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.
PROJECT NARATIVE The proposed research is relevant to public health because understanding the dynamics and mechanisms of neurochemical signaling and regulation in the lymph node is expected to inform the development of new types of treatments for chronic inflammation, autoimmunity, and even cancer. Tools to monitor rapid neurochemical signaling within intact immune tissue are lacking and we will fill this critical gap by developing tools to study neurochemical signaling in the lymph node with exquisite resolution. Thus, this proposal is relevant to part of the NIH?s mission that pertains to fostering fundamental creative discoveries and innovative research strategies as a basis for protecting and improving health.