Figure 1. tDCS produces large and synchronized Ca²⁺ surges in the cortex as visualized with G7NG817 mice.

(a) Sagittal brain section of the G7NG817 (upper panel) and C57BL/6 wild-type mice (lower panel). High expression of G-CaMP7 was seen in the cortex, hippocampus (particularly CA3), thalamus and striatum. Scale bar, 1 mm. (b) Immunostaining for G-CaMP7 (green) and astrocytes (S100B, red) shows a high degree of co-localization (arrows, co-stained astrocytes; arrowheads, G-CaMP7-positive neurons). Scale bar, 50 μm. (c) Bar graph that shows the layer-by-layer profile of G-CaMP7-positive cells in NeuN-labelled neurons (mean±s.e.m., n=9 slices from two mice) in the sensory cortex. (d) Sketch of the tDCS experiment configuration (left). The cathode is placed on the neck skin. On the right, the time course of an awake mouse transcranial imaging session during the initial 40 s of tDCS is displayed. The pseudocolour represents the range between the mean+1 s.d. and the peak value of the tDCS session. Three regions ROI1, 2 and 3 corresponds to the anodal, anterior and contra-anodal regions are illustrated for the following analyses. (e) Average peri-stimulus fluorescent intensity changes from multiple awake mice (n=8). Anodal (red), anterior (black) and contra-anodal (blue) regions are displayed. Shaded areas represent s.e.m. Arrowheads point to the initial large and long-lasting (≥10 s) Ca²⁺ surges triggered by tDCS. (f) Example traces of anodal cortical G-CaMP7 response during tDCS from three mice. Filled triangles (▾) indicate the occurrences of large and long-lasting (≥10 s) Ca²⁺ surges. (g) tDCS-induced Ca²⁺ surges have higher amplitudes than spontaneous Ca²⁺ events both on the anodal (red) and contra-anodal (blue) sites (anodal versus contra-anodal: P=0.30, sponta. versus tDCS: P=0.0001, two-way ANOVA). (h) Both anodal and contra-anodal sites have similar time to peak for the first Ca²⁺ surge since the onset of tDCS. (i) Long (≥10 s) Ca²⁺ surges are more frequent during tDCS (P=3.44E−7, two-way ANOVA), while shorter events occurred with a similar frequency (P=0.58, two-way ANOVA). Red and blue bars represent anodal and contra-anodal sites, respectively. (j) Distribution of the amplitudes of initial tDCS evoked Ca²⁺ surges in awake and urethane-anaesthetized mice. The amplitudes are similar between awake and urethane-anaesthetized mice. (k) Distribution of the onset of the initial tDCS evoked Ca²⁺ surge in awake and urethane-anaesthetized mice. The initial Ca²⁺ surge occurs within the first minute in the awake condition, whereas the onset time is more variable in urethane-anaesthetized mice. (l) tDCS-induced Ca²⁺ surges occur with a reduced frequency in urethane anesthesia compared with the awake condition (n=10 mice, cf. i). tDCS-induced Ca²⁺ surges do not occur in awake IP3R2 KO (IP₃R2^−/−;G7NG817^+/−) mice (n=4). tDCS-induced Ca²⁺ surges are blocked by i.p. injection of prazosin (10 mg kg⁻¹, 20 min before imaging) in G7NG817 mice (n=7) or in DSP-4-treated awake G7NG817 mice (n=7). ***P<0.0001. Error bars are defined as s.e.m.