Extended Data Fig. 8: Two-photon hippocampal imaging of nLightR2 during virtual spatial navigation.
From: Next-generation multicolor indicators for in vivo imaging of norepinephrine
a Two-photon imaging was performed in head-fixed awake mice navigating a virtual reality corridor. b Side view of the corridor and hippocampus schematic showing the imaged CA1 pyramidal layer (pink). c Five-day experimental protocol: water rewards at 85 cm on days 1–2 and first half of day 3; reward shifted to 145 cm for second half of day 3 and days 4–5. d Representative FOVs showing nLightR2 signal across days; images are temporal averages scaled to maximum intensity (scale bar 50 μm). e–g Event-triggered averages of nLightR2 signal (e), running speed (f), and normalized lick rate (g) aligned to running onset (gold, t = 0 s) or reward crossing (teal, t = 0 s); lines ± s.d. h Average nLightR2 responses vs running speed across five days; significance assessed by two-sided rank-sum test: day 1 P(run)=1.00, P(reward)=2.09×10−2; day 2 P(run)=0.248, P(reward)=1.00; day 3 P(run)=2.09×10−2, P(reward)=1.00; day 4 P(run)=2.09×10−2, P(reward)=2.09×10−2; day 5 P(run)=1.00, P(reward)=4.95×10−2. i Average nLightR2 responses vs normalized lick rate: P = 2.09×10−2, 2.09×10−2, 2.09×10−2, 0.248, 0.513 for days 1–5. j Representative FOV with overlaying ROI grid (scale bar 50 μm) used for trace extraction. k,m Event-triggered ΔF/F0 for running (k) and reward crossing (m) for all ROIs; l,n cross-correlation matrices (lower left triangles) and hierarchical clustering (upper-right triangles) for all traces extracted from the ROIs displayed in (k) and (m). o Pairwise Pearson’s correlation value of nLightR2 signals at reward crossing plotted as a function of Pearson’s correlation value upon running. Data from 386,120 ROI pairs from 3,920 ROIs in 4 mice over five sessions; diagonal indicated by white dashed line.
