ORIGINAL RESEARCH published: 26 November 2021 doi: 10.3389/fnagi.2021.725013 Multifocal Transcranial Direct Current Stimulation Modulates Resting-State Functional Connectivity in Older Adults Depending on the Induced Current Density Kilian Abellaneda-Pérez 1,2* , Lídia Vaqué-Alcázar 1,2 , Ruben Perellón-Alfonso 1,2 , Cristina Solé-Padullés 1,2 , Núria Bargalló 3,4 , Ricardo Salvador 5,6 , Giulio Ruffini 5,6 , Michael A. Nitsche 7,8 , Alvaro Pascual-Leone 9,10,11 and David Bartrés-Faz 1,2,11* 1 Department of Medicine, Faculty of Medicine and Health Sciences, Institute of Neurosciences, University of Barcelona, Barcelona, Spain, 2 Institute of Biomedical Research August Pi i Sunyer (IDIBAPS), Barcelona, Spain, 3 Section of Neuroradiology, Department of Radiology, Diagnostic Image Center, Hospital Clinic of Barcelona, University of Barcelona, Barcelona, Spain, 4 Magnetic Resonance Image Core Facility (IDIBAPS), Barcelona, Spain, 5 Neuroelectrics, Cambridge, MA, Edited by: United States, 6 Neuroelectrics, Barcelona, Spain, 7 Leibniz Research Centre for Working Environment and Human Factors, Wei Wu, Dortmund, Germany, 8 Department of Neurology, University Medical Hospital Bergmannsheil, Bochum, Germany, 9 Hinda Alto Neuroscience, United States and Arthur Marcus Institute for Aging Research and Deanna and Sidney Wolk Center for Memory Health, Hebrew SeniorLife, Boston, MA, United States, 10 Department of Neurology, Harvard Medical School, Boston, MA, United States, 11 Guttmann Reviewed by: Brain Health Institute, Guttmann University Institute of Neurorehabilitation, Autonomous University of Barcelona, Badalona, Virginia Conde, Spain Norwegian University of Science and Technology, Norway Helen Carlson, Combining non-invasive brain stimulation (NIBS) with resting-state functional magnetic University of Calgary, Canada resonance imaging (rs-fMRI) is a promising approach to characterize and potentially *Correspondence: Kilian Abellaneda-Pérez optimize the brain networks subtending cognition that changes as a function of age. kilian.abellaneda@ub.edu However, whether multifocal NIBS approaches are able to modulate rs-fMRI brain David Bartrés-Faz dynamics in aged populations, and if these NIBS-induced changes are consistent with dbartres@ub.edu the simulated electric current distribution on the brain remains largely unknown. In the Received: 14 June 2021 present investigation, thirty-one cognitively healthy older adults underwent two different Accepted: 02 November 2021 multifocal real transcranial direct current stimulation (tDCS) conditions (C1 and C2) and Published: 26 November 2021 a sham condition in a crossover design during a rs-fMRI acquisition. The real tDCS Citation: Abellaneda-Pérez K, conditions were designed to electrically induce two distinct complex neural patterns, Vaqué-Alcázar L, Perellón-Alfonso R, either targeting generalized frontoparietal cortical overactivity (C1) or a detachment Solé-Padullés C, Bargalló N, Salvador R, Ruffini G, Nitsche MA, between the frontal areas and the posteromedial cortex (C2). Data revealed that the two Pascual-Leone A and Bartrés-Faz D tDCS conditions modulated rs-fMRI differently. C1 increased the coactivation of multiple (2021) Multifocal Transcranial Direct functional couplings as compared to sham, while a smaller number of connections Current Stimulation Modulates Resting-State Functional Connectivity increased in C1 as compared to C2. At the group level, C1-induced changes were in Older Adults Depending on topographically consistent with the calculated electric current density distribution. At the the Induced Current Density. Front. Aging Neurosci. 13:725013. individual level, the extent of tDCS-induced rs-fMRI modulation in C1 was related with doi: 10.3389/fnagi.2021.725013 the magnitude of the simulated electric current density estimates. These results highlight Frontiers in Aging Neuroscience | www.frontiersin.org 1 November 2021 | Volume 13 | Article 725013 Abellaneda-Pérez et al. Multifocal tDCS Modulation of rs-fMRI in Aging that multifocal tDCS procedures can effectively change rs-fMRI neural functioning in advancing age, being the induced modulation consistent with the spatial distribution of the simulated electric current on the brain. Moreover, our data supports that individually tailoring NIBS-based interventions grounded on subject-specific structural data might be crucial to increase tDCS potential in future studies amongst older adults. Keywords: aging, electric current density, multifocal transcranial direct current stimulation, resting-state functional magnetic resonance imaging, non-invasive brain stimulation (NIBS), electric modeling, neuroimaging INTRODUCTION system rather than a concrete region. In this vein, a whole network modulation is particularly relevant in aging, since, The human brain is organized into complex neural networks as observed in numerous descriptive fMRI investigations, that can be studied through resting-state functional magnetic brain networks are less integrated and more segregated resonance imaging (rs-fMRI; Damoiseaux et al., 2006; Smith as a function of age (Cao et al., 2014; Chan et al., 2014; et al., 2009; van den Heuvel and Sporns, 2013; Petersen and Sala-Llonch et al., 2014; Grady et al., 2016; Spreng et al., Sporns, 2015). Some of these neural systems have been shown 2016), probably reflecting age-associated dedifferentiation to support cognition (Bressler and Menon, 2010) and change processes (Park et al., 2012). Notably, it has been proposed through the lifespan (Dosenbach et al., 2010; Betzel et al., 2014), within the cognitive neuroscience of aging literature that this as well as to be highly susceptible to aging (Tomasi and Volkow, loss of brain networks integration in the elderly relates to 2012; Ferreira and Busatto, 2013; Sala-Llonch et al., 2015; Nashiro poorer cognitive performance (i.e., Sala-Llonch et al., 2014; et al., 2017). Vidal-Piñeiro et al., 2014). Consequently, altering multiple Non-invasive brain stimulation (NIBS) protocols have been neural nodes within or between specific neural circuits used to modulate these functional systems subtending cognition based on a previously simulated electrical pattern on the in older adults (Abellaneda-Pérez et al., 2019a). The combination brain could be used to potentially restore a regular brain of NIBS with neuroimaging techniques in these populations has functioning and thus, hypothetically, ameliorate cognitive shed light into the network plasticity mechanisms underlying decline in advanced age. cognitive aging (i.e., Abellaneda-Pérez et al., 2019b) as well as The present study leverages from a previous investigation on the putative neurobiological mechanisms underlying NIBS- by our group and is based on its fMRI findings. In the induced phenotypic improvements in the elderly (i.e., Holland stated report, we observed that distinct groups of older adults, et al., 2011; Meinzer et al., 2013; Antonenko et al., 2018; with comparable