Transcranial Direct Current Stimulation (tDCS) Induces Adrenergic Receptor-Dependent Microglial Morphological Changes in Mice

Head-restraint tDCS experiment. A, Experimental setup for tDCS. B, Experimental schedule of immunohistochemical experiment. C, Top view of a BAC-GLT1-G7 Line 817 (G7NG817) mouse. Fluorescent Ca²⁺ signal is transcranially observable. Signals ∼3 mm anterior to the anodal site (1 × 1 mm² red square) are plotted from four mice (right, top traces). The bold trace on the bottom is the mean of the four traces, and the shaded areas represent SE. The red arrowhead and line indicate the onset of tDCS. Scale bar, 1 mm. tDCS-induced Ca²⁺ elevations were not observed in isoflurane-anesthetized mice (Extended Data Fig. 1-1).

Intensity analysis of microglial confocal images. A, B, Representative images of Cy3-labeled Iba1 IHC by maximum intensity projection obtained in Sham- and LPS-treated mice. Yellow scale bars: A, left, 100 μm; A, right, B, 20 μm. C, Cumulative pixel intensity distributions from unrestrained Ctl and head-restrained Sham groups were similar and distinct from the LPS-treated group. D–G, Intensity was compared between tDCS- and Sham-treated groups under the isoflurane-anesthetized (D, E) or awake (F, G) conditions, perfused at 30 min or 3 h after sham/tDCS. H, In awake mice, the pixel intensity histogram indicates that there is a cluster at z score >2 (i.e., mean + 2 SDs) region in the tDCS group (dotted red square). I, J, Representative images from a sham-treated mouse and a tDCS-treated mouse. Images in the red squares correspond to the thresholded images on the left at the mean + 2 SDs. Red scale bars, 20 μm. **p < 0.01, ***p < 0.001.

Quantification and comparison of microglial soma size. A, Example image of an Iba1 IHC confocal image stack collapsed by maximum intensity projection. Scale bar, 20 μm. B, Digitally processed image of A for soma extraction. C, Example of the elliptic approximation of soma (A, B, red dotted square). D, Comparison of median values of microglial soma areas between Sham-stimulated and unrestrained control mice (p = 0.1^a, Mann–Whitney Wilcoxon rank sum test). Scale bars: A, B, 10 μm; C, 20 μm. E, Comparison of microglial soma size in awake mice with/without tDCS treatment at different time points (30 min or 3 h) after tDCS. Microglial soma size was larger in the tDCS group in the awake 3 h experiment (p = 0.017^c, Mann–Whitney Wilcoxon rank sum test). Each group contains seven mice. F, Microglial soma size comparison in isoflurane-anesthetized mice (Isofl-3hr). **p < 0.03, N.S. not significant.

tDCS-induced microglial somatic enlargement depends on noradrenaline. A, Example of cortical image (inverted grayscale) from saline- (left) or DSP4- (right) pretreated mice stained with TH antibody. B, Mean intensity analysis of TH⁺ fiber. Each group contains data from three mice. Data from the same animals are plotted with the same symbol and color. Scale bars, 100 μm. C, Comparison between median glial soma size from sham- and tDCS-treated mice (Sham group: 7 mice; tDCS: 7 mice; p = 0.073^e, Mann–Whitney Wilcoxon rank sum test). N.S. not significant.

tDCS-induced microglial somatic enlargement depends on B2AR and A1AR pathways. A, Comparison between median microglial soma size between Sham- and tDCS-treated IP₃R2 KO mice (Sham group: 6 mice; tDCS group: 7 mice; p = 0.73^f). B, C, Comparison of microglial soma size between Sham- and tDCS-treated wild-type strain C57BL/6J with prazosin (B; Sham group: 7 mice; tDCS group: 7 mice; p = 0.8 × g), or ICI181551 pretreatment (C; Sham group: 6 mice; tDCS group: 6 mice; p = 0.48^h). N.S. not significant.

In vivo monitoring of microglial morphologic dynamics. A, B, Experimental setup (A) and time schedule of in vivo two-photon imaging (B). C, Representative images of a microglia overlaid from t = 0 to respective time points (10, 30, and 40 min). D, Normalized surveillance area curve during 60 min imaging period. Red arrowheads show the time points for the images in C. Scale bar, 10 μm. E, Initial microglial area at t = 0 of Before and After sessions are similar in Sham mice (13 cells from 8 mice; p = 0.82ⁱ). Blue lines represent data from individual microglia, and the black line represents averaged data. F, Normalized surveillance area curves during the 60 min imaging period before (blue) and after (red) stimulation in the sham (left) and tDCS (right) mice. Data are represented as the mean ± SEM. G, Normalized surveillance area at t = 40 min in Before and After sessions in tDCS-treated mice (normalized by surveillance area at t = 0/Before). Red lines represent data from individual microglia, and the black line represents averaged data. p = 0.014^j, paired t test. **p < 0.03.