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 brain cell, yet we lack methods to simultaneously record many such signals in real time. We developed a probe to track ten or more neuropeptide and neuromodulator concentrations using spatial multiplexing of genetically encoded fluorescent sensors. Cultured cells, each expressing one sensor type, are immobilized at the front of a gradient refractive index 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 activation of the choroid plexus epithelium. Our method introduces a new approach to quantitative, real-time, high-dimensional tracking of brain extracellular fluid composition.