educational attainment but different levels of Nilakantan et al., 2019). white matter hyperintensities burden, can engage dissimilar In the latest years, transcranial direct current stimulation brain activity patterns to successfully solve a particular working (tDCS) has been widely employed, particularly in advancing memory paradigm (for further details on the original brain age (Perceval et al., 2016; Tatti et al., 2016). In conventional patterns, see Fernández-Cabello et al., 2016). In the present tDCS studies, which aim to target discrete cortical regions, study, two real tDCS conditions were designed based on a single anode accompanied by its corresponding cathode is those previous fMRI findings to induce two distinct electrical used. According to our current mechanistic understanding, distribution configurations, which were expected to produce during tDCS, neural membrane potentials are depolarized under either generalized frontoparietal cortical overactivity (i.e., the anode, leading to an increase in cortical excitability, condition 1, or C1) or an antero-posterior dissociation aiming while they are hyperpolarized under the cathode, thus to enhance frontal areas whereas reducing the posteromedial diminishing cortical excitability at the macroscopic level cortex activity (i.e., condition 2, or C2; see section “tDCS (Nitsche and Paulus, 2000; Nitsche et al., 2008). More parameters” for detailed information). It is worth noting that recently, novel multifocal or network-based tDCS protocols there is a correspondence between those brain functional have been developed in order to target multiple brain configurations observed during activation and at rest (Smith areas simultaneously (Ruffini et al., 2014). In multifocal et al., 2009). Hence, while C1 patterns are consistent with the tDCS, multiple electrodes with differential intensities and classical frontoparietal circuits observed in rs-fMRI studies, the polarities are employed such that the resulting field aims C2 anatomically resemble both the frontoparietal as well as to maximally target a specific distributed brain network the default-mode networks (see also Abellaneda-Pérez et al., and can result in higher modulatory efficacy than an 2020). The goals of the present study were: (I) to explore the otherwise similar two-electrode tDCS approach in younger impact of two distinct multifocal tDCS montages on rs-fMRI samples (Fischer et al., 2017). However, in what manner associated connectivity changes in older adults; (II) to examine multifocal tDCS entails capability to influence rs-fMRI the topographical correspondence between the tDCS-induced rs- dynamics in the aging brain, and whether this modulation fMRI effects and the simulated electric current distribution on is consistent with a previously targeted neural pattern, has the brain at the group level; and (III) to individually determine not been previously investigated, despite the potential of whether, and how, the observed rs-fMRI changes are associated network-based approaches to directly modulate a complex with the calculated electric current values. Frontiers in Aging Neuroscience | www.frontiersin.org 2 November 2021 | Volume 13 | Article 725013 Abellaneda-Pérez et al. Multifocal tDCS Modulation of rs-fMRI in Aging MATERIALS AND METHODS duration of the experimental sessions was ∼ 40 min, whereof the first ∼ 5 min were used for tDCS-MRI setting preparation, Participants and the remaining ∼ 35 min were entirely conducted within As in our previous studies (Vidal-Piñeiro et al., 2014; Vaqué- the MRI scanner. Out of the total in-scanner time, the initial Alcázar et al., 2017, 2020), subjects participating in the present 25 min were acquired during brain stimulation, and the final investigation were recruited from the Fundació Institut Català variable minutes were dedicated to additional acquisitions. In de l’Envelliment. We contacted thirty-seven subjects and initially the experimental sessions, multifocal tDCS was applied to all included thirty-three participants fulfilling the following criteria: participants using the two different montages referred above (i.e., aged older than 65 and neuropsychological assessment within C1 and C2) with real stimulation as well as a sham stimulation range of normality (see below). Selected exclusion criteria condition (see section “tDCS parameters” for further details). were Hamilton Depression Rating Scale > 13, history of The order of the experimental sessions was counterbalanced. epilepsy, neurological or psychiatric disorders and any NIBS- There was a minimum one-month wash-out between each related contraindication (Rossi et al., 2009, 2021; Antal et al., experimental session to avoid possible prevalence of after- 2017) as well as for the MRI. One subject was discarded effects (Figure 1). Further, a questionnaire of tDCS-related for a previous single episode of absence seizure and another adverse events was administered at the end of each experimental volunteer was excluded due to morphine pump implantation to session and compared between the experimental conditions treat chronic pain. [all p-values > 0.05; for further details see Supplementary Finally, thirty-one subjects met criteria to participate in this Table 1 and Supplementary Material]. In addition, a very brief study. All participants were tDCS naïve and right-handed older assessment of the quality of sham was administered (for further adults [mean age ± standard deviation (SD), 71.68 ± 2.5 details, see Supplementary Material). years; age range, 68 – 77 years; 19 females; years of education mean ± SD, 12.29 ± 4.0 years]. All volunteers provided informed Neuropsychological Assessment consent in accordance with the Declaration of Helsinki (1964, A comprehensive battery of neuropsychological tests covering last revision 2013). All study procedures were approved by the all cognitive domains was administered, including the Rey- Institutional Review Board (IRB 00003099) at the University Osterrieth Complex Figure (ROCF), Rey Auditory Verbal of Barcelona. For all participants, MRI images were examined Learning Test (RAVLT), Boston Naming Test (BNT), Semantic by a senior neuroradiologist for any clinically significant category evocation of animals, Number location and incomplete pathology (none found). letters from the Visual Object and Space Perception Battery (VOSP), Trail Making Test (TMT), parts A and B, Phonemic fluency (FAS), Stroop Color Word Test, Symbol Digit Modalities Experimental Design Test (SDMT), and Digit span forward and backward from The present study was conducted in a randomized single- WAIS-III. Finally, the Vocabulary Subtest from WAIS-III was blind sham-controlled crossover design that consisted of four also administered to have a measure of premorbid intelligence. visits to our center. On the first visit (i.e., pre-experimental All participants presented a normal cognitive profile with session; day 0), all participants underwent a comprehensive