Comparative Efficacy of Dual- vs. Single-Node tACS in Amnestic Mild Cognitive Impairment: Behavioral and EEG Evidence

Comparative Efficacy of Dual- vs. Single-Node tACS in Amnestic Mild Cognitive Impairment: Behavioral and EEG Evidence | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Comparative Efficacy of Dual- vs. Single-Node tACS in Amnestic Mild Cognitive Impairment: Behavioral and EEG Evidence Li Wang, Yuxian Li, Yiwen Jiang, Zhixin Piao, Lin Gu, Fenglin Zhu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7785019/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background The efficacy of current transcranial stimulation in cognitive disorders is limited by single-node intervention. Recent evidence indicates that amnestic mild cognitive impairment (aMCI) is associated with dysconnectivity in the frontoparietal network (FPN) and theta oscillations; modulating the FPN with theta-frequency stimulation represents a promising intervention for aMCI. Methods We developed a noninvasive transcranial alternating current stimulation (tACS) protocol for modulating long-range theta interactions within the FPN in aMCI patients. Thirty patients with aMCI were randomly assigned to 2 mA, 6 Hz, 25 min, and 10 sessions of dual-node tACS applied over right FPN (i.e., the DLPFC and the posterior parietal cortex) or single-node tACS over right DLPFC, followed by clinical visits at 4 weeks after treatment. Participants also undergone EEG recordings during resting state, 2-back working memory, and associative memory task before and after intervention. Results Compared with single-node stimulation, dual-node stimulation produced more sgnificant improvements in global cognition, as measured by Montreal Cognitive Assessment. Dual-node stimulation enhanced resting-state theta power in dorsolateral and midline prefrontal cortices. Furthermore, dual-node stimulation was also superior to single-site stimulation in improving memory performance and network dynamics, including theta-gamma phase-amplitude coupling in right dorsolateral prefrontal cortex during the working memory task and right frontal-to-parietal theta-phase synchronization during the associative memory task. Conclusion This study demonstrates behavioural benefits and neural mechanisms of dual-node stimulation in ameliorating cognitive impairment, providing a promising approach for achieving network-level intervention in cognitive disorders. Trial registration Chinese Clinical Trial Registry, ChiCTR2200058652 Registration Date Biological sciences/Neuroscience/Learning and memory Health sciences/Diseases Alzheimer’s disease Theta oscillations Transcranial alternating current stimulation Mild cognitive impairment Frontoparietal network Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Alzheimer’s disease (AD) is the most common neurodegenerative disorder, characterized by progressive cognitive decline. Amnestic mild cognitive impairment (aMCI) is widely regarded as the prodromal stage of AD, making effective intervention at this stage crucial for preventing the onset of dementia. However, clinical trials of pharmacological treatments have yielded disappointing results in slowing the progression from aMCI to AD [ 1 ]. Although cognitive training demonstrated some benefits, its application in aMCI populations is limited by the participants’ comprehension and compliance. There is an urgent need for innovative intervention. In recent years, non-invasive neuromodulation techniques have garnered increasing attention as potential intervention strategy for aMCI. Repetitive Transcranial Magnetic Stimulation (rTMS) and transcranial Direct Current Stimulation (tDCS) are two commonly used technologies. Studies applied high-frequency rTMS or anodal tDCS over the dorsolateral prefrontal cortex (DLPFC), posterior parietal cortex (PPC), or precuneus in aMCI patients, with mixed outcomes reported [ 2 – 4 ]. Recently, transcranial Alternating Current Stimulation (tACS) has emerged as a promising alternative after tDCS. In contrast to the excitatory or inhibitory effects of rTMS and tDCS, tACS modulates not only the amplitude but also the frequency and phase of neural oscillations, offering a unique tool to entrain endogenous brain rhythms [ 5 ]. Increased evidence suggests that tACS may boost cognitive function in healthy adults [ 6 , 7 ] and those with cognitive impairment [ 8 , 9 ]. In healthy individuals, theta-frequency tACS applied over the parietal cortex enhanced associative memory (AM) under high task demands, and gamma-tACS over the left DLPFC improved working memory (WM) performance more effectively than the tDCS [ 6 , 7 ]. A meta-analysis including 102 published studies in healthy aging and neuropsychiatric populations found modest-to-moderate improvements in several cognitive domains with tACS treatment, including WM, attention, executive control, and fluid intelligence [ 10 ]. However, these studies targeted a single node within either the prefrontal or parietal cortex, which may be a major reason limiting the efficacy. A plenty of neuroimaging evidence advances the understanding of human brain function from the perspective of large-scale neural networks [ 11 ]. The cognitive decline has been associated with functional and connectivity disturbances in multiple brain networks [ 12 , 13 ], which makes sense multiple-node network-level intervention to maximize therapeutic potential. The frontoparietal network (FPN), including the DLPFC and posterior parietal cortex, plays a central role in coordinating neural systems in response to task demands [ 14 , 15 ]. The FPN is consistently activated during various memory tasks [ 16 , 17 ], and a recent meta-analysis of neuroimaging studies highlighted altered FPN function in aMCI patients across multiple metrics, including low-frequency oscillations, regional homogeneity, and functional connectivity [ 18 ]. Further, a prior work of our group reported altered resting-state functional dynamics of the FPN in aMCI patients [ 19 ]. Together, these studies position the FPN as a promising target for network-level intervention for aMCI. Synchronization of neural oscillations across brain networks is essential for cognitive function [ 20 , 21 ]. Theta oscillations, in particular, facilitate both local processing and long-range communication, serving as a fundamental mechanism for memory processing [ 21 ]. Studies