Individual target extraction and experimental design.

(A) Methodological workflow used to extract the individualized target for the neuromodulation protocol used in experiments 1, 3 and 4. Participants underwent fMRI scanning to individualize the stimulation sites and permit neuronavigation. Target individualization was derived by computing a PC functional connectivity profile for each participant, thus obtaining a map of positively correlated voxels, respectively representing the DMN (Panel A, left). The individual stimulation targets were defined as the centroid of the strongest PC activation being on the top of a cortical gyrus and representing the shortest perpendicular path connecting the stimulating TMS coil on the scalp and the cortex (Panel A, center). PC coordinates for each participant are represented in red on an MNI brain template, showing the overlap with the DMN (Panel A, right). (B) Biophysical modeling was computed for each subject acquiring T1w and T2w MRI and using the Simnibs toolbox for the T1w segmentation and the 3D-mesh transformation (Panel B, left). The mean Norm E-field was extracted from a target ROI-sphere (10 mm radius) centered on the individual coordinates of the PC (Panel B, center). The simulated induced electric field is shown for a representative subject produced by iTBS (e-field modeling, left) and tACS (e-field modeling, right). EEG source activity reconstruction induced by the TMS pulse over the precuneus in a representative subject (Panel B, right). Experimental design of experiments 1(C), 2 (D), 3 (E), 4 (F). The effect of simultaneous iTBS+γtACS on memory performances was investigated in experiments 1(C) and 2 (D) through two memory tasks: the face-name associative task (FNAT), which required the memorization of 12 faces with corresponding faces and occupations, and the visual short-term memory binding test (STMB), which consisted in a change detection task. In the main experiment 1 (C), subjects were involved in a cross-over design with different experimental sessions of neuromodulation separated by a washout week. Every session corresponded to a different balanced and randomized stimulation condition (i.e., iTBS+γtACS, iTBS+sham-tACS, sham-iTBS+sham-γtACS) immediately followed by the FNAT learning phase and immediate recall, the STMB and the FNAT delayed recall (15-minute delayed) and recognition.

In experiment 2 (D) subjects were involved in a cross-over design with two balanced and randomized stimulation conditions (i.e., iTBS+γtACS, iTBS+sham-tACS) separated by a washout week. During the first session (day 1), participants received the neuromodulation protocol and then performed the learning phase and immediate recall FNAT, the STMB and the FNAT delayed recall. In the second session (day 2), participants performed FNAT recall with a 24-hour delay from the neuromodulation protocol, while in the third session (day 7), participants performed FNAT recall and recognition with a 1-week delay. In experiment 3 (E) participants were involved in two randomized and balanced experimental sessions of neuromodulation (i.e., iTBS+γtACS, iTBS+sham-tACS) separated by a washout week. TMS-EEG recordings were performed before (T0), immediately after (T1), and 20 minutes after the neuromodulation (T2). In experiment 4 (D), after the first MRI scanning used for neuronavigation, participants were involved in two randomized and balanced experimental sessions of neuromodulation (i.e., iTBS+γtACS, iTBS+sham-tACS) separated by a washout week. The fMRI scanning was performed before (T0) and immediately after (T1) the neuromodulation protocol.

Photographs reported represent the author Michele Maiella performing the task and an example of the face item used in the task taken from the FACES database (Ebner et al., 2010).

Memory performance outcome.

(A) Face-name associative task (FNAT) accuracy in immediate and delayed trials for FNAT TOTAL (left), NAME (center), and OCCUPATION (left) association resulting from experiment 1 after iTBS+sham-tACS (grey), sham-iTBS+sham-tACS (blue), iTBS+γtACS (red). N=20. (B) Short-term memory binding task RTs (left) and accuracy (right) resulting from the three conditions of experiment 1: iTBS+sham-tACS (grey), sham-iTBS+sham-tACS (blue), iTBS+γtACS (red). N=20. (C) FNAT’s long-lasting effect resulted from two conditions of experiment 2: iTBS+sham-tACS (grey) and iTBS+γtACS (red). The results are shown over time (day 1, day 2, day 7) dividing the performance between FNAT TOTAL (right), NAME (center), and OCCUPATION (left) association. N=10. * = p<0.05; bars depict standard error.

Experiment 3 neurophysiological outcome.

(A) Precuneus (PC) oscillatory activity elicited by iTBS+γtACS (up) and iTBS+sham-tACS (down) when testing PC over the three time points (T0, T1, T2 from left to right). (B) Gamma oscillation changes from baseline after iTBS+γtACS (red) and iTBS+sham-tACS (grey). (C) TMS-evoked potential (TEP) produced over the PC when performing TMS-EEG over PC in the two stimulation conditions: iTBS+γtACS (up-left) and iTBS+sham-tACS (up-right) over the three time points (T0, T1, T2). (Down) Topographies and statistical differences in TEPs amplitude after the different stimulation conditions (iTBS+γtACS, left; iTBS+sham-tACS, right) over the three time points (T0, T1, T2, from left to right). N=14; * = p<0.05; bars depict standard error.

rsFC changes after iTBS+γtACS and correlation with DTI.

(A) The standard MNI brain (left) and the bar plot (right) display the positive correlation between the PC and the bilateral HIP after the iTBS+γtACS resulted from the ROI-to-ROI analysis. (B) Seed-to-voxel analysis results from each significant ROI (i.e. left HIP, left; PC, center; right HIP, right) are overlaid to a standard MNI brain. (C) MdLF extracted (left); positive correlation between MdLF integrity and functional connectivity changes between the PC and bilateral HIP after iTBS+γtACS (up right); the absence of correlation in the iTBS+sham-tACS condition and functional connectivity (down right). N=16; * = p<0.05; bars depict standard error.

Demographic characteristics.