mini-mental state examination (MMSE) scores of ≥27 and neuropsychological assessment to ensure cognitive functioning performance scores not more than 1.5 SD below normative within the normal range according to age and years of education data (adjusted for age and years of education) on any of the (see section “Neuropsychological assessment”). Subsequently, administered neuropsychological tests (i.e., they did not fulfill three experimental sessions with distinct multifocal tDCS the criteria for mild cognitive impairment; Petersen and Morris, conditions (days 1, 2, and 3) were conducted while acquiring 2005). MRI data during brain stimulation. These MRI acquisitions comprised, firstly, an arterial spin labeling (ASL) dataset (∼ 6 min), secondly, an rs-fMRI acquisition (∼ 8 min), and finally, Transcranial Direct Current Stimulation a task-based fMRI dataset (∼ 11 min). The present study Parameters focuses on rs-fMRI images to investigate in which manner the Two distinct multifocal tDCS montages were designed with simulated electric current distributions are associated with the the Stimweaver montage optimization algorithm (Ruffini et al., actual experimentally induced effects on the aged brain when 2014). The latter determines the positions and currents of the not engaged in any particular task. Furthermore, on day 1, electrodes over the scalp that induce an electric field in the brain a high-resolution three-dimensional (hr-3D; ∼ 8 min) dataset that better approximates a weighted target electric field map. We was acquired for functional data preprocessing purposes and optimized for the electric field component normal (orthogonal, to estimate the distribution of the induced electric current. In En ) to the cortical surface, assuming a first order model for the addition, the mentioned hr-3D acquired on day 1, and a fluid- interaction of the electric field with neurons in the cortex: when attenuated inversion recovery (FLAIR) sequence obtained on En points into/out of the cortical surface (positive/negative values day 2 (∼ 3 min), were used for neuroradiological assessment to of En in our convention), this leads to an increase/decrease in the exclude any brain structural abnormalities in study participants. membrane potential of the soma of pyramidal cells (and hence, Finally, a diffusion tensor imaging was obtained on day 3 (∼ cortical excitability). As mentioned, the weighted target En -maps 9 min; not used in this investigation). Therefore, the total used in this study were designed based on the findings obtained in Frontiers in Aging Neuroscience | www.frontiersin.org 3 November 2021 | Volume 13 | Article 725013 Abellaneda-Pérez et al. Multifocal tDCS Modulation of rs-fMRI in Aging FIGURE 1 | Study protocol and timeline of the procedures accomplished before and during multifocal tDCS-MRI. On the pre-experimental session, all the subjects undertook a neuropsychological screening session to verify a normal cognitive functioning. Then, during the three consecutive experimental sessions, transcranial stimulation was delivered using differential tDCS montages (i.e., C1, C2 and sham) in a counterbalanced fashion while rs-fMRI data was acquired. A hr-3D and a FLAIR datasets were acquired for additional examinations. tDCS, transcranial direct current stimulation; MRI, magnetic resonance imaging; rs-fMRI, resting-state functional MRI; hr-3D, high-resolution three-dimensional; FLAIR, fluid-attenuated inversion recovery; C1, condition 1; C2, condition 2. our previous fMRI study (Fernández-Cabello et al., 2016). More MRI acquisition. The current was delivered to each electrode precisely, on the one hand, the C1 montage was grounded on an with a wireless neurostimulator (Starstim, by Neuroelectrics fMRI pattern of extended cortical activity, including the bilateral Barcelona) connected to a computer via Bluetooth. For safety middle frontal gyri, the paracingulate gyri, the precuneus cortex, issues, the maximum current delivered by any electrode was the bilateral supramarginal gyri and/or intraparietal sulcus area 2 mA, while the maximum current injected through all the and the lingual gyri (Figure 2A). On the other hand, the C2 electrodes was 4 mA. In the real intervention conditions, the montage was derived from a second fMRI pattern including current was supplied during the whole rs-fMRI acquisition, moderate activity increases in the bilateral middle frontal and starting just before the ASL acquisition and concluding at the the paracingulate gyri, altogether with brain activity decreases end of the task-based fMRI dataset. In all groups, the current was of the posterior cingulate gyrus, the ventral precuneus, and the initially increased and finally decreased in a 30 s ramp-up and precentral gyri (Figure 2B). In the optimization, the regions that ramp-down fashion, carefully configured to not overlap with the registered a brain activity increase/decrease were targeted with MRI acquisitions. For the sham condition, the current dosage was an excitatory/inhibitory (positive/negative) target En field (see composed of an initial ramp-up of 30 s immediately followed by Figure 2C for C1, and Figure 2D for C2). Cortical maps of the a 1 min ramp-down, and a final ramp-down of 30 s immediately magnitude of the induced electric field, in which we focused in preceded by a ramp-up of 1 min. this investigation, are shown framed in Figure 2E for C1 and in Figure 2F for C2, in the template head model used in the optimization (Colin27; Miranda et al., 2013). Stimulation was Magnetic Resonance Imaging delivered via an MRI-compatible Starstim Neuroelectrics device, Acquisition using 8 circular MRI Sponstim electrodes with an area of 8 cm2 . All participants were scanned with a Siemens Magnetom Trio These MRI-compatible electrodes consist of a carbon rubber Tim Syngo 3 Tesla system at the MRI Core Facility (IDIBAPS) core and a sponger cover, both translucid materials. Hence, the of the Hospital Clinic of Barcelona, Barcelona, Spain. In the core electrodes were located on the participant’s scalp by fitting succeeding, only the acquisitions used in this investigation, both them inside the sponge and into the holes of a neoprene cap for research purposes and clinical screening, are detailed. Three corresponding to the 10/10 international system for electrode identical rs-fMRI datasets [T2∗ -weighted GE-EPI sequence; placement. The central Cz position was aligned to the vertex of interleaved acquisition; repetition time (TR) = 2,700 ms; the head in every subject to ensure an accurate cap placement. echo time (TE) = 30 ms; 40 slices per volume; slice Based upon the foregoing, in the C1 montage the electrodes thickness = 3.0 mm; interslice gap = 15%; voxel size = 3.0 x were placed in the AF7, F4, FC5, P3, P4, P7, P8 and Cz positions 3.0 x 3.0 mm; field of view (FOV) = 216 mm; 178 volumes] (see Figure 2G and Table 1), while in the C2 montage the were acquired, one each experimental day. Furthermore, a electrodes were