Monitoring neurochemical signaling dynamics in the lymph node

项目总结 免疫器官内的神经化学信号，如淋巴结，仍然是具有挑战性的，用现有的 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的影响 豁免权。我们的长期目标是了解炎症过程中神经化学调节的免疫， 自身免疫力，甚至抑郁症。为了实现这一目标，需要新的分析工具来捕捉快速 完整免疫器官中的神经化学信号具有高空间分辨率。这项建议的具体目标是 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 检测肠系膜淋巴结切片中的去甲肾上腺素、三磷酸腺苷和褪黑素。所有三种神经化学物质 对于触发或抑制肠道免疫系统内的免疫反应都很重要；然而， 它们发挥作用的动力学和机制尚不清楚。这项建议的理由是 开发新的工具来监测完整的MLN中的快速神经化学信号将提供关于 肠道中的神经免疫通讯动力学可能导致复杂的神经化学靶向 胃肠道炎症的治疗和对肠道-脑轴的更好的了解。这项提议将 将通过以下三个具体目标完成：(1)开发创新的电化学方法来检测和 验证神经元去甲肾上腺素在活体脑片中的释放，(2)开发阴离子交换掺杂碳纤维 微电极用于灵敏地检测液氮中的三磷酸腺苷，以及(3)开发用于多路复用器的耐污染传感器。 用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与检测相结合 在淋巴结活切片中。这项工作也是创新的，因为新的碳电极和表面 将开发用于靶向分析物检测的化学方法。这项工作意义重大，因为开发的工具 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 通讯机制和动态，特别是在肠道免疫系统内。检测快速反应的工具 去甲肾上腺素、三磷酸腺苷和褪黑素的浓度波动也很显著，因为它们不仅 参与免疫系统的免疫调节，但在很大程度上参与全身的信号传递。 这些工具可以移植到任何生物系统中。预期结果是一个高时态的新工具箱 对淋巴结中神经化学物质的分辨率检测将有助于更好地理解 健康状态下MLN空间分辨区域神经化学信号的机制和功能 和炎症。这项工作将对如何研究神经免疫沟通产生积极影响，并将 神经递质调节免疫的先进知识导致靶向免疫的进展 针对自身免疫、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.