Intracranial electric fields induced by Cb-tACS.

a Experimental design for combined in vivo electrophysiology and transcranial alternating current stimulation (tACS) in crus I-II of cerebellar cortex in awake mice, showing silver-ring active and reference (Ref.) electrode locations. Inset (i.) shows a schematic sagittal view of the recording chamber design. b Schematic representation of a sagittal section of the brain showing the reference (Ref.) and active electrodes location (gray bar) and a representative track in the lateral cerebellum highlighting the depths where the electric field was measured (color dots). c tACS stimulation (top trace) applied over the scalp and exemplary recording of the actual field potentials generated at different depths (from 0 to 4 mm) in a representative animal. The traces were overlapped to facilitate amplitude comparison. d Average (filled symbols) and individual (empty symbols) electric field strength recorded at different depths for ± 2 (circles), ± 20 (squares) and ± 200 μA (triangles) tACS.

tDCS modulation of PC activity in crus I/II of awake mice.

a, b Recording of spontaneous firing activity of two PCs showing the presence of SS and CS. c,d Z-score-transformed average PSTH (bin size: 0.1 s) of the SS activity of the 2 PCs shown in a,b before, during and after anodal (red trace) or cathodal (blue trace) tDCS. e,f Statistical comparison of the SS firing rate of the 2 PCs shown in a,b, measured in 5 s windows before, during and after tDCS (RM-ANOVA or Friedman tests, p < 0.05). Error bars represent SEM. **p < 0.01; ***p < 0.001. g Modulation of SS (p) of individual PCs (circles) during anodal (red) and cathodal (blue) tDCS. Filled circles represent statistically significant modulation during tDCS (n = 24, RM-ANOVA or Friedman tests, p < 0.05). h Schematic representation of the recording sites and active electrode location (gray bar) during tDCS.

tDCS modulation of non-PC activity in crus I/II of awake mice.

a, b Recording of spontaneous firing activity of two non-PCs. c, e Z-score-transformed average PSTH (bin size: 0.1 s) of firing rate for the 2 neurons in a,b, before, during and after anodal (red trace) or cathodal (blue trace) tDCS. d, f Statistical comparison of the firing rate of the 2 neurons in a,b, measured in 5 s windows before, during and after tDCS (RM-ANOVA or Friedman tests, p < 0.05). Error bars represent SEM. *p < 0.05; **p < 0.01; ***p < 0.001. g Modulation of firing rate of individual neurons (circles) during anodal (red) and cathodal (blue) tDCS. Filled circles represent statistically significant modulation during tDCS (n = 50, RM-ANOVA or Friedman tests, p < 0.05). h Schematic representation of the recording sites and active electrode (gray bar) location during tDCS.

tDCS modulation of PC and non-PC activity in the vermis of anesthetized mice.

a Schematic representation of the recording sites and active electrode (gray bar) location during tDCS. b A representative coronal section of the vermis immunofluorescently stained with Calbindin (green). The magnification inset highlights the distinct orientation of PCs in different layers, which is indicated by the drawings of PC somas and dendrites (with dendrites always extending into the molecular layer, shown in green). c,d Modulation of SS firing rate of individual PCs (c) and firing rate of individual non-PCs (d) during anodal (red) and cathodal (blue) tDCS over cerebellar vermis. Filled circles represent statistically significant modulation during tDCS (n = 31 PCs and 25 non-PCs, RM-ANOVA or Friedman tests, p < 0.05). GCL: granular cell layer, ML: molecular layer, PCL: Purkinje cell layer.

Relationship between tDCS-driven modulation of PC firing rate and somatodendritic axis orientation in anesthetized mice.

a-d (Left) Confocal images of labeled neurons with different somatodendritic angles relative to the electric field (dotted white vertical line), (Right) z-score of their firing rate modulation during anodal (red) or cathodal (blue) tDCS and statistical analysis (RM-ANOVA or Friedman tests, p < 0.05). Error bars represent SEM. *p < 0.05; **p < 0.01; ***p < 0.001. e Relationship between firing rate modulation and somatodendritic angle for all juxtacellularly-labelled PCs (n = 8). Arrow length represents firing rate modulation during anodal (red arrows, at left) or cathodal (blue arrows, at right) tDCS at 200 μA, relative to the firing rate during control condition (represented by 100% solid circle). f Average change in firing rate during anodal (red) and cathodal (blue) tDCS for individual PCs with different somatodendritic orientations.

Impact of tDCS on PCs with opposite axodendritic orientations simultaneously recorded in the awake mice.

a Probe location marked with Dil in the cerebellar vermis stained with Hoechst 33342 dye. b Magnification of square area in “a” showing the location of two oppositely oriented PCs recorded at Ch#55 and Ch#42. The orientation of the PCs in each of the 2 layers is indicated with drawings of PC soma and dendrites (yellow), in which the soma appear at the interface between the granule cell layer (GCL, shown in blue) and the molecular layer (ML, shown in green). c Z-score-transformed average PSTH (bin size: 0.1 s for SS) of the SS activity before, during and after anodal and cathodal tDCS pulses for each of the 2 simultaneously recorded PCs shown in b. d Anatomical localization of the different PCs recorded. The inset shows the recorded places marked with Dil (red) and stained with anti-Calbindin antibody (green, molecular layer) and Hoechst 33342 (blue, granule layer) (scale bar: 300 µm). PCs in which dendrites are pointed toward or away from the active tDCS electrode are denoted with triangles pointing upward or downward respectively. The color of the triangle indicates whether the modulation was an increase (red) or a decrease (blue) in firing rate. e Modulation of SS firing rate of individual PCs during anodal and cathodal tDCS. Filled symbols represent statistically significant modulation during tDCS, with the meaning of color and shape as in d (n = 9, RM-ANOVA or Friedman tests, p < 0.05). ML: molecular layer, PCL: Purkinje cell layer, GCL: granular cell layer.