reported disturbed theta-band power spectrum in populations with aMCI and AD, as indexed by absolute and relative power, amplitude global field power, global field synchronization, and phase lag index (PLI) [ 22 , 23 ]. By leveraging the advantage of tACS in modulating phase relations between brain nodes [ 24 ], researchers employed theta in-phase tACS to regulate the frontoparietal or frontotemporal networks in healthy individuals, demonstrating superior performance in cognitive function and electrophysiological domains [ 25 , 26 ]. However, there is currently a lack of research on populations with cognitive impairment. Against this background, we conducted a randomized single-blind clinical trial to compare the therapeutic effects and related electrophysiological mechanisms underlying dual-node tACS applied over right FPN. Thirty individuals with aMCI were randomly assigned to receive either theta-band in-phase tACS over right FPN or single-node theta-band tACS over right DLPFC. Clinical ratings and neuropsychological tests were administered before and after tACS intervention. The EEG signals were recorded during resting state, 2-back WM task, and AM task to investigate treatment-related mechanisms. We hypothesized that theta-band tACS over the FPN would yield a stronger improved effect on cognitive function than the single-node stimulation, which may be mediated by enhanced network dynamics of the theta oscillations. Materials and methods Study design The study was conducted at the Beijing Institute of Technology Hospital between April 2022 and October 2023 (ChiCTR2200058652). Ethical approval was obtained from the Beijing Institute of Technology in accordance with the principles of the Declaration of Helsinki. All participants signed written informed consent. As illustrated in Fig. 1 A, 30 patients with aMCI were randomly assigned to either dual-node or single-node tACS. Each participant completed 10 tACS sessions within two consecutive weeks. Although a double-blind crossover design might be methodologically preferable, it was not adopted due to concerns regarding potential long-term cumulative effects after multiple tACS, as with the 10 sessions in this study, which complicate the definition of an appropriate washout period. Participants Key eligibility criteria were as follows: age between 65 and 85 years; right-handedness; a diagnosis of aMCI based on Petersen’s criteria [ 27 ]; a Mini-Mental State Examination (MMSE) score ≥ 24; a Montreal Cognitive Assessment (MoCA) score between 18 and 26; essentially preserved activities of daily living, as indicated by an Activity of Daily Living Scale (ADL) score > 26; a Clinical Dementia Rating (CDR) score of 0.5; no use of cognitive-enhancing medications within the preceding 6 weeks; and agreement to forgo any other psychoactive treatments during the study. Exclusion criteria comprised comorbid psychiatric disorders such as depression or schizophrenia; neurological conditions including epilepsy or Parkinson’s disease; significant visual or hearing impairment; history of neuromodulation treatment within the last 6 months; non-native Chinese speakers; or contraindications to tACS. This study aimed to compare the therapeutic effects of dual-node versus single-node stimulation in patients with aMCI. Based on a pilot study, the anticipated group difference for changes in MoCA scores following tACS treatment was 2.5, with a standard deviation of 2. Using a power (1 – β) of 0.8 and a significance level of 0.05, a minimum of 11 participants per group was required. To account for the potential loss to follow-up, the sample size was increased to 15 per group. tACS Intervention tACS was delivered using a transcranial electrical stimulator (Ruier Weikang, Hangzhou, China). The experimental group received dual-node theta-band in-phase tACS, while the control group received single-node theta-band tACS. Although studies in healthy individuals used single-session anti-phase tACS as a control condition to demonstrate modulatory effects by in-phase tACS, such an approach was ethically unsuitable for the current study, given that administering 10 sessions of anti-phase tACS to patients with cognitive impairment could potentially exacerbate cognitive decline. In the dual-node stimulation, two active electrodes (5×5 cm) were placed over the right DLPFC (F4) and the right PPC (P4) according to the 10–20 EEG system, and a larger reference electrode (10×10 cm) was positioned over the right shoulder (Fig. 1 B). The single-node stimulation was applied only in the right DLPFC. The right hemisphere was selected based on evidence that the memory tasks engage the FPN’s activity and connectivity predominantly in the right hemisphere [ 28 ]. Both the dual-node and single-node groups received 10 sessions of bipolar sinusoidal tACS at 6 Hz with a current intensity of ± 2 mA. Each session of tACS lasted 25 minutes and was administered five times per week over two consecutive weeks. Electrodes were affixed to the scalp using Ten20 conductive paste (Weaver and Company, Aurora, Colorado), and impedance was maintained below 15 kΩthroughout stimulation. For the dual-node stimulation, the alternating currents delivered to the two sites were synchronized with a relative phase difference of 0°(Fig. 1 C). Electric field distributions for both stimulation protocols were simulated using SimNIBS (Simulation of Non-Invasive Brain Stimulation; http://www.simnibs.org/ ) (Fig. 1 C, D). Participants were withdrawn from the study if they experienced severe adverse reactions, missed two or more consecutive sessions, failed to complete required assessments, or voluntarily requested to discontinue. EEG Recording The EEG signals were acquired using a 64-channel Ag/AgCl electrode cap and a DC-coupled amplifier system (SynAmps2, Neuroscan, Compumedics, Texas, USA). The ground electrode was positioned between FPz and Fz, and the reference electrode was placed between Cz and CPz. Data were sampled at 1000 Hz, and electrode impedance was kept below 10 kΩthroughout the recording. In this study, the EEG signals were recorded for patients before and after 10 treatment sessions during resting state, 2-back WM and AM tasks. Working Memory Task WM refers to the cognitive system responsible for temporarily maintaining and manipulating information, and is considered a fundamental component of higher-order cognitive