Norepinephrine and dopamine sensor crosstalk depends on local innervation density

G-protein-coupled receptor-based fluorescent sensors are widely used to correlate neuromodulatory signaling with brain function. Although experiments in transfected cells often reveal selectivity for individual neurotransmitters, sensor specificity in the brain frequently remains uncertain. Using imaging in mouse brains, we find that norepinephrine and dopamine cross-activate the respective sensors. Non-specific activation occurred when innervation of the cross-reacting transmitter was high, and silencing of specific innervation was indispensable for interpreting sensor fluorescence.