Fig. 6: nLightG2 reveals spatially structured NE dynamics in the VC during sensory stimulation and behavioral state transitions in mice.
From: Next-generation multicolor indicators for in vivo imaging of norepinephrine
a, Schematic of viral injection in the VC and two-photon imaging setup with nLightG2 expression in layer 2/3 of the primary VC (V1) in head-fixed mice on a running wheel. b, Individual ΔF/F0 traces from two example FOVs illustrating loom-evoked activity in nLightG2 (average across ROIs with ΔF/F0 > 3σ; green) and nLightG2-ctr (gray) mice, highlighting a single ROI with a strong loom-evoked response in the nLightG2 animal to a single loom, whereas only a small, albeit detectable, change was observed in the control, potentially reflecting hemodynamic signals. c, Example ΔF/F0 traces from representative nLightG2 (green), GRABNE2m (blue) and nLightG2-ctr (gray) animals over the course of individual recordings. Orange triangles in the traces highlight spontaneous NE events in the absence of locomotion. d, An example FOV from an nLightG2-expressing mouse. e, Same as in d for a GRABNE2m-expressing animal. Peak responses are indicated in red. f, Example spatial distribution of peak response latency after loom onset across tile ROIs with a response ΔF/F0 > 3σ within a single FOV. g, Time-to-peak analysis for data plotted in f. h, Scatter plots of loom-evoked changes in ΔF/F0 (%) per ROI, including all ROIs across all mice within each group (nLightG2 ROIs (1,075 ROIs, n = 7 mice), GRABNE2m (751 ROIs, n = 3 mice) and nLightG2-ctr (375 ROIs, n = 3 mice)). i, Cumulative distributions of ΔF/F0 values in the 20-s postloom window compared to preloom baseline across all tile ROIs. nLightG2 showed a significant shift for all tile ROIs pooled across mice (two-sided Kolmogorov–Smirnov test, P = 3.8 × 10−5), whereas GRABNE2m (P = 0.093) and nLightG2-ctr (D = 0.093, p = 0.076) did not. j, Scatter plots of mean ΔF/F0 during stationary versus forced running epochs from the same ROIs and animals shown in f. k, Cumulative distributions of running-evoked ΔF/F0 changes showed a rightward shift in nLightG2 compared to GRABNE2m (two-sided Kolmogorov–Smirnov test, P = 4 × 10−4) and nLightG2-ctr (P = 3.2 × 10−3) during forced locomotion. l, GLM quantifying the variance in NE dynamics explained by looming stimuli (loom), running speed (run) and their interaction (inter) in nLightG2-expressing animals (n = 7 mice). Left, the full model explained 4.4 ± 2.3% (mean ± s.e.m., n = 7 mice) of the total fluorescence variance, whereas excluding the running term reduced explained variance to 0.37 ± 0.09%, excluding the looming term to 4.2 ± 2.2% and excluding the interaction term to 4.3 ± 2.3% (mean ± s.e.m., n = 7 mice). Right, example tiled map showing the spatial distribution of the total explained variance (ΔR2) for an nLightG2-expressing mouse; Expl., explained. m, Leave-one-out GLM analysis validating the contribution of running, looming and their interaction to explained variance in NE dynamics (mean ± s.e.m.; relative ΔR2; running: 68.0 ± 5.7%, looming: 20.7 ± 3.5%, interaction: 10.5 ± 2.8%; Friedman P = 9.1 × 10−4; Wilcoxon P = 0.016, n = 7 mice).