placed in the AF3, C3, C4, F4, FC6, Fpz, Oz and hr-3D structural dataset [T1-weighted magnetization-prepared Cz positions (see Figure 2H and Table 1). For sham stimulation, rapid gradient-echo (T1-weighted MPRAGE); sagittal plane either C1 or C2 montages were randomly used. The stimulator acquisition; TR = 2,300 ms; TE = 2.98 ms, inversion time was situated outside the MRI room and the electrodes were (IT) = 900 ms; slice thickness = 1.0 mm; voxel size = 1.0 soaked with saline solution and a thin layer of Ten20 conductive x 1.0 x 1.0 mm; FOV = 256 mm; 240 slices] was acquired paste to ensure good conductivity and stability throughout the on experimental day 1. In addition, an axial FLAIR sequence Frontiers in Aging Neuroscience | www.frontiersin.org 4 November 2021 | Volume 13 | Article 725013 Abellaneda-Pérez et al. Multifocal tDCS Modulation of rs-fMRI in Aging FIGURE 2 | Multifocal tDCS montages. Targeted fMRI patterns in (A) C1 and (B) C2, from Fernández-Cabello et al. (2016, adapted with permission). Distribution of the En field in the cortical surface in (C) C1 and (D) C2 (in V/m units). Distribution of the electric field’s magnitude in the cortical surface in (E) C1 and (F) C2 (in V/m units). Representation of the electrode locations and shape on the scalp (left) and the gray matter (right) based on a Montreal Neurological Institute (MNI) head in (G) C1 and (H) C2, obtained for visual purposes. fMRI, functional magnetic resonance imaging; C1, condition 1; C2, condition 2. (TR = 9,000 ms; TE = 96 ms; slice thickness = 3.0 mm; Functional Connectivity Preprocessing FOV = 240 mm; 40 slices) was obtained on experimental day 2 Resting-state functional magnetic resonance imaging (rs-fMRI) (see Figure 1). data preprocessing included the removal of the first five volumes, motion correction, skull stripping, spatial smoothing [Full Width Functional Connectivity Analyses at Half Maximum (FWHM) = 7 mm], grand mean scaling and The FMRIB Software Library (FSL; version 6.001 ) and the filtering with both high-pass and low-pass filters (0.01- and 0.1- Analysis of Functional NeuroImages (AFNI2 ) were used for Hz thresholds, respectively). Data were then regressed with six preprocessing and analyzing functional neuroimaging data. rigid-body realignment motion parameters, mean white matter, and mean cerebrospinal fluid signal. No global signal regression 1 http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/ was used. Normalization to MNI standard space was also applied. 2 https://afni.nimh.nih.gov/ A visual inspection of the preprocessed rs-fMRI images was Frontiers in Aging Neuroscience | www.frontiersin.org 5 November 2021 | Volume 13 | Article 725013 Abellaneda-Pérez et al. Multifocal tDCS Modulation of rs-fMRI in Aging TABLE 1 | Electrode positions and current intensities in C1 and C2. These head models contain representations of the scalp, skull, cerebrospinal fluid (including the ventricles), eyeballs, gray- C1 C2 matter and white-matter. Second, the electrode positions (i.e., AF7: 363 µA AF3: 864 µA the center coordinates of the modeled electrodes) were placed on F4: 539 µA C3: 665 µA each subject head mesh, according to the locations established for FC5: 385 µA C4: 1,291 µA each montage (i.e., C1 and C2). Then, electric current simulations P3: 1,493 µA F4: 1,179 µA were computed for each condition separately. Following the P4: 1,220 µA FC6: −966 µA specific characteristics of the MRI Sponstim electrodes from P7: −1,705 µA Fpz: −998 µA Neuroelectrics, the electrode shape was set as elliptical, and the P8: −1,525 µA Oz: −396 µA size was defined as 2.3 cm of diameter and 1 mm of thickness. Cz: −770 µA Cz: −1,639 µA The electrode’s sponge size was defined as 3.2 cm of diameter and 3 mm of thickness. Tissue and electrode conductivity values were set as default in SimNIBS software (Thielscher et al., 2011; thoughtfully completed before conducting further functional Saturnino et al., 2015). Third, individual results were averaged connectivity analyses. Moreover, as head movement may affect together for each condition, resulting in group averages and rs-fMRI results (Power et al., 2012, 2015; Van Dijk et al., 2012), SDs of the electric current density distribution. Finally, for in-scanner head motion was calculated for every subject. More each montage, data on the peak fields (99.9th percentile) and precisely, two standard measures to estimate in-scanner head the electric current magnitude values within the selected ROIs, motion were obtained in a similar manner as described elsewhere based on fMRI findings, were obtained (see sections “Statistical (Power et al., 2012). Displacement relative to a single reference analyses” and “Multifocal tDCS effects on rs-fMR” for further volume (absolute displacement) and relative to the precedent information). The magnitude of the current density (normJ) volume (relative displacement) were calculated for every subject. was used in all the subject-based analyses of this investigation In our sample, no significant differences were found between (i.e., those computed with SimNIBS). Current density seems the three conditions (i.e., C1, C2 and sham), considering both to be particularly useful for dosage determination in terms absolute and relative displacement (all p-values > 0.05; for of the regional quantity of current reaching the brain with further data, see Supplementary Table 2). advancing age (i.e., Indahlastari et al., 2020). Hence, if not otherwise specified (i.e., as in the originally designed models Regions of Interest-Based Functional Connectivity using a template brain), figures and analysis results reflect the Analyses electric current density in all cases, and are expressed as amperes Functional connectivity analyses were implemented based on a per square meter (A/m2 ). Due to technical issues during the whole-brain atlas that parcels the brain into a set of anatomical generation of the head mesh, related to poor quality of the regions of interest (ROIs). The selected atlas was the one T1 MRI data, a subject was discarded from these analyses. developed for the CONN toolbox (Whitfield-Gabrieli and Nieto- The segmentations quality from all generated brain images was Castanon, 2012). This atlas includes a rich set of regions to individually examined and deemed appropriate. perform comprehensive whole-brain analyses using ROI-based approaches. More specifically, this atlas includes 132 ROIs, Statistical Analyses combining the FSL Harvard-Oxford cortical (91 ROIs) and Data analyses were performed using IBM SPSS (IBM Corp. subcortical atlases (15 ROIs) and the cerebellar areas from Released 2017. IBM SPSS Statistics for Windows, Version 25.0. the Anatomical Automatic Labeling (AAL) atlas (26 ROIs). Armonk, NY: IBM Corp.) and MATLAB (Version R2019a, The Individualized time-series of the different ROIs were extracted MathWorks Inc., Natick, MA, United States). from the preprocessed and regressed images. In order to obtain a resting-state