Multiplexed neurochemical methods to understand adenosine neuromodulation

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PROJECT SUMMARY What is the role of adenosine as a rapid modulator of neurotransmission and how can we harness its power for potential therapeutic use? To answer questions such as these, we need analytical tools that can measure multiple neurochemicals simultaneously with high temporal and spatial resolution. Our lab pioneered fast-scan cyclic voltammetry (FSCV) for adenosine, and discovered spontaneous, transient adenosine signaling that lasts only a few seconds. However, the range and effects of rapid adenosine neuromodulation are not well understood. Genetically-encoded sensors have recently been developed for neurotransmitter and calcium detection that offer high sensitivity, selectivity, and spatial resolution. While they can monitor a wide variety of neurochemicals, and not just electroactive molecules, there are still limited colors to detect different analytes. FSCV combined with genetically-encoded sensors would be advantageous to detect the neuromodulator adenosine and measure its downstream effects on dopamine and glutamate neurotransmission, as well as neuronal activity. The long-term goal of my lab is to develop new tools for monitoring real-time neuromodulation in the brain. The goal of this project is to develop multiplexed tools to understand neurochemical interactions and apply these tools to understand adenosine modulation of glutamate, dopamine, and calcium. The central hypothesis is that rapid adenosine release provides transient, but spatially localized, modulation of neurotransmitters in the brain. In the first Aim, we will develop multichannel FSCV, with an array of four electrodes, to determine how far adenosine diffuses in brain slices and the range of its neuromodulatory effects on dopamine release. In the second Aim, we will combine FSCV with genetically-encoded fluorescent sensors to probe the spatial and temporal profile of adenosine (measured with FSCV) modulation of dopamine (measured with GRABDA) or glutamate (measured with iGluSnFR). In the third Aim, we will combine multichannel FSCV and in vivo fiber photometry measurements of genetically-encoded sensors. We will demonstrate in vivo detection of adenosine, dopamine, and calcium changes to probe adenosine neuromodulation of neurotransmission and neuronal activity simultaneously. This research is significant because it develops tools that are broadly applicable for multiplexing neurotransmitter and neuromodulator measurements, harnessing the combined strengths of FSCV and genetically-encoded sensors. It is also significant because multiplexed tools will provide an unprecedented picture of the temporal and spatial dynamics of adenosine neuromodulation. The biological impact is understanding the rapid and local nature of adenosine neuromodulation, which is important for designing adenosine-based therapeutics for diseases such as Parkinson’s, ischemia, or traumatic brain injury where adenosine could be neuroprotective. The multiplexed tools could be applied to monitoring many other neurochemical interactions, in brain slices or in vivo, and will advance the field of neurochemical monitoring beyond one neurochemical at a time sensing.