functions [ 29 ]. The task consisted of 130 trials, with target stimuli accounting for 25% of the trials. In each trial, a black letter was presented on a white background for 0.5 s, followed by a fixation cross displayed for 2000 ms (Fig. 1 E). Participants were instructed to press a button whenever the current letter matched the letter presented two trials earlier. Response accuracy and reaction time were recorded. Associative Memory Task AM reflects the ability to form connections between unrelated items and store them as integrated representations, which is a core mechanism underlying episodic memory and plays a vital role in daily cognitive functioning [ 30 ]. In this study, AM was assessed using an object-word paired recall task, comprising encoding and retrieval phases (Fig. 1 F). During encoding, participants were presented with 20 grayscale object-word pairs on printed cards, each shown for approximately 3 seconds with a 500 ms inter-stimulus interval. Participants were instructed to memorize each object-word association. During retrieval, two cards were presented: each contained the same object paired with a different word. Participants were asked to identify the correct pairing based on the encoding phase. Data Preprocessing The EEG data were preprocessed using EEGLAB and custom MATLAB scripts. The signals were first re-referenced to the bilateral mastoids (M1 and M2). Resting- and task-state EEG data were bandpass-filtered at 1–40 Hz and 0.5–45 Hz, respectively. All the data were then downsampled to 250 Hz. The resting-state data were segmented into 5-second epochs, while the task-state data were epoched relative to stimulus onset, spanning from 0 to 2000 ms. Artifacts were removed via independent component analysis (ICA). Power Analysis Segmented EEG epochs were converted from the time domain to the frequency domain using Welch’s method. For each subject and electrode, spectral power within the theta band was summed for each epoch. Power values were then averaged across epochs for each electrode to enhance the signal-to-noise ratio. Subsequently, the power values were averaged for each subject and condition across electrodes within six regions of interest (ROIs): mid-frontal cortex (Fz, FCz), medial parietal cortex (CPz, Pz, POz), left prefrontal cortex (F1, F3, F5, AF3, FC3), right prefrontal cortex (F2, F4, F6, AF4, FC4), left parietal cortex (P1, P3, P5, CP5, PO5), and right parietal cortex (P2, P4, P6, CP4, PO4). These ROIs were selected based on established relevance to cognitive processes in prior literature [ 14 , 15 , 31 , 32 ]. Phase Synchronization Phase synchronization refers to the consistency of the phase angles between rhythmic neural signals originating from distinct cortical regions oscillating at a specific frequency. The Weighted PLI (wPLI) was used to quantify phase synchrony, accounting for non-zero phase lag interactions between signals while mitigating the effects of volume conduction [ 33 ]. In this study, resting- and task-state EEG data were filtered within the theta band (4–8 Hz), and phase information was extracted using the Hilbert transform. The cross-spectrum between electrode pairs was calculated for each epoch, and the wPLI values were averaged across trials to enhance the reliability. Higher wPLI values indicate stronger phase consistency between brain regions. The fronto-parietal connectivity was assessed by averaging the wPLI values between the frontal and parietal ROIs. Phase-Amplitude Coupling Phase-amplitude coupling (PAC) between low-frequency phase and high-frequency amplitude was quantified using the mean vector length (MVL) method [ 34 ], which yields a modulation index (MI). This approach measures PAC by assessing the extent to which high-frequency amplitude is modulated by the phase of a lower frequency rhythm. The MVL is defined as: where \(\:\theta\) is the total number of data points, \(\:\text{t}\) is a data point, \(\:{\text{a}}_{\text{t}}\) is the amplitude at data point, \(\:\text{t}\) and \(\:{\theta}_{\text{t}}\) is the phase angle at data point \(\:\text{t}\) . The theta-gamma PAC was computed at the electrode level by coupling the phase of theta activity (4–8 Hz, in 2 Hz steps) with the amplitude of high-gamma activity (30–45 Hz, in 2 Hz steps). The resulting values were aggregated across trials and time points for each subject and stimulation condition. Assessments and Outcomes The MoCA was used to evaluate global cognition, supplemented by a battery of neuropsychological tests to assess specific cognitive domains, including the Trail Making Test (TMT) Parts A and B to assess processing speed, attention, and cognitive flexibility, the Coding test to assess executive function, and the Stroop test to measure response inhibition. During the Stroop-Word test, participants were presented with color words printed in incongruent inks and asked to read the word (target) while ignoring the color (distractor). The total time taken to name all words was recorded. Conversely, in the Stroop-Color test, participants were required to name the ink color (target) while ignoring the word itself (distractor). The number of correct responses made within 90 seconds was recorded. At the end of the treatment, adverse events related to electrical stimulation—such as dizziness, headache, and scalp discomfort—were recorded. Participants were invited to complete a follow-up assessment of MoCA at four weeks after the intervention. Statistical Analysis Statistical analyses were conducted using SPSS (version 25.0; IBM Corp.). Demographic and clinical data were compared between groups: continuous variables were analyzed using independent-sample t-tests if normally distributed, or the Mann–Whitney U test otherwise; and categorical variables were compared using Fisher’s exact test. The effects of tACS on behavioral and neuroelectrophysiological measures were evaluated with simple contrasts (pre versus post stimulation) within each group using paired t tests or Wilcoxon signed-rank tests. To control for time-related confounds, we also performed double contrasts to compare the differences in pre- to post-intervention changes between the two groups using independent-sample t-tests or Mann–Whitney U tests, as appropriate. Pearson correlation analysis was then performed to examine the relationships between changes in EEG-derived metrics and improvements in