Fig. 1.

Design of NE-FFNs. a The design of NE-FFNs combines structural features of NE with the coumarin fluorescent core. b NE-FFNs trace NE uptake from the extracellular space, packaging into vesicles, and exocytosis as they are designed to be substrates of NET and VMAT2. c Representative illustration of NE neuron distribution in the brain (Allen Institute)⁵¹. Many NE neurons originate from the locus coeruleus (LC) and project to the majority of brain regions. d General synthetic scheme for preparation of 3-series aminoethyl-7-hydroxycoumarins as potential NE-FFN candidates. See Methods for experimental conditions. e Focused series of candidate NE-FFNs. f Total cellular fluorescence after loading of FFN candidates (5 µM) in hNET-HEK cells. Signal-to-Basal ratio (S/B ± SEM) was determined by comparing FFN fluorescence in the presence and absence of 2 µM nomifensine (NET inhibitor) after a 30 min incubation period. Blue arrows highlight previously described DA-FFNs, and a magenta arrow highlights FFN270, the leading pH-sensitive NE-FFN candidate (S/B: 8.5 ± 0.4, mean ± SEM, n = 3 independent experiments run in triplicate). Representative images of FFN270 without inhibitor (g) and with inhibitor (h). Scale bar: 20 µm