Developing genetically-encoded detectors for neuropeptide release based on class B G-protein coupled peptide receptors

摘要 突触传递主要由经典的神经递质如谷氨酸和g-氨基丁酸介导 谷氨酸（GABA），它能加速大脑中的信息流动。这一过程受到神经调节素的严格调控。 包括单胺类和神经肽类的因子。在神经调质中，特别是神经肽， 很难研究，因为它们通常是化学惰性的（不可氧化的），通常会激活7个反式- 膜G蛋白偶联受体（GPCR）依赖于延迟（秒至分钟）的第二信使 信号，排除了他们的研究，通过传统的电生理学或与氧化探针。此外，endog- 静脉神经肽通常从大的多聚蛋白前体中分选出来， 囊泡（DCV），这表明需要一种检测策略，不干扰生物发生， 内源性DCV的天然分选。因此，目前缺乏适当的工具来研究空间 以及神经肽释放和肽能神经回路的时间动力学。为了满足这一需求， 我们建议开发一种新的遗传编码光学传感器家族，称为Neu嵌合检测器， 通过利用B类（分泌素样）GPCR的独特结构特征， 它们只识别肽作为它们的天然配体。基于最近解决的蛋白质结构和 新的强大的遗传模型的可用性，以应用和验证我们的方法，我们将特别关注葡聚糖， 促性腺激素样肽-1（GLP-1）和促肾上腺皮质激素释放激素/因子（CRH/CRF）， 复杂的神经行为，如进食和压力。验证CDNR用于检测神经肽 我们将使用最先进的病毒介导的基因在定义的GLP-1和CRF回路中表达CDNR 在特定的转基因小鼠模型中进行转导，并进行高分辨率光学记录， 内源性GLP-1和CRF的tect释放，在脑切片中离体和在行为动物中体内。Suc- 这项工作的成功实施将提供一组新的和经过充分验证的基因编码的CDNR， 可以立即应用于解剖脑中GLP-1和CRF的肽能回路。此外，这将 同时也为未来其他几种神经元探测器的开发奠定了概念和技术基础， 肽结合B类GPCR中包括的肽（PACAP、VIP、CGRP和胰泌素）。 !

Abstract Synaptic transmission is mainly mediated by classical neurotransmitters such as glutamate and g-amino butyric acid (GABA) which transduce fast information flow in the brain. This process is tightly regulated by neuromodu- lators including monoamines and neuropeptides. Among neuromodulators, neuropeptides in particular, have been difficult to study because they are often chemically inert (non-oxidizable) and typically activate 7 transmem- brane G-protein coupled receptors (GPCRs) which rely on delayed (seconds to minutes) second messenger signaling, precluding their study by conventional electrophysiology or with oxidizable probes. Moreover, endog- enous neuropeptides are typically sorted from large polyprotein precursors into specific pools of dense core vesicles (DCVs), suggesting the need for a detection strategy that does not interfere with the biogenesis and native sorting of endogenous DCVs. As a result, there is currently a lack of suitable tools for studying the spatial and temporal dynamics of neuropeptide release and peptidergic neurocircuitry. Toward addressing this need, we propose to develop a new family of genetically-encoded optical sensors, termed Chimeric Detectors for Neu- ropeptide Release (CDNRs), by harnessing the unique structural signatures of Class B (Secretin-like) GPCRs that exclusively recognize peptides as their native ligands. Based on recently solved protein structures and the availability of new robust genetic models to apply and validate our approach, we will specifically focus on gluca- gon-like peptide-1 (GLP-1) and corticotropin-releasing hormone/factor (CRH/CRF) which have important func- tions in complex neurobehaviors such as feeding and stress. To validate CDNRs for detection of neuropeptide release, we will express CDNRs in defined GLP-1 and CRF circuitry using state-of-the-art viral mediated gene transduction in specific genetically modified mouse models and perform high-resolution optical recording to de- tect release of endogenous GLP-1 and CRF, both ex vivo in brain slices and in vivo in behaving animals. Suc- cessful implementation of this work will deliver a set of novel and well validated genetically-encoded CDNRs that can be immediately applied to dissect the peptidergic circuitry of GLP-1 and CRF in the brain. Moreover, this will also lay a conceptual and technical foundation for the future development of detectors for several other neuro- peptides (PACAP, VIP, CGRP and secretin) included among peptide-binding class B GPCRs. !