functional connectivity (rs-FC) measure for each Functional Connectivity Statistical Analyses ROI-to-ROI connection in each subject, the acquired ROI time- Resting-state functional connectivity (rs-FC) correlation series were correlated with one another to create correlation matrices, permutation testing and pixel correction for multiple matrices, using Pearson product-moment correlations. comparisons were performed using custom made MATLAB scripts. Functional connectivity differences were compared Electric Current Computations between conditions using non-parametric permutation testing SimNIBS 3.0.7 was used to individually calculate the electrical (Nichols and Holmes, 2002). We chose this method because it current induced by tDCS based on the finite element method does not rely on assumptions about the distribution of the data (FEM) and individualized head models derived from the and correction methods for multiple comparisons can be easily structural MRI datasets (3 Windhoff et al., 2013; Thielscher implemented (Theiler et al., 1992). et al., 2015). First, T1-weighted anatomical images were used Time-series data for each subject, ROI and condition to create individualized tetrahedral FE head meshes of each were concatenated into a 4D array (i.e., subject x time- subject, using MATLAB toolboxes (Nielsen et al., 2018), MeshFix point x ROI x condition). Then, correlations between all ROIs for (Attene, 2010), and Gmsh (Geuzaine and Remacle, 2009). each subject and condition were computed, taking time-points as individual observations in each ROI-to-ROI correlation. This 3 www.simnibs.org resulted in three correlation matrices (i.e., one for each condition; Frontiers in Aging Neuroscience | www.frontiersin.org 6 November 2021 | Volume 13 | Article 725013 Abellaneda-Pérez et al. Multifocal tDCS Modulation of rs-fMRI in Aging FIGURE 3 | Rs-fMRI analyses. (A) Mean correlation matrices for (left to right): C1, C2 and sham. (B) Mean difference matrices for (left to right): C1 – Sham, C2 – Sham, C1 – C2. (C) Thresholded and pixel-corrected matrices for (left to right): C1 – Sham, C2 – Sham, C1 – C2 (dark: sub-threshold; yellow: supra-threshold; red: pixel-corrected). Bar-plots showing the mean with standard error of the mean (SEM) of rs-FC comparing (D) C1 vs. Sham and (E) C1 vs. C2, computed for visually inspecting the group tendencies in each coupling. Representation of the significant connections for (F) C1 vs. Sham and (G) C1 vs. C2 on a standard map (left to right: axial, sagittal and coronal view). Note that in Panel (F), selected main hubs (i.e., those ROIs entailing ≥2 significant rs-FC connections when comparing C1 vs. Sham) are displayed with a correspondingly larger size to emphasize them. C1, condition 1; C2, condition 2; toMTG, Middle temporal gyrus, temporo-occipital part; Cereb, Cerebellum; Ver, Vermis; PT, Planum temporale; Amy, Amygdala; pSMG, Supramarginal gyrus, posterior division; pPaHC, Parahippocampal gyrus, posterior division, pSTG, Superior temporal gyrus, posterior division; SCC, Supracalcarine cortex; LG, Lingual gyrus; L, left; R, right; A, anterior; P, posterior. Figure 3A) with three dimensions each (i.e., ROI x ROI x subject). and compared with the null-hypotheses distribution generated Then, differences between the means of the correlation matrices, by randomly shuffling the condition labels over subjects and for each pair of conditions and across subjects, were computed repeating this procedure for 1000 iterations (Figure 3C, α = 0.05, for each ROI x ROI correlation matrix point (Figure 3B) supra-threshold: in yellow). The resulting comparison matrices Frontiers in Aging Neuroscience | www.frontiersin.org 7 November 2021 | Volume 13 | Article 725013 Abellaneda-Pérez et al. Multifocal tDCS Modulation of rs-fMRI in Aging were corrected for multiple comparisons using pixel correction When contrasting C1 against C2, two connections were detected (Cohen, 2014; Figure 3C, pixel-corrected: in red). This procedure to be significantly different (Figures 3E,G). These results consists in picking the largest and smallest test statistic values of represented occipital-cerebellar couplings. Particularly, we each permutation -which results in two distributions of extreme observed significant modification of the connections between values- and then setting the thresholds for statistical significance the right lingual gyrus (LG.R) and the right supracalcarine to be the values corresponding to the 97.5 percentile of the largest cortex (SCC.R) and the seventh lobule of the vermis (Ver7). No value and the 2.5 percentile of the smallest value. Pixel correction differences were observed between C2 and sham. was preferred as compared to the more common cluster-based methods because the nature of our data (i.e., correlation matrices) Electric Current Simulations does not require that significant differences are spatially clustered. The means and SDs of electric current density distributions induced by C1 and C2 are displayed in Figure 4A (for C1) and Electric Current and Further Statistical Examinations Figure 4B (for C2). Individually modeled electric current density Furthermore, differences regarding the magnitude of the peak distributions are displayed in Supplementary Figure 1 (for C1) fields (99.9th percentile) between C1 and C2 were compared and Supplementary Figure 2 (for C2). The electric current using a paired sample t-test. Additional paired sample t-tests density distribution induced by C1 predominantly included the were used to test for electric current magnitude differences inferior parietal lobule as well as temporal and occipital regions. between experimental conditions for each of the main ROIs In contrast, the electric current density induced by C2 showed a selected based on rs-fMRI analyses. ROIs were selected and more anteriorly centered distribution principally encompassing considered as main hubs when they involved ≥ 2 significant the precentral, superior and, in a lesser extent, the middle frontal rs-FC couplings that survived multiple comparisons using pixel gyri. Moreover, as expected for the C2, the electric current correction. Moreover, Pearson product-moment correlation was magnitude within the posteromedial and occipital areas was very used to explore the association between tDCS-induced rs- low. As can be further observed, the originally designed patterns fMRI effects and the magnitude of calculated current density created with the Colin27 template and the average simulated estimates in the selected ROIs. More specifically, tDCS-induced configurations obtained with our aged individuals were entirely functional changes in each particular coupling that survived pixel anatomically consistent (see Figures 2E,F, 4A,B). correction were linked to the selected ROIs’ electric current Furthermore, C1 reached statistically significant higher peak magnitude values when this fell within the rs-FC connection. fields (99.9th percentile) as compared to C2 (t = 10.716; p < 0.001; Finally, to compare head movement and tDCS-related adverse Figure 4C). Additionally, the electric current magnitude values events differences between the experimental groups, a one-way extracted from the three main ROIs identified on the rs-fMRI repeated measures ANOVA was used. In these analyses, data analyses (i.e., toMTG.L, toMTG.R and PT.R), were, as expected, distribution was tested for normality with the Shapiro-Wilk test significantly higher in C1 when compared to C2 (toMTG.L: (p > 0.05; Shapiro and Wilk, 1965; Razali and Wah, 2011). Non- t = 19.986, p < 0.001; toMTG.R: t = 18.473, p < 0.001; PT.R: parametric tests were used in cases where parametric tests were Wilcoxon signed-rank test, Z = 4.000; p < 0.001; Figure 4D). not appropriate. These non-parametric tests are explicitly stated when necessary. No adjustment for multiple comparisons was applied in these exploratory statistical analyses. All these tests Associations Between Resting-State were two-tailed and α was set at 0.05. Functional Magnetic Resonance Imaging Modulation and Induced Electric Current Lastly, correlation analyses between tDCS-induced rs-fMRI RESULTS changes and the magnitude of the calculated current density values were performed. We observed that those subjects who Multifocal Transcranial Direct Current showed increased coactivation in C1 compared to sham in the Stimulation Effects on Resting-State toMTG.R – Ver3 coupling also presented lower induced electric Functional Magnetic Resonance Imaging current magnitude estimates in C1 (r = −0.401, p = 0.028; The two tDCS conditions differently influenced rs-fMRI Figure 4E). In addition, those subjects who showed higher connectivity. When comparing C1 against sham, some coactivation at C1 compared to sham in the toMTG.R – Cereb9.L specific connections (i.e., a total of 10 resting-state couplings) also presented a negative association with the induced electric significantly increased their coactivation (Figures 3D,F). Many current magnitude values in C1, however, this association was not of the affected connections involved temporal and temporo- statistically significant (r = −0.358, p = 0.052; Figure 4F). Thus, occipital areas and distinct cerebellar regions. In particular, it was observed that, although in the majority of cases (67.7% for three temporal areas emerged as main hubs (i.e., those with ≥ 2 the toMTG.R – Ver3 coupling; 64.5% for toMTG.R – Cereb9.L significant rs-FC couplings). These regions were the left and coupling) an increase in coactivation happened with certain right temporo-occipital middle temporal gyri (toMTG.L and levels of induced current, in those subjects with higher current toMTG.R, respectively) and the right planum temporale (PT.R), density estimates, a functional coactivation reduction most likely which fall in the posterior part of the temporal lobe. Remarkably, occurred (see Figures 4E,F). Of note, since no significant C1 was also able to modulate structures entailing the limbic differences were observed between C2 and sham, we focused system, such as the amygdala and the hippocampal formation. these analyses to C1, which was significantly different from sham. Frontiers in Aging Neuroscience | www.frontiersin.org 8 November 2021 | Volume 13 | Article 725013 Abellaneda-Pérez et al. Multifocal tDCS Modulation of rs-fMRI in Aging FIGURE 4 | Multifocal tDCS simulated electric current distributions. Anatomical pattern of the electric current density mean (top) and SD (bottom) for all subjects in (A) C1 and (B) C2 (in A/m2 units). (C) Comparison between C1 and C2 of the mean magnitude of the peak fields (99.9th percentile; in A/m2 units). (D) Comparison between C1 and C2 of the electric current magnitude extracted from the three main hubs detected on the rs-fMRI analyses (i.e., toMTG.L, toMTG.R and PT.R; in A/m2 units). (E,F) Scatter plots showing the associations between multifocal tDCS-induced changes in rs-fMRI connectivity estimates and electric current magnitude values (in A/m2 units). Data in (C,D) are presented with mean with SEM. C1, condition 1; C2, condition 2; toMTG, Middle temporal gyrus, temporo-occipital part; PT, Planum temporale; Ver, Vermis; Cereb, Cerebellum; L, left; R, right; Diff, difference. DISCUSSION calculated electric current estimates, which might be in line with the stochastic resonance hypothesis (see below). This represents, to the best of our knowledge, the first In this investigation, we observed that multifocal tDCS study investigating the impact of two distinct multifocal tDCS can modulate brain dynamics as measured through rs-fMRI montages on rs-fMRI in healthy aging. Our results showed that: connectivity amongst older adults. Furthermore, network-based (I) multifocal tDCS modulates rs-FC in a montage-dependent tDCS appears to anatomically modulate functional connectivity manner in older adults. (II) Moreover, the functional impact is in a manner dependent with the simulated electric current consistent with the spatial distribution of the induced electric distribution. In this vein, the impact of transcranial stimulation current on the brain. (III) Finally, specific individual tDCS- was particularly evident when comparing C1 to sham. Three induced rs-fMRI responses are related with the magnitude of the temporal regions emerged as the principal modulated regions: the Frontiers in Aging Neuroscience | www.frontiersin.org 9 November 2021 | Volume 13 | Article 725013 Abellaneda-Pérez et al. Multifocal tDCS Modulation of rs-fMRI in Aging left and right toMTG and the right PT. A relevant modulatory magnitude estimates within the designated main ROIs. We effect between these cortical nodes and the cerebellum was observed that, in specific functional couplings, the higher detected, though the modulation of cerebellar areas was less the magnitude of calculated current values, the lower the specific. Moreover, in other cortical temporo-parietal areas, coactivation increment. Thus, it is conceivable that aged such as the posterior divisions of the superior temporal individuals with dissimilar neuroanatomical characteristics and the supramarginal gyri, rs-FC was also modulated. In (which relates to distinct simulated current density parameters) addition, the connectivity of subcortical regions, such as the may require differential stimulation intensities to result in amygdala and the hippocampal formation, which are core optimal modulations of functional brain connectivity. The areas of the limbic system, was also modified. Of note, hypothesis of the stochastic resonance might provide a this system has been associated with emotion, motivation, mechanistic explanation of this state-dependency observation. and memory (Morgane et al., 2005), which are processes According to Polanía et al. (2018), the stochastic resonance of particular significance in advancing age (i.e., Liu et al., is a phenomenon referring to a situation in which a signal 2020). In the present investigation, the main rs-fMRI