behavioral and electrophysiological measures. Results Participants Among the 40 patients initially recruited, 30 met the eligibility criteria and were enrolled into the study. One participant from each group discontinued the intervention due to adverse effects (head tension or sleep disturbance). One patient in the dual-node group and four in the single-node group withdrew due to loss of interest or COVID-19-related lockdown restrictions. Finally, a total of 13 patients in the dual-node group and 10 in the single-node group completed all the tACS sessions. Seventeen participants (10 in the dual-node group and 5 in the single-node group) were lost to the 4-week follow-up due to pandemic-related constraints. Given the substantial attrition during follow-up, only pre- and post-treatment data were included. Among the 23 patients who completed the treatment, valid resting-state EEG data were available for 9 in the dual-node group and 8 in the single-node group. For the WM task, usable EEG data were obtained from 8 participants per group. For the AM task, data from 8 participants per group were included in the analysis. The two groups were balanced in demographic or clinical characteristics (Table 1). Treatment effects on cognitive function and memory performance There were no significant differences between the dual-node and single-node groups on baseline clinical or neuropsychological measures (Table 1). Regarding global cognition, the MoCA scores were increased in the dual-node (versus single-node) group from pre- to post-stimulation (t = 2.482, p = 0.022). Specifically, the dual-node group increased significantly in the MoCA scores after tACS (t = 5.148, p < 0.001), while no change was observed in the single-node group (t = 2.236, p = 0.052) (Fig. 2 A). For specific cognitive domains, the dual-node group improved in the Stroop-Word test (Stroop-Word: T = 12, p = 0.034), Stroop-Color test (Stroop-Color: t = 3.504, p = 0.004), and number completed in the Coding test (t = 2.198, p = 0.048), which, however, failed to survive the double contrast (Fig. 2 B-D). The two groups did not differ at baseline behavioral performance for the WM task (accuracy: p = 0.535; reaction time: p = 0.623) and the AM task (recall accuracy: p = 0.072; reaction time: p = 0.385). Simple contrasts indicated improved WM accuracy after dual-node stimulation (T = 5, p = 0.005) but no change in the single-node group (t = 0.054, p = 0.958), which, however, failed to survive the double contrast (Fig. 2 E). The dual-node group also exhibited an increase in recall accuracy during the AM task (T = 0, p < 0.001), which was again absent in the single-node group (t = 0, p = 1.000). A double contrast confirmed that this increase was specific to the dual-node group (t = 3.822, p < 0.001) (Fig. 2 F). Treatment effects on resting-state theta power and connectivity Topographic distributions of theta power before and after tACS treatment are shown in Fig. 3 A. Dual-node group demonstrated increased theta power over the right DLPFC electrodes after tACS (T = 4, p = 0.028), while the single-node group showed no changes (T = 12, p = 0.401). A double contrast confirmed the specific increase in the dual-node (versus single-node) group (t = 2.452, p = 0.027) (Fig. 3 B). The theta power in the mid-frontal cortex showed an increased trend after dual-node stimulation (T = 8, p = 0.086), which was again absent in the single-node group (T = 14, p = 0.575); this increase was specific to the dual-node group (t = 2.277, p = 0.038) (Fig. 3 C). The dual-node group increased in the right frontoparietal wPLI (T = 5, p = 0.022) but not the single-node group (t = 0.749, p = 0.478), which, however, failed to survive the double contrast (Fig. 3 D). Treatment effects on theta activity during the WM task The dual-node group (t = 2.595, p = 0.036), but not the single-node group (t=-0.226, p = 0.827), increased in the wPLI values for right frontal-to-parietal ROI connectivity during the WM task, which, however, failed to survive the double contrast (Fig. 4 A). For cross-frequency coupling, the dual-node group demonstrated increased theta-gamma PAC in the right DLPFC electrodes after tACS (t = 2.759, p = 0.028), which was absent in the single-node group (T = 17, p = 0.889); the double contrast confirmed a specific increase in the dual-node group (t = 2.784, p = 0.015) (Fig. 4 B). Treatment effects on theta activity during the AM task During the AM task, the wPLI values for right frontoparietal connectivity was increased in the dual-node group (T = 4, p = 0.049) but not in the single-node group (t=-1.046, p = 0.330), which survived the double contrast (t = 2.784, p = 0.015) (Fig. 4 C). The theta-gamma PAC (MVL-MI) in right DLPFC electrodes was elevated in the dual-node group (t = 3.683, p = 0.008) but remained unchanged in the single-node group (T = 11, p = 0.327), which, however, failed to survive the double contrast (Fig. 4 D). Behavioral-neurophysiological correlations Although within-group analyses did not reveal significant correlations between changes in behavioral measures and electrophysiological indicators, several correlations showed statistical significance when data from both groups were combined. Specifically, increased theta-gamma PAC in the right prefrontal electrodes was positively correlated with improved accuracy on the WM task (R = 0.59, p = 0.016) (Fig. 5 A). Increased wPLI values for right frontoparietal theta-phase synchronization were positively correlated with improvements in MoCA scores (R = 0.80, p < 0.001) and AM task accuracy (R = 0.55, p = 0.028), but was negatively correlated with Trail Making Test-Part B (TMT-B) completion time (R=-0.68, p = 0.0034) (Fig. 5 B-D). Discussion This study investigated the therapeutic effects of dual-node network-targeted tACS versus single-node intervention in patients with aMCI. The results demonstrate that dual-node tACS targeting the right DLPFC and PPC significantly improved global cognition compared with single-node stimulation applied solely over the right DLPFC. These behavioral benefits were accompanied by improvements in neurophysiological measures, including resting-state theta power, frontoparietal connectivity, and theta-gamma PAC during memory tasks. Dual-node stimulation led to a greater improvement in global cognition relative to single-node stimulation, as measured by the MoCA. This finding aligns