Brain fluids carry large quantities of information in the form of soluble factors, including hormones, neurotransmitters and neuromodulators. An average neuron expresses a dozen or more receptors for a specific subset of these factors, thereby receiving information from its extracellular fluid that acts in parallel with its synaptic inputs to determine neural activity. Although major advances have been made in characterizing synaptically wired networks, the rules by which neurons interpret such combinatorial neurohumoral inputs remain poorly understood, and no methods yet exist to rapidly, comprehensively and non-destructively record their molecular concentrations.
To determine how the multidimensional code of soluble factor concentrations in brain fluids shapes the activity of adjacent neurons, new tools are needed to measure these dynamic molecular signals on the spatial and temporal scale of neural activity. Recent development of ligand-responsive genetically encoded fluorescent proteins, such as G-protein-coupled receptor activation-based (GRAB) sensors, has greatly expanded the palette of soluble factors that can be recorded in the brain. However, these tools rely on biological chromophores with broad emission spectra, making it challenging to differentiate more than two or three co-expressed sensors of different colours, and standard methods track just one sensor per colour.
To expand the number of factors that can be simultaneously tracked in small fluid volumes in the brain, we developed multiplexed optical recording of sensors on a micro-endoscope (MORSE). Our approach involved co-expressing ten or more GRAB sensors in the single green colour channel in distinct pools of cultured cells. Several hundred of these sensor-expressing cells were then mixed and immobilized in a ~40-nl transparent hydrogel at the front circular face of a gradient refractive index lens — a cylindrical glass ‘micro-endoscope’.
This probe design allowed us to place the cell-laden hydrogel deep into a volume of fluid or mouse brain tissue and capture the 3D image of the green cells in the hydrogel at the back face of the micro-endoscope using two-photon microscopy. Every MORSE probe underwent an in vitro calibration process, which involved imaging the cell-laden hydrogel through the micro-endoscope as it was robotically dipped into fluids containing known concentrations and mixtures of the soluble factors that matched each sensor. This produced both a spatial fingerprint of the subsets of cells expressing each sensor and the dose–response relationships linking each factor’s concentration to each sensor’s fluorescence intensity.
Each calibrated probe could then be used to determine the multiplexed set of soluble factor concentrations present in the immediate vicinity of the hydrogel corresponding to all expressed GRAB sensors simultaneously. This is a preview of subscription content, access via your institution Access Nature and 54 other Nature Portfolio journals Get Nature+, our best-value online-access subscription Receive 12 print issues and online access Prices may be subject to local taxes which are calculated during checkout Kalugin, P. Simultaneous, real-time tracking of many neuromodulatory signals with multiplexed optical recording of sensors on a micro-endoscope.
Neuron https://doi.org/10.1016/j.neuron.2026.07.008 (2026) Article PubMed PubMed Central Google Scholar Department of Anesthesiology and Critical Care, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA P.N.K. is a co-author of US Patent Office application number 63/481,367, docket number 01948-288001 concerning this work. In situ capture of combinatorial neuromodulatory codes. Neurosci. (2026). https://doi.org/10.1038/s41583-026-01091-9 DOI: https://doi.org/10.1038/s41583-026-01091-9
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