Fig. 5: Simultaneous two-photon imaging of local NE release and astrocytic calcium dynamics in awake mice during virtual navigation.

a, Simultaneous two-photon imaging was performed in head-fixed mice navigating a virtual corridor to collect water rewards delivered at 85 cm from the start of the corridor. b, Schematic of the HPC highlighting the imaged region (pink and green), indicating the expression of nLightR2 (neurons) and GCaMP6f (astrocytes) in the pyramidal layer. c, Representative images showing nLightR2-labeled neurons (left, magenta) and GCaMP6f-labeled astrocytes (middle, green) in the CA1 pyramidal layer. Images are normalized fluorescence intensity (F_norm) projection of recorded t-series, and contrast has been adjusted to aid visualization. nLightR2 and GCaMP6f signals were simultaneously imaged in the red and green channels, respectively, using the excitation wavelengths (λ_ex) indicated in the figure. White lines on the images indicate ROIs (for nLightR2, we show only 16 square ROIs in close proximity to one astrocyte, square ROI size ~17 μm × 17 μm (ref. ³⁷); for GCaMP6f, anatomically defined ROIs are as in ref. ⁶³). Right, the two images on the left are shown overlaid; scale bars, 50 µm; AU, arbitrary units. d, Representative functional traces of nLightR2 and GCaMP6f fluorescence extracted from the ROIs shown in c. Markers indicate either run (gold) or reward (teal) behavioral epochs. Traces in d have been smoothed with a 500-ms rolling mean filter. e, Event-triggered averages showing mean ΔF/F₀ of nLightR2 signal when the mouse started to run (Run, start of running bout at t = 0 s) for all the ROIs identified in c. f, Bottom left triangle, correlation (corr.) matrix for all traces extracted from the ROIs displayed in e. Top right triangle, corresponding hierarchical clustering. g,h, Same as in e and f, but for event-triggered averages following animals crossing the reward position (Reward, crossing time at t = 0 s). i–l, The same metrics as in e–h but referring to ΔF/F₀ computed for astrocytic ROIs and astrocytic GCaMP6f signals. m, Maximum ΔF/F₀ of GCaMP6f event-triggered average traces for astrocytic ROIs expressed as a function of the maximum ΔF/F₀ of nLightR2 event-triggered average recorded in the nLightR2 ROI nearest to the considered astrocytic ROI during either the run (gold) or reward (teal) behavioral epoch (P = 0.030 and P = 0.326 for reward and run epochs, respectively; two-tailed permutation test with 10⁴ repetitions, n = 114 pairs recorded in seven sessions from four mice). n, Mean ΔF/F₀ of GCaMP6f event-triggered average traces for astrocytic ROIs expressed as function of the mean ΔF/F₀ of nLightR2 event-triggered averages recorded in the nLightR2 ROI nearest to the considered astrocytic ROI during either the run (gold) or reward (teal) behavioral epoch (P = 0.025 and P = 0.144 for reward and run epochs, respectively; two-tailed permutation test with 10⁴ repetitions, n = 114 pairs recorded in seven sessions from four mice). o, Pearson’s correlation between nLightR2 and GCaMP6f signals computed either during the run (gold) or reward (teal) behavioral epoch as a function of pairwise distance (P = 0.001 for epoch effect (run versus reward), distance–epoch interaction not significant for all distances (Supplementary Table 1), linear mixed-effects model, n = 7 sessions from four mice). Lines and shaded areas in m–o indicate mean ± s.d. and mean ± s.e.m., respectively.

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