with the established role of the FPN in supporting executive control and memory functions [ 14 – 17 ]. Prior studies applying rTMS or tDCS to individual nodes within the prefrontal or parietal cortex [ 2 – 4 ] have demonstrated limited efficacy in aMCI patients. Neuroimaging evidence showed the FPN disconnection in AD and aMCI [ 18 ], making network-targeted stimulation a more scientifically grounded approach for cognitive enhancement than the single-node intervention. Although research in clinical populations remains vacant, the superiority of dual-node network-targeted to single-node interventions has been confirmed in improving memory performance among healthy individuals [ 26 ]. Of note, in this study, the group differences in clinical improvements was statistically significant in global cognition rather than in isolated cognitive domains, suggesting the global cognition a more sensitive indicator of stimulation effects. In terms of neurophysiological outcomes, the dual-node group exhibited a more significant increase after tACS in resting-state theta power over the right DLPFC and the mid-frontal cortex compared with the single-node group. These results indicate that, at the dimension of resting-state spectral power, the FPN-targeted intervention primarily affects the PFC functioning. The PFC is involved in a range of cognitive processes including memory and executive control, with dysfunction implicated in various cognition-related disorders [ 35 ]. Theta oscillations originate from the mid-frontal cortex and reflect a common computational mechanism underlying cognitive control [ 32 ]. Theta-tACS applied over the mid-frontal cortex has enhanced memory consolidation [ 36 ], and in-phase theta stimulation between the mid-frontal cortex and the DLPFC increased inter-regional phase synchronization and cognitive control ability in healthy volunteers [ 37 ]. Therefore, we posit that that dual-node stimulation applied over the FPN may exert effects not only on the targeted prefrontal regions but also propagate to upstream cortical areas involved in cognitive control. During the WM task, we observed a preferential increase in theta-gamma PAC at the right prefrontal electrodes areas following dual-node stimulation. The increase in theta-gamma PAC suggests that modulating slower rhythmic activity within the FPN can regulate faster, local oscillatory processes associated with memory function. The PAC reflects the supervisory influence of large-scale network oscillations operating at lower frequencies over localized high-frequency activity [ 38 ]. Theta-gamma PAC is a fundamental mechanism for integrating localized neural information into a coherent stream that supports complex memory processes [ 38 , 39 ]. Impairments in this coupling have been documented in memory disorders including AD and aMCI [ 40 ], indicating its potential as an intervention target. In healthy volunteers, in-phase theta-tACS applied over the DLPFC-PPC [ 41 ] or frontotemporal [ 26 ] pathways preferentially enhanced theta-gamma PAC compared with anti-phase dual-node or single-node stimulation. The positive correlation between increased PAC and improved WM accuracy further supports its role as a quantitative indicator of WM enhancement. Notably, increased theta-gamma PAC were observed specifically during task performance but not at rest, suggesting that the stimulation effects are state-dependent and may become more evident when the cognitive resources are engaged. Dual-node stimulation was superior to single-site stimulation in improving AM performance and right frontal-to-parietal theta-phase synchronization during the AM task. Moreover, the increase in theta phase synchronization was positively correlated with improvements in AM recall accuracy and MoCA scores, but negatively correlated with shorter completion times on the TMT-B. AM decline is a recognized marker of cognitive impairment and an early sign of AD [ 42 ]. Age-related cognitive decline has been linked to reduced cortical information transmission, exemplified by diminished theta phase synchronization [ 43 ]. While studies of transcranial electrical stimulation (tES) targeted the prefrontal or temporal regions to improve AM in older healthy or cognition-impaired adults, results have been inconsistent in treatment efficacy [ 3 ]. In contrast, although fewer studies applied parietal stimulation, most of them report beneficial effects on AM [ 3 ], which may suggest a stronger memory enhancement effect for the parietal stimulation than those of the frontotemporal cortex. Successful associative retrieval recruits the PPC [ 44 ]. Recent work applying high-frequency rTMS to the left PPC that showed strongest functional connectivity with the hippocampus demonstrated enhanced AM, hippocampal network connectivity, and parietal activity during retrieval [ 30 , 45 ]. Based on the importance of the PPC in AM, we propose that synchronous stimulation over the FPN may produce reinforcement effects on the network dynamics than single-node stimulation by leveraging the functional role of the parietal cortex. The correlations between increases in right frontal-parietal theta phase synchronization and performance in cognitive function (MoCA, AM accuracy, and TMT-B) may suggest that a better synchronization within the FPN leads to more cognitive benefits, further indicating the importance of network-level intervention. Several issues should be considered in future research. Larger sample sizes and the inclusion of a sham control group will be helpful. An ideal control might incorporate anti-phase stimulation; however, ethical considerations arise when applying such protocols to cognitively impaired populations. Longitudinal follow-ups with regular assessments on cognitive function were required to determine the long-term duration of treatment effects. Furthermore, neuromodulation may be more effective when is personalized to entertain intrinsic brain rhythms and ongoing brain states. Future research should account for inter-individual variability in brain physiology and anatomy. The development of individualized tACS protocols is expected to improve the efficacy of dual-site interventions. In conclusion, this study demonstrates that modulating the FPN functioning by theta-band in-phase stimulation rather than a local region is more effective for cognitive disorders. These findings