Fig. 5: Pixel-by-pixel analysis proved significant pH change in the brain during visual stimulation.

a A representative example of the spatial distribution of pixels with statistically significant changes in pH during visual stimuli. The data in Fig. 2c were analyzed here. A one-sample t-test was used at each pixel to statistically compare the difference between mean pH change for each direction of drifting gratings (a total of 30 trials for each direction) and mean pH change for all eight directions (i.e. the mean over 8 directions × 30 trials). Pixels with a significant decrease (acidic change, p < 0.05) are shown in blue, while pixels with a significant increase (alkaline change, p < 0.05) are marked in yellow. The spatial distribution differed depending on the direction of the stimulation. White dotted bars demarcate the region of the pH-sensing area inserted into the brain and the region of the pH-sensing area left outside of the brain (see Supplementary Fig. 2). b The number of pixels with statistically significant changes in brain pH was summarized for all observations in mice (n = 9) or in HEPES-buffered saline (n = 3) for each direction of visual stimulation. A two-way ANOVA test indicated a statistically significant difference between mouse brain and HEPES-buffered saline measurements (**p < 0.01). In the box and whisker plots, the center line indicates the median; the box defines the 25–75th percentiles; the whiskers represent minimum and maximum values while the red crosshairs represent outliers. Filled circles are individual data points. Green plots correspond to HEPES-buffered saline while blue plots correspond to mouse brains. c The number of pixels of significant changes in pH was pooled across all eight drifting gratings directions. An unpaired t-test indicated a significant difference between brain and HEPES-buffered samples (***p < 0.001). Definitions of box and whisker plots and n numbers are same as in that stated in b. The source data underlying b and c are provided as a Source Data file.