results that is too weak to be detected by a sensor might be when contrasting C1 against sham (i.e., considering the enhanced by adding an optimal level of noise. This assumption main hubs; Figure 3F) topographically correspond with the proposes that in a non-linear system, as the brain is, there estimated electric current distribution on the brain, as the are optimal noise levels for neural processing, and only largest electric current values in C1 were observed also in intermediate, but not high or low levels of noise, can led the inferior parietal and particular temporo-occipital brain to higher discriminability of the signal of interest. In this regions (see Figure 4A). Furthermore, these results were vein, the presence of neural noise (that can be added by consistent with the position of the electrodes that delivered means of NIBS) might confer to neurons more sensitivity to the highest stimulation intensities in C1 (see Figure 2G a given range of weak inputs, thereby rendering the signal and Table 1), namely P3 and P4 (with positive polarity), stronger, or even synchronized (for a review, see Miniussi as well as P7 and P8 (with negative polarity). Interestingly, et al., 2013). This phenomenon has been reported in previous P3 and P4 electrode positions in the international 10-10 experimental NIBS studies, both using transcranial magnetic system are anatomically located above the inferior parietal stimulation (TMS; Abrahamyan et al., 2011) and conventional lobule and principally encompass the Brodmann’s area 39 tDCS (Peña-Gómez et al., 2011). More precisely, Abrahamyan (BA39; Koessler et al., 2009). Moreover, the P7 and P8 et al. (2011) demonstrated that visual sensitivity can be improved electrodes are situated above the middle occipital and inferior with the right amounts of noise induced by means of TMS temporal gyri and mainly include Brodmann’s areas 37 and to the visual cortex. Further, in a previous study from our 19, respectively (BA37 and BA19; Koessler et al., 2009). group, it was observed that distinct personality traits (introverts Hence, the highest intensity electrodes (regardless of the vs. extraverts), which might entail distinct baseline levels of polarity) topographically covered the temporo-parieto-occipital brain activity, responded behaviorally different to the same junction (i.e., De Benedictis et al., 2014). In consequence, tDCS protocol. The authors explained these results within the in the C1 montage, we observed, at the group level, a stochastic resonance paradigm, claiming that introverts, with clear topographical association between the electrode’s assembly higher levels of intrinsic neural activity, could have reached and injected current intensities, the corresponding calculated the threshold more easily than extraverts with a relatively electric current density distribution in the cortical surface, weak electrical stimulation (see Peña-Gómez et al., 2011). and the neuroimaging results obtained with rs-fMRI data in Our data, using multifocal tDCS, appear to be consistent our experimental design. Of note, no clear multifocal tDCS with the hypothesis that specific amounts of current density effects were observed at the rs-fMRI level in C2, potentially are required to produce an optimal neural effect (i.e., an because the delivered stimulation was not sufficient to induce increase in functional coactivation). On the contrary, both an observable functional connectivity modulation in the studied not delivering enough electrical current in the targeted brain aging brains (for a visual inspection, compare Figures 4A,B, and structures (i.e., as could have occurred in C2), as well as a see Figures 4C,D plots). supplying supposedly excessive electrical current, could result In previous literature, considerable inter-individual variability in suboptimal neural effects (i.e., a functional coactivation in response to distinct NIBS protocols has been observed reduction). In this vein, our results shows that the induced (i.e., Hamada et al., 2013; López-Alonso et al., 2014; Wiethoff electric current magnitude estimates, which are contingent to et al., 2014). These reports highlight the importance of the individual head and brain anatomy (Thielscher et al., 2011; identifying the individual predictors of NIBS effects, particularly Miranda et al., 2013), might entail a feasible predictive value in older adults, where substantial attempts have been made regarding NIBS effects in aging when considered within this noise to modulate and optimize brain function and the associated generation hypothesis. cognitive performance (i.e., Antonenko et al., 2018; Nilakantan Altogether, our data show that multifocal non-invasive et al., 2019). In the present study we focused on the stimulation models and protocols are capable to effectively magnitude of the simulated electric current density as a modulate precise fMRI configurations in older adults. These potential factor contributing to such variability. In this vein, observations may have important future implications for the we investigated whether dissimilar individual differences in cognitive neuroscience of aging field, since many of the neural rs-fMRI modulation were related to the electric current basis regarding cognitive functioning, longitudinal trajectories Frontiers in Aging Neuroscience | www.frontiersin.org 10 November 2021 | Volume 13 | Article 725013 Abellaneda-Pérez et al. Multifocal tDCS Modulation of rs-fMRI in Aging and inter-individual differences, including the development of the elderly. Moreover, this functional modulation is related theoretical models, have been mainly based on studies employing with the simulated electric current distribution on the brain. this imaging technology (Grady, 2012; Cabeza et al., 2018; Vaqué- Thus, applying network-based procedures might entail a Alcázar et al., 2020). Furthermore, fMRI changes can effectively novel feasible approach to accurately target and modulate track the positive impact of behavioral interventions aimed to specific fMRI networks and connections critically involved ameliorate cognition in the elderly (Duda and Sweet, 2020), in the cognitive aging process. Further, we have shown that including its combined effects with NIBS (Antonenko et al., the estimated magnitude of current density is a relevant 2018). Therefore, and as opposed to the use of conventional tDCS factor accounting for the individual variability to NIBS montages, the possibility of a priori designing particular NIBS- in aged populations. Gathering knowledge about these based interventions of regional-specific fMRI patterns may offer variables will allow us to ultimately refine the parameters a valuable and refined approach for studies intended to optimize of transcranial stimulation to boost the brain and the potential complex brain configurations amongst older individuals, or to cognitive benefits derived from NIBS-based interventions investigate the impact of interventions in clinical