support the potential of network-targeted dual-node tACS as a non-invasive therapeutic approach for cognitive impairment. Abbreviations AD, Alzheimer’s dementia; tACS, transcranial alternating current stimulation; rTMS, repetitive transcranial magnetic stimulation; tDCS, transcranial direct current stimulation; DLPFC, dorsolateral prefrontal cortex; WM, working memory; FPN, fronroparietal network; AM, associative memory; MMSE, Mini Mental State Examination; MoCA, Montreal Cognitive Assessment; CDR, Clinical Dementia Rating Scale; ICA, independent component analysis; wPLI, weighted phase lag index; MVL, mean vector length; MI, modulation index; PAC, phase-amplitude coupling; TMT, Trail Making Test. Declarations Acknowledgements The authors would like to thank the participants in this trial. Author contributions Li Wang: Conceptualization, Investigation, Methodology, Project administration, Funding acquisition, Supervision, Writing original draft, Writing-review & editing. Zhixin Piao, Hongfang Su: Project administration. Yiwen Jiang: Data curation, Methodology. Anshun Kang, Fenglin Zhu: Methodology. Tianyi Yan: Funding acquisition. Funding This research was funded by the 2030 Major Project of the Ministry of Science and Technology (2022ZD0208500), the Beijing Institute of Technology Research Fund Program for Young Scholars (3160012222109), and the National Natural Science Foundation of China (U20A20191). Data availability The datasets used during the current study are available from the corresponding or first author upon reasonable request. Ethics approval and consent to participate Ethical approval was received from Beijing Institute of Technology (BIT-EC-H-2021112). The informed consent from all participants was obtained. Competing interests The authors declare no financial or non-financial interests. References Yu T-W, Lane H-Y, Lin C-H. Novel Therapeutic Approaches for Alzheimer’s Disease: An Updated Review. Int J Mol Sci 2021; 22 : 8208. Chou Y-H, Ton That V, Sundman M. 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Additional Declarations The authors have declared there is NO conflict of interest to disclose Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: revise 16 Dec, 2025 Review # 1 received at journal 01 Nov, 2025 Review # 2 received at journal 30 Oct, 2025 Reviewer # 2 agreed at journal 24 Oct, 2025 Reviewer # 1 agreed at journal 18 Oct, 2025 Reviewers invited by journal 13 Oct, 2025 Editor assigned by journal 08 Oct, 2025 Submission checks completed at journal 08 Oct, 2025 First submitted to journal 07 Oct, 2025 Unknown event 07 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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04:18:18","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":126632,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7785019/v1/47fd90bd119cba19b162b158.html"},{"id":94622205,"identity":"5b0c6401-358b-4143-a1c1-d1046ff1b409","added_by":"auto","created_at":"2025-10-29 04:18:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1067089,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResearch procedure.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: The flow chart of the experiment. B: For the dual-node stimulation, 6-Hz in-phase tACS was applied over the right dorsolateral prefrontal cortex (F4, 10-20 EEG system) and right posterior parietal cortex (P4, 10-20 EEG system). For the single-node stimulation, 6-Hz tACS was applied over the right dorsolateral prefrontal cortex alone. C, D: The current-flow models are shown on 3D reconstructions of the cortical surface. E: 2-back working memory (WM) task. F: Associative memory (AM) task.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7785019/v1/f31ef73512bc47efa2a529b0.png"},{"id":94622305,"identity":"bfee4f53-6210-4aa5-bbe2-14629ad43e41","added_by":"auto","created_at":"2025-10-29 04:18:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":132509,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreatment-related effects on clinical scales and neuropsychological tests.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-F: Bar chart shows the differences in clinical scales and neuropsychological test scores between and within the dual-node group and the single-node group before and after treatment, including the MoCA, Stroop-Word Test, Stroop-Color Test, Coding Test, 2-back working memory (WM) task, and associative memory (AM) task. n.s.: not significant. The data are presented as mean ± standard deviation, with individual values shown for each subject.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7785019/v1/bbf89fc59805a3c72375eed5.png"},{"id":94622386,"identity":"107c00d4-bb12-450d-a414-0ceb1b2b719c","added_by":"auto","created_at":"2025-10-29 04:18:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":518914,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreatment-related effects on resting-state theta-band activity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Topography of theta power before and after treatment. B-D: Bar charts show the differences between and within the dual-node group and the single-node group in theta power of the right dorsolateral prefrontal electrodes and mid-frontal cortex, and in the weighted phase lag index (wPLI) calculated between right frontal and parietal electrodes.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7785019/v1/acbb456545eb7f06b60074b9.png"},{"id":94622231,"identity":"c467a063-2ac5-474c-87aa-1c1a5ae24586","added_by":"auto","created_at":"2025-10-29 04:18:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":229611,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreatment-related effects on theta-band activity during the memory task.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-D: Bar charts show differences between and within the two groups in treatment-related changes in the wPLI between right frontal and parietal electrodes and the mean vector length - modulation index (MVL-MI) in the right prefrontal electrodes during working memory (WM) task and associative memory (AM) tasks.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7785019/v1/b84a1bfb4c9b0c71e6cc637b.png"},{"id":94622347,"identity":"bfe95af4-de56-42cd-ac1b-b348c6e9803b","added_by":"auto","created_at":"2025-10-29 04:18:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":224170,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBehavioral-electrophysiological correlations.