Characterizing the Cocaine-responsive peptidome in mammalian telecephalon

 描述(由申请人提供)：滥用药物对公众健康的影响是巨大和广泛的；不良后果包括死亡、发病率，包括衰弱的身体和心理症状，以及生产力丧失。现有的药物并不是完全有效的，而且往往具有副作用的特点，因此，迫切需要寻找新的神经化学靶点来开发成瘾疗法。作为神经化学的一类，相对于多巴胺等“经典”神经调节剂，对多肽的研究还不够深入。然而，对数量相对较少的先验识别多肽的研究表明，它们在成瘾周期中发挥着深远的功能作用。下一代抗成瘾药物很可能是基于尚未发现的在成瘾循环中具有特定功能作用的多肽。在这项提案中，我们的目标是应用一个基于尖端质谱学(MS)的分析平台来发现新的多肽(多肽发现)，并以前所未有的精度表征已知的多肽，跨越两种药物状态：急性可卡因中毒和可卡因戒断高峰期。该技术平台包括以下方法：1.基于气相串联MS碎片法和等压标记的多肽组学方法，以快速定量大量神经肽；2.基质辅助激光解吸/电离质谱学成像(MALDI-MSI)，使用新型的金纳米颗粒基质，与原位杂交相关联，绘制多肽通量的空间分布，并实现与多巴胺信号的共定位；3.亲和力增强的微透析，使用创新的磁性纳米颗粒，大规模放大从中枢神经系统(CNS)细胞外空间释放的多肽，从而提高灵敏度和时间分辨率；4.任何新发现的多肽的合成，并在经过良好验证的大脑刺激-奖励阈值程序中对这些序列进行行为测试。这些方法非常先进，一些新的创新以前从未在哺乳动物组织中尝试过。因此，考虑到R21奖励机制的时间限制，我们的第一个目标是通过专门关注急性可卡因中毒和可卡因戒断高峰期伏核和前额叶皮质同时传递阿片、阿片和多巴胺的技术平台来提炼和优化技术平台。在两个大脑部位进行这种类型的组合分析从未被尝试过。我们的工作有可能揭示成瘾生物学中一个重要的对手-过程理论的关键见解，在成瘾周期中假设与-阿片/多巴胺作用相关的不同和相反的-阿片作用。选择急性可卡因中毒和高峰戒断是对这一理论的最有力考验。从这些分析中获得的见解可以转化为独特有效的组合治疗策略。我们的第二个目标是在时间允许的情况下，在我们平台的后续阶段测试在基于MS的“鸟枪式”多肽分析中发现的任何新的多肽序列。这一计划将使我们能够完善一个强大的、技术先进的多肽类发现平台，并实现从转录到行为水平的史无前例的垂直整合。与此同时，我们的研究将提出一个具有重大科学意义的离散问题，即在不同的可卡因相关状态下，阿片肽和多巴胺之间的相互作用。

 DESCRIPTION (provided by applicant): The public health impact of substance abuse is enormous and widespread; adverse consequences include mortality, morbidity including debilitating physical and psychological symptoms, and loss of productivity. Existing drugs are not completely effective and often characterized by adverse side effects; hence, there is a great need to discover new neurochemical targets for the development of addiction therapeutics. As a neurochemical class, peptides are understudied relative to "classic" neuromodulators such as dopamine. Yet, work on a relatively small number of a priori-identified peptides show that they play profound functional roles across the addiction cycle. The next generation of anti-addiction medications could very conceivably be based on yet undiscovered peptides with specific functional roles in the addiction cycle. In this proposal, we aim to apply a cutting-edge mass spectrometry (MS)-based analytic platform to discover new peptides (peptidomic discovery), and characterize already-known peptides with unprecedented precision, across two drug states: acute cocaine intoxication and peak cocaine withdrawal. The technological platform consists of the following methods: 1. Shotgun peptidomics approach based on gas-phase tandem MS fragmentation methods coupled with isobaric tagging to rapidly quantify a large number of neuropeptides; 2. Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) of thin tissue slices using a novel gold-nanoparticle matrix, in correlation with in situ hybridization, to map the spatial distribution of peptide fluxes and enable co-localization wih dopamine signals; 3. Affinity-enhanced microdialysis using innovative magnetic nanoparticles to massively amplify the recovery of peptide efflux from the central nervous system (CNS) extracellular space, thereby improving sensitivity and temporal resolution; 4. Synthesis of any novel peptides discovered, and behavioral testing of these sequences in a well- validated brain stimulation-reward threshold procedure. These methods are highly advanced, and some new innovations have never been tried before in mammalian tissues. Hence, considering the time constraints of the R21 grant mechanism, our first goal is to refine and optimize the technological platform by specifically focusing on simultaneous -opioid, -opioid, and dopamine co-transmission in the nucleus accumbens (Acb) and prefrontal cortex (PFC) during acute cocaine intoxication and peak cocaine withdrawal. This type of combinatorial analysis in both brain sites has never been attempted. Our work has the potential to reveal crucial insights regarding an important opponent-process theory in addiction biology, positing diverse and opposing - opioid actions relative to -opioid/dopamine actions across the addiction cycle. Choosing acute cocaine intoxication and peak withdrawal offers the strongest test of the theory. Insights gained from these analyses could translate into uniquely effective combinatorial treatment strategies. Our second goal is to test any new peptide sequences discovered in the MS-based "shotgun" peptidomic analysis in the subsequent stages of our platform, as time permits. This plan will allow us to refine a powerful, technologically advanced platform for peptidomic discovery, and achieve unprecedented 'vertical integration' from the transcriptional to the behavioral levels. At the same time, our studies will ask a discrete question of great scientific importance regarding the interplay among opioid peptides and dopamine in distinct cocaine-associated states.