trials. Moreover, in advanced age. the obtained tDCS results at the individual level are aligned with the notion that an exhaustive neuroanatomical characterization is critical to determine the right amount of stimulation required DATA AVAILABILITY STATEMENT to produce optimal neurophysiological effects in each subject. The raw data and data processing code supporting the conclusions of this article will be made available by the authors, LIMITATIONS without undue reservation. The present study is not without limitations. First, it is worth noting that the present investigation focused on the large-scale ETHICS STATEMENT brain networks that hypothetically sustain cognitive aging, The studies involving human participants were reviewed and but not in cognitive performance itself. Hence, forthcoming approved by the Ethics Committee of the University of Barcelona studies should make an effort to unify the various levels of (IRB 00003099). The patients/participants provided their written brain description (i.e., cells, networks, behavior) toward a informed consent to participate in this study. comprehensive characterization of brain–behavior relationships in advanced age. Furthermore, although the observed results in fMRI data are unlikely to be due to poor control of the AUTHOR CONTRIBUTIONS experimental conditions, our study could have improved methodologically by several different means, for instance, by KA-P, LV-A, and DB-F designed and conducted the study and using a sham condition for each real stimulation condition. prepared the manuscript draft with important intellectual input Moreover, it is worth noting that our original modelings from RP-A, CS-P, NB, RS, GR, MN, and AP-L. KA-P and LV-A designed with Stimweaver were based on the template Colin27. completed the data collection. KA-P, LV-A, and RP-A performed Models based on aged brains or on individual MRIs could have the data analysis. All authors reviewed the manuscript. improved the multifocal tDCS montage for both conditions. Furthermore, even though our individually generated models topographically matched the originally designed, future FUNDING experimental investigations should incorporate both a T1- and a T2-weigthed acquisitions to optimize the electric current This work was supported by grants from the Spanish Ministry simulation procedures. Further in this line, it is pertinent for T1 of Economy and Competitiveness (MINECO/FEDER; PSI2015- images to be acquired without the stimulation cap assembled 64227-R) and the Spanish Ministry of Science, Innovation to avoid potential confounders in the segmentation process. and Universities (MICIU/FEDER; RTI2018-095181-B-C21) Finally, it must be recognized that in all simulation-based to DB-F, which was also supported by an ICREA Academia electric current planning systems, there is uncertainty about the 2019 grant award. KA-P was supported by a postdoctoral precise conductivity values that should be used for the different fellowship associated with the MICIU/FEDER; RTI2018- tissues and materials when creating and analyzing a model for a 095181-B-C21 grant. LV-A was supported by a postdoctoral particular montage (see for further detail Miranda et al., 2013; fellowship associated with the MINECO/FEDER PSI2015- Saturnino et al., 2019). Notwithstanding, notable advances are 64227-R grant (reference number, BES-2016-077620). MN was being achieved in this vein (Huang et al., 2017). supported by the German Ministry of Education and Research (TRAINSTIM, grant 01GQ1424E). AP-L was supported by the Sidney R. Baer Jr. Foundation, the National Institutes of Health CONCLUSION (NIH R01 MH100186, R01 NS073601, R01 HD069776, R21 MH099196, R21 NS082870, R21 NS085491, and R21 HD07616), Present results highlight that multifocal electrical stimulation Harvard Catalyst | The Harvard Clinical and Translational protocols are capable of modulating neural dynamics in Science Center (NCRR and the NCATS NIH, UL1 RR025758), Frontiers in Aging Neuroscience | www.frontiersin.org 11 November 2021 | Volume 13 | Article 725013 Abellaneda-Pérez et al. Multifocal tDCS Modulation of rs-fMRI in Aging DARPA (via HR001117S0030) and the Football Players ACKNOWLEDGMENTS Health Study at Harvard University. The project that gave rise to these results also received the support of a We are indebted to the Magnetic Resonance Imaging fellowship from “la Caixa” Foundation (ID 100010434). Core Facility (IDIBAPS) and the SimNIBS developers for The fellowship code is LCF/BQ/DI19/11730050. 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Brain Imaging Behav. 11, 318–332. doi: 10.1007/s11682-016-9584-8 The remaining authors declare that the research was conducted in the absence of Vidal-Piñeiro, D., Valls-Pedret, C., Fernández-Cabello, S., Arenaza-Urquijo, E. M., any commercial or financial relationships that could be construed as a potential Sala-Llonch, R., Solana, E., et al. (2014). Decreased default mode network conflict of interest. connectivity correlates with age-associated structural and cognitive changes. Front. Aging Neurosci. 6:256. doi: 10.3389/fnagi.2014.00256 Publisher’s Note: All claims expressed in this article are solely those of the authors Whitfield-Gabrieli, S., and Nieto-Castanon, A. (2012). Conn: a functional and do not necessarily represent those of their affiliated organizations, or those of connectivity toolbox for correlated and anticorrelated brain networks. Brain the publisher, the editors and the reviewers. Any product that may be evaluated in Connect. 2, 125–141. doi: 10.1089/brain.2012.0073 this article, or claim that may be made by its manufacturer, is not guaranteed or Wiethoff, S., Hamada, M., and Rothwell, J. C. (2014). Variability in response to endorsed by the publisher. transcranial direct current stimulation of the motor cortex. Brain Stimul. 7, 468–475. doi: 10.1016/j.brs.2014.02.003 Copyright © 2021 Abellaneda-Pérez, Vaqué-Alcázar, Perellón-Alfonso, Solé- Windhoff, M., Opitz, A., and Thielscher, A. (2013). Electric field calculations in Padullés, Bargalló, Salvador, Ruffini, Nitsche, Pascual-Leone and Bartrés- brain stimulation based on finite elements: an optimized processing pipeline Faz. This is an open-access article distributed under the terms of the for the generation and usage of accurate individual head models. Hum. Brain Creative Commons Attribution License (CC BY). The use, distribution or Mapp. 34, 923–935. doi: 10.1002/hbm.21479 reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication Conflict of Interest: RS and GR works for Neuroelectrics. MN serves on the in this journal is cited, in accordance with accepted academic practice. No scientific advisory board for Neuroelectrics and NeuroDevice. AP-L serves use, distribution or reproduction is permitted which does not comply with on the scientific advisory boards for Starlab Neuroscience, Neuroelectrics, these terms. Frontiers in Aging Neuroscience | www.frontiersin.org 14 November 2021 | Volume 13 | Article 725013