PROJECT SUMMARY A comprehensive understanding of immunity necessitates a thorough exploration of the intricate interactions between immune cells and neurotransmitters, thereby clarifying their impact on communication at the neuron- immune synapse. The long-term goal of my research program is to develop new electrochemical and microfluidic tools to study dynamic neuroimmune communication to understand the molecular mechanisms of neuroinflammation. To achieve this goal, new approaches are needed which can measure immune cell communication with higher temporal resolution. The specific objective of this proposal is to demonstrate and determine the mechanism of subsecond dopamine signaling from CD4+ CD25+ T-cells (Tregs) and to establish how dopamine release from senescent Tregs impacts neuronal health. Tregs are important immune cells that function to suppress the immune system and have been implicated in several age-associated neurodegenerative diseases. Tregs contain the machinery to synthesize and respond to dopamine; however, to date, direct real-time measurements of dopamine release and how this subsecond signaling influences neuron-immune crosstalk has not been possible. Current paradigms hold that immune cell secretion of neurotransmitters does not occur with subsecond dynamics seen in neurons and is therefore dispensable in neuronal biology. The evidence supporting this viewpoint is limited by the lack of experimental systems to measure physiological secretion of neurotransmitters by immune cells on this timescale. Our unprecedented preliminary studies using fast-scan cyclic voltammetry (FSCV) reveals that T cells are capable of rapid subsecond secretion of dopamine. This proposal is based on the premise that employing traditional neuroscience methods to investigate neurochemical signaling in isolated immune cells can yield valuable insights into the dynamic mechanisms of subsecond immune cell communication during aging. The proposal will be completed by the following two specific aims: (1) Ascertain the mechanism of subsecond dopamine release from Tregs and (2) Develop a neuron-Treg co-culture platform to probe the consequence of subsecond Treg-derived dopamine signaling on neuronal health with age. We will pursue these aims with an innovative approach combining the power of FSCV’s subsecond detection capabilities with traditional immunology approaches in co-cultures of Tregs and cholinergic neurons. This work is significant because we will reveal the ability of Tregs to release dopamine on a subsecond timescale and demonstrate that this rapid immune- derived neurochemical signaling influences neuronal health for the first time. The expected outcome is a new understanding of the dynamics of immune cell signaling and a deeper understanding of the molecular changes which occur at the neuron-immune cell synapse as a function of age. This work will have a positive impact on how we study immune cell signaling, and will advance current knowledge o...