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: The increase in theta-gamma phase-amplitude coupling (MVL-MI) of the right prefrontal electrodes was positively correlated with the improvement in WM accuracy rate. B-D: The increase in right frontal-to-parietal theta phase synchronization (wPLI) was positively correlated with the improvements in the MoCA and the accuracy rate of AM, and negatively correlated with the TMT-B scores. These correlations were calculated after integrating the two groups.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7785019/v1/9c5762b04a27bacb2b3d8daa.png"},{"id":94640455,"identity":"4dbd207a-669c-4a49-897d-8d6bf83ad7bb","added_by":"auto","created_at":"2025-10-29 07:49:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3233547,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7785019/v1/d004c749-37ba-4ab4-b303-8a166a5da022.pdf"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Comparative Efficacy of Dual- vs. Single-Node tACS in Amnestic Mild Cognitive Impairment: Behavioral and EEG Evidence","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlzheimer\u0026rsquo;s disease (AD) is the most common neurodegenerative disorder, characterized by progressive cognitive decline. Amnestic mild cognitive impairment (aMCI) is widely regarded as the prodromal stage of AD, making effective intervention at this stage crucial for preventing the onset of dementia. However, clinical trials of pharmacological treatments have yielded disappointing results in slowing the progression from aMCI to AD [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although cognitive training demonstrated some benefits, its application in aMCI populations is limited by the participants\u0026rsquo; comprehension and compliance. There is an urgent need for innovative intervention.\u003c/p\u003e\u003cp\u003eIn recent years, non-invasive neuromodulation techniques have garnered increasing attention as potential intervention strategy for aMCI. Repetitive Transcranial Magnetic Stimulation (rTMS) and transcranial Direct Current Stimulation (tDCS) are two commonly used technologies. Studies applied high-frequency rTMS or anodal tDCS over the dorsolateral prefrontal cortex (DLPFC), posterior parietal cortex (PPC), or precuneus in aMCI patients, with mixed outcomes reported [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Recently, transcranial Alternating Current Stimulation (tACS) has emerged as a promising alternative after tDCS. In contrast to the excitatory or inhibitory effects of rTMS and tDCS, tACS modulates not only the amplitude but also the frequency and phase of neural oscillations, offering a unique tool to entrain endogenous brain rhythms [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Increased evidence suggests that tACS may boost cognitive function in healthy adults [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and those with cognitive impairment [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In healthy individuals, theta-frequency tACS applied over the parietal cortex enhanced associative memory (AM) under high task demands, and gamma-tACS over the left DLPFC improved working memory (WM) performance more effectively than the tDCS [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. A meta-analysis including 102 published studies in healthy aging and neuropsychiatric populations found modest-to-moderate improvements in several cognitive domains with tACS treatment, including WM, attention, executive control, and fluid intelligence [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, these studies targeted a single node within either the prefrontal or parietal cortex, which may be a major reason limiting the efficacy.\u003c/p\u003e\u003cp\u003eA plenty of neuroimaging evidence advances the understanding of human brain function from the perspective of large-scale neural networks [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The cognitive decline has been associated with functional and connectivity disturbances in multiple brain networks [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], which makes sense multiple-node network-level intervention to maximize therapeutic potential. The frontoparietal network (FPN), including the DLPFC and posterior parietal cortex, plays a central role in coordinating neural systems in response to task demands [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The FPN is consistently activated during various memory tasks [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and a recent meta-analysis of neuroimaging studies highlighted altered FPN function in aMCI patients across multiple metrics, including low-frequency oscillations, regional homogeneity, and functional connectivity [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Further, a prior work of our group reported altered resting-state functional dynamics of the FPN in aMCI patients [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Together, these studies position the FPN as a promising target for network-level intervention for aMCI.\u003c/p\u003e\u003cp\u003eSynchronization of neural oscillations across brain networks is essential for cognitive function [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Theta oscillations, in particular, facilitate both local processing and long-range communication, serving as a fundamental mechanism for memory processing [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Studies reported disturbed theta-band power spectrum in populations with aMCI and AD, as indexed by absolute and relative power, amplitude global field power, global field synchronization, and phase lag index (PLI) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. By leveraging the advantage of tACS in modulating phase relations between brain nodes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], researchers employed theta in-phase tACS to regulate the frontoparietal or frontotemporal networks in healthy individuals, demonstrating superior performance in cognitive function and electrophysiological domains [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, there is currently a lack of research on populations with cognitive impairment.