technology with broad general applications. The contribution is innovative, because it combines expertise from cell biology, molecular genetics and neural circuit analysis to develop a novel methodology. The work proposed in this application will therefore benefit the field of neuroscience as a whole, and also enable studies of neural chemistry and circuitry whose dysfunction may underlie psychiatric disorders.