Simultaneous tracking of many neuromodulatory signals in the awake mouse brain

Abstract

Most neurons express receptors for dozens of neuromodulatory molecules. Concentrations of these molecules vary widely in the brain interstitial space across time and space, and their influences overlap and combine to modulate neural activity. While current tools allow for small numbers of such signals (usually one or two) to be tracked at the same time, no real-time methods exist to scale up this molecular dimensionality and reach a complete accounting of dynamic changes in local neuromodulator concentrations in the awake brain. To begin to address this gap, we have developed a probe to track a dozen or more neuropeptide and neuromodulator concentrations in various brain regions in mice. Our probe consists of an emerging family of genetically encoded fluorescent sensors, the G protein-coupled receptor activation-based (GRAB) sensors, expressed in cultured cells and immobilized at the front of a gradient refractive index (GRIN) lens for 3D two-photon imaging. We validate this probe both in vitro, by sequentially placing it cells-first into small volumes (≤20 μL) of fluids with known concentrations of each sensor ligand while imaging the cells through the lens, as well as ex vivo by pressing it against a live brain slice and evoking neuromodulator release, detecting parallel molecular concentrations down to nanomolar levels. We have also validated the probe in vivo by acutely implanting it in the lateral ventricles of awake, behaving mice. We observed rapid shifts in cerebrospinal fluid signal concentrations across seconds to minutes, both spontaneously and in response to peripheral drug administration. Our novel, scalable approach enables rapid in situ profiling of a panel of molecules of interest in very small fluid samples and tissue regions in vivo and ex vivo. In future, this method can be used to study how the rich ensemble of time-varying concentrations of chemical signals correlates with and controls the activity of various cell types throughout the brain.