Monitoring neurochemical signaling dynamics in the lymph node

项目摘要 免疫器官（如淋巴结）内的神经化学信号传导仍然具有挑战性， 然而，了解这种信号的机制和功能将对我们的理解产生积极影响。 豁免权。我们的长期目标是了解炎症过程中神经化学物质调节的免疫， 自身免疫甚至抑郁症为了实现这一目标，需要新的分析工具， 神经化学信号在完整的免疫器官具有高空间分辨率。本提案的具体目标是 是开发和验证在碳基微电极上使用快速扫描循环伏安法（FSCV）的方法， 检测肠系膜淋巴结（mLN）切片中的去甲肾上腺素、ATP和褪黑激素。这三种神经化学物质 对于触发或抑制肠道免疫系统内的免疫应答是重要的;然而， 它们发挥作用的动力和机制尚不清楚。提出这一建议的理由是， 开发新的工具来监测完整mLN中的快速神经化学信号传导将提供以下知识： 肠道中的神经免疫通讯动力学，这可能导致复杂的神经化学靶向 治疗胃肠道炎症和提高对肠-脑轴的理解。该提案将 通过以下三个具体目标来完成：（1）开发创新的电化学方法来检测和 在活体mLN切片中验证神经元去甲肾上腺素释放，（2）开发阴离子交换掺杂的碳纤维 用于mLN中敏感ATP检测的微电极，以及（3）开发用于多路复用的抗污染传感器。 用mLN中的儿茶酚胺检测褪黑素。我们将以创新的方式实现这些目标 将快速扫描循环伏安法的高时间分辨率和空间分辨率的能力与检测相结合， 在淋巴结的活体切片中。这项工作也是创新的，因为新的碳电极和表面 将开发用于目标分析物检测的化学品。这项工作意义重大，因为开发的工具 这将有助于改变免疫调节缓慢的范式，并将影响我们对神经免疫的理解。 沟通机制和动力学，特别是在肠道免疫系统。快速检测工具 去甲肾上腺素、ATP和褪黑激素的浓度波动也是显著的，因为它们不仅 参与免疫系统的免疫调节，但在整个身体的信号转导中也有很大的参与。 这些工具可以应用于任何生物系统。预期的结果是一个新的工具箱， 淋巴结中神经化学物质的分辨率检测，这将有助于更好地了解 健康状态下mLN空间分辨区域神经化学信号传导的机制和功能 和炎症。这项工作将对神经免疫通讯的研究产生积极的影响， 推进神经递质调节免疫的现有知识，从而促进靶向免疫 自身免疫、癌症和疾病的免疫疗法。

PROJECT SUMMARY Neurochemical signaling within immune organs, like the lymph node, remains challenging to probe with existing technology yet knowing the mechanisms and function of this signaling would positively impact our understanding of immunity. Our long-term goal is to understand neurochemical regulated immunity during inflammation, autoimmunity, and even depression. To achieve this goal, new analytical tools are needed which can capture rapid neurochemical signaling in intact immune organs with high spatial resolution. The specific objective of this proposal is to develop and validate methods using fast-scan cyclic voltammetry (FSCV) at carbon-based microelectrodes to detect norepinephrine, ATP, and melatonin in slices of the mesenteric lymph node (mLN). All three neurochemicals are important for either triggering or suppressing immune responses within the gut-immune system; however, the dynamics and mechanisms by which they function are not understood. The rationale for this proposal is that the development of new tools to monitor rapid neurochemical signaling in an intact mLN will provide knowledge of neuroimmune communication dynamics in the gut which could lead to sophisticated neurochemical-targeted therapies for gastrointestinal inflammation and an improved understanding of the gut-brain axis. The proposal will be completed by the following three specific aims: (1) Develop innovative electrochemical methods to detect and validate neuronal norepinephrine release in live mLN slices, (2) Develop anion-exchange doped carbon-fiber microelectrodes for sensitive ATP detection in the mLN, and (3) Develop fouling-resistant sensors for multiplexed detection of melatonin with catecholamines in the mLN. We will pursue these aims with an innovative approach combining the power of fast-scan cyclic voltammetry’s high temporal resolution and spatial resolution with detection in live slices of the lymph node. This work is also innovative because new carbon electrodes and surface chemistries will be developed for targeted-analyte detection. This work is significant because the tools developed will help shift the paradigm that immunomodulation is slow and will impact our understanding of neuroimmune communication mechanisms and dynamics, specifically within the gut-immune system. Tools to detect rapid concentrations fluctuations in norepinephrine, ATP, and melatonin are also significant because they are not only involved in immunomodulation in the immune system, but are heavily involved in signaling throughout the body. The tools are translatable to any biological system. The expected outcome is a new toolbox for high temporal resolution detection of neurochemicals in the lymph node which will lead to an improved understanding of the mechanism and function of neurochemical signaling in spatially-resolved regions of mLN during conditions of health and inflammation. This work will have a positive impact on how neuroimmune communication is studied, and will advance current knowledge of neurotransmitter regulated immunity leading to advancements in targeted immunotherapies for autoimmunity, cancer, and disease.