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

 描述（由申请人提供）：描述大脑的常见比喻是它像一台超级计算机。因此，目前致力于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，大脑是一台电化学机器。叠加在其突触连接网络上的是"化学连接体"，这是一种基本上不可见的神经调节剂网络，如血清素和神经肽（NP），对大脑功能产生深远的影响。神经调质影响大脑状态，改变神经回路执行的计算，并对情绪，情绪和影响至关重要。因此，对神经调节影响的理解与人类精神疾病有关。如果没有测量和操纵特定神经调质释放的能力，我们对神经元回路功能的理解将是根本不完整的。然而，在我们监测脑电活动的能力和我们以相当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大脑中单个突触处特定NP的释放。为了填补这一空白，我们建议开发一种方法，用于在体内神经末梢水平上成像特定NP的释放。长期目标是开发新的方法，用于可视化，检测和抑制体内NP释放，并应用这些方法来了解特定的，行为相关的神经回路的神经调节的动力学。这项建议的总体目标是开发一种新的方法，用于神经末梢NP释放的时间分辨成像。该提案的中心目标是标记大致密核心囊泡（LDCV）和特定NP的组分，并使用pH敏感性荧光蛋白，以确定这些报告者是否可用于神经分泌颗粒释放成像。果蝇提供了一个有用的试验台，因为它的遗传操纵和先进的成像方法。为了实现我们的目标，在目标1中，我们将不同的pH敏感的荧光报告基因融合到几种LDCV特异性蛋白和NP的蛋白编码序列上，并产生转基因果蝇。在目标2中，我们将使用体内含有这些报告基因的特定神经肽能神经元的光遗传学激活，以确定是否可以检测到荧光的激活依赖性增加，并与突触囊泡（SV）释放区分开来。贡献将是确定所提出的方法的可行性，并实现该方法的原理应用的证明。这一贡献意义重大，因为它有可能创造一种具有广泛通用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、分子遗传学和神经回路分析的专业知识，开发了一种新的方法。因此，本申请中提出的工作将有益于整个神经科学领域，并且还使得能够研究神经化学和电路，其功能障碍可能是精神障碍的基础。

 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.