Fig. 3: Reliable pan-cortical expression of JEDI-1P-Kv.

a Sagittal slide illustrating that JEDI-1P-Kv (top left) and mCherry (bottom left) are expressed pan-cortically. Middle, coronal section. Right, zoomed-in and overlaid image of JEDI-1P-Kv and mCherry. Adeno-associated viruses encoding soma-localized JEDI-1P (JEDI-1P-Kv) and a cytosolic red-emitting reference fluorescent protein (mCherry) were introduced bilaterally by neonatal intracerebral ventricular (ICV) injection. b Imaging setup schematic. All experiments were conducted at 200 Hz. c Representative image of pan-cortical JEDI-1P-Kv fluorescence acquired with the system depicted in (b). The numbered squares show the 6 × 6-pixel (600 × 600 µm) regions of interest (ROIs) analyzed in (d) and (e). d, e JEDI-1P-Kv (d) and mCherry (e) can be robustly detected over the background. Bars represent the mean fluorescence intensity at different ROIs from mice expressing JEDI-1P-Kv and mCherry (green and black bars, respectively; n = 10) or control mice expressing neither (gray bars, n = 4). Lines show data from individual mice expressing JEDI-1P-Kv or mCherry (solid) or control mice (dashed). Mean intensities of all ROIs from mice expressing JEDI-1P-Kv and mCherry are significantly higher than those from control mice. p = 0.002 for each ROI, two-sided Mann–Whitney U test. f–i JEDI-1P-Kv is photostable under wide-field illumination in vivo. Excitation power was 0.05 mW/mm², lower than used in vitro (Fig. 2g, h). Data shows mean JEDI-1P-Kv fluorescence intensity over a single trial (f), a daily session (g), all 10 imaging sessions (h), and 1-h continuous illumination (i). Bottom schematics illustrate the illumination programs. Inter-trial intervals (ITI) were ~20 s. Total imaging time per session was 34 min. During continuous illumination, images were acquired at 0, 30, and 60 min; 5 trials/animal at each timepoint. Data were normalized to the beginning of the trial (f), the mean intensity of the first trial (g), the mean intensity of the first session of the first day (h), and the mean intensity at 0 min (i). n = 300 trials from 3 mice (100 trials/animal) (f), 27 sessions from 3 mice (g), 3 mice (h), and 4 mice (i). For (d–i), mean intensities are shown. Error bands (f, g) and bars (other panels) denote the 95% CI. Panel (b) was created using Biorender.com.