Characterizing the Cocaine-responsive peptidome in mammalian telecephalon

哺乳动物端脑可卡因反应肽组的表征

• 批准号: 9036371
• 负责人: BRIAN A BALDO
• 金额: $ 22.51万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9036371
• 关键词: Acute; Adverse effects; Affinity; Animals; Antibodies; Attention; Behavioral; Biogenic Amines; Biology; Brain; Brain region; Cocaine; Cocaine Dependence; Collection; Coupled; Dependence; Development; Dopamine; Drug Addiction; Drug abuse; Dynorphins; Enkephalins; Extracellular Space; Gases; Gene Expression; Goals; Gold; Grant; Guns; Health; Image; Imaging Techniques; In Situ Hybridization; Injection of therapeutic agent; Intoxication; Label; Leucine; Magnetic nanoparticles; Maps; Mass Spectrum Analysis; Methods; Microdialysis; Morbidity - disease rate; Neuraxis; Neuromodulator; Neurons; Neuropeptides; Nucleus Accumbens; Opioid; Pharmaceutical Preparations; Pharmacotherapy; Phase; Play; Prefrontal Cortex; Procedures; Process; Proteins; Public Health; Recovery; Relapse; Resolution; Rewards; Role; Running; Sampling; Self Administration; Shotguns; Signal Transduction; Site; Slice; Spatial Distribution; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staging; Stimulus; Substance abuse problem; Technology; Testing; Therapeutic; Time; Tissues; Translating; Withdrawal; Work; addiction; adverse outcome; base; behavior test; beta-Endorphin; combinatorial; comparative; design; gene discovery; gene product; improved; innovation; insight; mRNA Expression; mortality; nanoparticle; neurochemistry; next generation; novel; physical symptom; productivity loss; psychological symptom; response; success; tandem mass spectrometry; temporal measurement; theories; transmission process; treatment strategy

 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.

 描述（由适用提供）：滥用药物的公共卫生影响是巨大而广泛的；不利后果包括死亡率，发病率，包括使身体和心理症状衰弱以及生产力丧失。现有药物不是完全有效的，并且经常以不利的副作用为特征。因此，非常需要发现成瘾疗法发展的新神经化学靶标。作为一种神经化学类别，辣椒是相对于“经典”神经调节剂（例如多巴胺）的理解的。但是，在相对较少的先验识别肽上进行工作表明，它们在整个成瘾周期中扮演着强烈的功能角色。下一代的抗添加药物可能基于尚未发现的肽，在成瘾周期中具有特定的功能作用。在此提案中，我们旨在应用尖端的质谱法（MS）的分析平台，以发现新的肽（肽症发现），并在两个药物状态下以前所未有的精度来表征已经众所周知的肽：急性可卡因肠毒性毒性毒性和峰值可卡因。技术平台由以下方法组成：1。基于气相串联MS片段化方法的shot弹枪肽方法，并与等速标记相结合，以迅速量化大量神经肽； 2。使用一种新型的金纳米颗粒基质与原位杂交相关，以映射肽通量的空间分布并启用共融合毒素dopopamine sage s映射薄纳米粒子基质，以矩阵辅助激光解吸/电离激光解吸/电离质谱成像（MALDI-MSI（MALDI-MSI））与原位杂交相关。 3。使用创新的磁性纳米颗粒的亲和力增强的微透析，以大大扩增中枢神经系统（CNS）细胞外空间的肽外排回收，从而提高灵敏度和临时分辨率； 4。发现的任何新型胡椒的合成，以及在验证良好的大脑模拟 - 奖励阈值程序中对这些序列进行的行为测试。这些方法是高度先进的，并且在哺乳动物组织中从未尝试过一些新的创新。因此，考虑到R21赠款机制的时间限制，我们的第一个目标是通过专门关注核，阿片，-阿片类药物和多巴胺在核酸核（ACB）和前额叶皮层（PFC）中的酸，阿片类药物和多巴胺共转移来完善和优化技术平台。这两个大脑部位中的这种组合分析从未尝试过。我们的工作有可能揭示有关重要选择过程理论的关键见解，此外，在整个成瘾周期中，相对于-阿片/多巴胺的行动，广告潜水员和相反的阿片类动作。选择急性可卡因中毒和峰值戒断提供了对该理论的强烈检验。从这些分析中获得的见解可以转化为独特的有效组合治疗策略。我们的第二个目标是在时间允许的情况下测试在平台的后续阶段中基于MS的“ shot枪”肽分析中发现的任何新肽序列。该计划将使我们能够完善一个功能强大的技术高级平台，以进行嗜血群发现，并从转录到行为层面实现前所未有的“垂直整合”。同时，我们的研究将提出一个关于阿片类药物宠物和多巴胺在不同可卡因相关状态中的相互作用的离散性科学重要性的问题。