In vivo multiplex imaging of dynamic neurochemical networks with designed far-red dopamine sensors

Dopamine (DA) plays a crucial role in a variety of brain functions through intricate interactions with other neuromodulators and intracellular signaling pathways. However, studying these complex networks has been hindered by the challenge of detecting multiple neurochemicals in vivo simultaneously. To overcome this limitation, we developed a single-protein chemigenetic DA sensor, HaloDA1.0, which combines a cpHaloTag–chemical dye approach with the G protein–coupled receptor activation–based (GRAB) strategy, providing high sensitivity for DA, subsecond response kinetics, and a far-red to near-infrared spectral range. When used together with existing green and red fluorescent neuromodulator sensors, calcium indicators, cyclic adenosine 5′-monophosphate sensors, and optogenetic tools, HaloDA1.0 showed high versatility for multiplex imaging in cultured neurons, brain slices, and behaving animals, facilitating in-depth studies of dynamic neurochemical networks.