Fig. 3: Benchmarking NE indicators in vivo with fiber photometry.
From: Next-generation multicolor indicators for in vivo imaging of norepinephrine
a–c, Injection scheme (a) and histological verification of nLightR2 expression in the dHPC (b) and ChR2 expression in the LC (c). Data for the brain schematics obtained from ref. 73 under a Creative Commons license CC BY 4.0. Data in b and c were repeated independently five times with similar results. d, Color-coded heat maps of z-scored ΔF/F0 of nLightR2 in the dHPC following optogenetic activation of the LC. The blue bar indicates the duration of optogenetic stimulation. e, Mean ± s.e.m. of z-scored indicator fluorescence in the dHPC (top) and relative pupil diameter (bottom) in exemplary mice expressing nLightR2 (left), nLightR (center) and nLightR2-ctr (right). Blue bars indicate optogenetic LC stimulation (n = 15 trials for nLightR2 and nLightR, n = 50 trials for nLigthR2-ctr). Iso., isoemissive. f, Mean ± s.d. of optogenetically evoked indicator fluorescence (left) and pupil dilation (right) of animals expressing nLightR2 (n = 6 animals), nLightR (n = 4 animals) and nLightR2-ctr (n = 3 animals). Data were analyzed by one-way ANOVA with a Tukey’s test: nLightR2 versus nLightR (P = 0.05), nLightR2 versus nLightR2-ctr (*P = 0.024) and nLightR versus nLightR2-ctr (P = 0.80). g,h, Experimental schematics for nLightG2, nLightG and GRABNE2m viral injections in the pBLA. i, Behavioral task design for the cued fear conditioning paradigm, consisting of a baseline (BL) session with 20-s tone cues, an association (AS) session with the same tone cues co-terminating with 1-s footshocks and a re-exposure (RE) session with tone cues only in the absence of footshocks (n of trials in each session is 10). j,k, Baseline session. j, Heat maps showing trial-averaged z-score fluorescence responses across the ten tone trials for nLightG2 (top), nLightG (middle) and GRABNE2m (bottom) in the pBLA. k, Corresponding mean ± s.e.m. z-score fluorescence traces for nLightG2 (green), nLightG (gray) and GRABNE2m (blue). l,m, Association session. l, Heat maps of trial-averaged z-score fluorescence responses for each indicator type during ten tone–footshock pairings. m, Corresponding mean ± s.e.m. traces showing tone-evoked activity (0- to 20-s tone) co-terminating with a 1-s footshock. n,o, Re-exposure session. n, Heat maps of trial-averaged z-score fluorescence responses for each indicator type during ten tone presentations. o, Corresponding mean ± s.e.m. traces for tone-evoked responses (0- to 20-s tone) in the absence of footshock. Dashed vertical lines mark cue onset and footshock timing. p, Quantification of CS-evoked peak z-score responses in nLightG2, nLightG and GRABNE2m mice across all conditions (baseline, association and re-exposure). Data were analyzed by two-way ANOVA (Psession = 0.0037; Pindicator type = 0.0121), post hoc Tukey’s comparisons (baseline: PnLightG2 versus nLightG = 0.0460, PnLightG2 versus GRABNE2m = 0.1163, PnLightG versus GRABNE2m = 0.9002; association: PnLightG2 versus nLightG = 0.0129, PnLightG2 versus GRABNE2m = 0.0061, PnLightG versus GRABNE2m = 0.9553; re-exposure: PnLightG2 versus nLightG = 0.0318, PnLightG2 versus GRABNE2m = 0.0161, PnLightG versus GRABNE2m = 0.9574). q, Quantification of footshock-evoked peak z-score responses in nLightG2, nLightG and GRABNE2m mice (Welch’s one-way ANOVA: P = 0.0002; post hoc Dunnett’s T3 comparisons (two-sided): PnLightG2 versus nLightG = 0.0002, PnLightG2 versus GRABNE2m = 0.0270, PnLightG versus GRABNE2m = 0.1096; nLightG2, n = 5 mice; nLightG, n = 5 mice; GRABNE2m, n = 5 mice); WT, wild-type.
