Fig. 3: Ex vivo two-photon imaging of iAdo dynamics in acute mouse brain slices using HypnoS.

a Schematic illustration depicting the experimental design for (c–h). b Representative images showing HypnoS expression in the hippocampus CA1. Scale bar, 1 mm. Representative images (c), representative traces (d, top), and group summary (d, bottom) of the change in HypnoS fluorescence to indicated concentration of Ado or 100 μM inosine (Ino) in sensor-expressing brain slices. Scale bar, 100 μm (c, top) and 20 μm (c, middle and bottom). n = 20, 30, 30, 30, and 30 neurons from 3 slices of 2 mice for 1 μM, 10 μM, 100 μM, 1 mM Ado, and 100 μM Ino. Two-tailed Student’s t-test: P = 3.4 × 10⁻¹³ between Ino and Ado. Representative images (e), representative traces (f, top), and group summary (f, bottom) of the change in HypnoS fluorescence to 25 mM 2-DG. Scale bar, 100 μm (e, top) and 20 μm (e, middle and bottom). n = 60 neurons from 6 slices of 4 mice. Two-tailed Student’s t-test: P = 1.2 × 10⁻¹⁸ between ACSF and 2-DG. Representative images (g), representative traces (h, left), and group summary (h, right) of fluorescence response of HypnoS to electrical stimulation either in artificial cerebrospinal fluid (ACSF) or in 25 mM 2-DG. White dashed line, electrode placement. Scale bar, 100 μm. n = 5 slices from 5 mice. Paired two-tailed Student’s t-test: P = 0.0086 between ACSF and 2-DG. Representative images (i), representative traces (j, left), and group summary (j, right) of fluorescence response of astrocytic HypnoS to electrical stimuli. White dashed line, electrode placement. Scale bar, 100 μm. n = 38 cells of 5 slices from 5 mice. Two-tailed Student’s t-test: P = 0.22 between Basal and 20 pulses; P = 0.61 between Basal and 600 pulses. Data are shown as mean ± s.e.m. in (d, f, h, j) with the error bars or shaded regions indicating the s.e.m. **P < 0.01; ***P < 0.001; ns not significant. Source data are provided as a Source Data file.