Simultaneous, real-time tracking of many neuromodulatory signals with Multiplexed Optical Recording of Sensors on a micro-Endoscope

Dozens of extracellular molecules jointly impact a given neuron, yet we lack methods to simultaneously record many such signals in real time. We developed a probe to track ten or more neuropeptides and neuromodulators using spatial multiplexing of genetically encoded fluorescent sensors. Cultured cells expressing one sensor at a time are immobilized at the front of a gradient refractive index (GRIN) lens for 3D two-photon imaging in vitro and in vivo. The sensor identity and detection sensitivity of each cell are determined via robotic dipping of the probe into wells containing various ligands and concentrations. Using this probe, we detected stimulation-evoked release of multiple neuromodulators in acute brain slices. We also tracked endogenous and drug-evoked changes in cerebrospinal fluid composition in the awake mouse lateral ventricle, which triggered downstream activation of the choroid plexus epithelium. Our approach offers a first step towards quantitative, real-time, high-dimensional tracking of brain fluid composition.