\u003c/p\u003e\u003cp\u003eAgainst this background, we conducted a randomized single-blind clinical trial to compare the therapeutic effects and related electrophysiological mechanisms underlying dual-node tACS applied over right FPN. Thirty individuals with aMCI were randomly assigned to receive either theta-band in-phase tACS over right FPN or single-node theta-band tACS over right DLPFC. Clinical ratings and neuropsychological tests were administered before and after tACS intervention. The EEG signals were recorded during resting state, 2-back WM task, and AM task to investigate treatment-related mechanisms. We hypothesized that theta-band tACS over the FPN would yield a stronger improved effect on cognitive function than the single-node stimulation, which may be mediated by enhanced network dynamics of the theta oscillations.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy design\u003c/h2\u003e\u003cp\u003eThe study was conducted at the Beijing Institute of Technology Hospital between April 2022 and October 2023 (ChiCTR2200058652). Ethical approval was obtained from the Beijing Institute of Technology in accordance with the principles of the Declaration of Helsinki. All participants signed written informed consent.\u003c/p\u003e\u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, 30 patients with aMCI were randomly assigned to either dual-node or single-node tACS. Each participant completed 10 tACS sessions within two consecutive weeks. Although a double-blind crossover design might be methodologically preferable, it was not adopted due to concerns regarding potential long-term cumulative effects after multiple tACS, as with the 10 sessions in this study, which complicate the definition of an appropriate washout period.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eParticipants\u003c/h3\u003e\n\u003cp\u003eKey eligibility criteria were as follows: age between 65 and 85 years; right-handedness; a diagnosis of aMCI based on Petersen\u0026rsquo;s criteria [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]; a Mini-Mental State Examination (MMSE) score\u0026thinsp;\u0026ge;\u0026thinsp;24; a Montreal Cognitive Assessment (MoCA) score between 18 and 26; essentially preserved activities of daily living, as indicated by an Activity of Daily Living Scale (ADL) score\u0026thinsp;\u0026gt;\u0026thinsp;26; a Clinical Dementia Rating (CDR) score of 0.5; no use of cognitive-enhancing medications within the preceding 6 weeks; and agreement to forgo any other psychoactive treatments during the study.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eExclusion criteria comprised\u003c/strong\u003e\u003cp\u003ecomorbid psychiatric disorders such as depression or schizophrenia; neurological conditions including epilepsy or Parkinson\u0026rsquo;s disease; significant visual or hearing impairment; history of neuromodulation treatment within the last 6 months; non-native Chinese speakers; or contraindications to tACS.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eThis study aimed to compare the therapeutic effects of dual-node versus single-node stimulation in patients with aMCI. Based on a pilot study, the anticipated group difference for changes in MoCA scores following tACS treatment was 2.5, with a standard deviation of 2. Using a power (1 \u0026ndash; β) of 0.8 and a significance level of 0.05, a minimum of 11 participants per group was required. To account for the potential loss to follow-up, the sample size was increased to 15 per group.\u003c/p\u003e\u003cp\u003e\u003cb\u003etACS Intervention\u003c/b\u003e\u003c/p\u003e\u003cp\u003etACS was delivered using a transcranial electrical stimulator (Ruier Weikang, Hangzhou, China). The experimental group received dual-node theta-band in-phase tACS, while the control group received single-node theta-band tACS. Although studies in healthy individuals used single-session anti-phase tACS as a control condition to demonstrate modulatory effects by in-phase tACS, such an approach was ethically unsuitable for the current study, given that administering 10 sessions of anti-phase tACS to patients with cognitive impairment could potentially exacerbate cognitive decline.\u003c/p\u003e\u003cp\u003eIn the dual-node stimulation, two active electrodes (5\u0026times;5 cm) were placed over the right DLPFC (F4) and the right PPC (P4) according to the 10\u0026ndash;20 EEG system, and a larger reference electrode (10\u0026times;10 cm) was positioned over the right shoulder (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The single-node stimulation was applied only in the right DLPFC. The right hemisphere was selected based on evidence that the memory tasks engage the FPN\u0026rsquo;s activity and connectivity predominantly in the right hemisphere [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBoth the dual-node and single-node groups received 10 sessions of bipolar sinusoidal tACS at 6 Hz with a current intensity of \u0026plusmn;\u0026thinsp;2 mA. Each session of tACS lasted 25 minutes and was administered five times per week over two consecutive weeks. Electrodes were affixed to the scalp using Ten20 conductive paste (Weaver and Company, Aurora, Colorado), and impedance was maintained below 15 kΩthroughout stimulation. For the dual-node stimulation, the alternating currents delivered to the two sites were synchronized with a relative phase difference of 0\u0026deg;(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Electric field distributions for both stimulation protocols were simulated using SimNIBS (Simulation of Non-Invasive Brain Stimulation; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.simnibs.org/\u003c/span\u003e\u003cspan address=\"http://www.simnibs.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D).\u003c/p\u003e\u003cp\u003eParticipants were withdrawn from the study if they experienced severe adverse reactions, missed two or more consecutive sessions, failed to complete required assessments, or voluntarily requested to discontinue.\u003c/p\u003e\n\u003ch3\u003eEEG Recording\u003c/h3\u003e\n\u003cp\u003eThe EEG signals were acquired using a 64-channel Ag/AgCl electrode cap and a DC-coupled amplifier system (SynAmps2, Neuroscan, Compumedics, Texas, USA). The ground electrode was positioned between FPz and Fz, and the reference electrode was placed between Cz and CPz. Data were sampled at 1000 Hz, and electrode impedance was kept below 10 kΩthroughout the recording. In this study, the EEG signals were recorded for patients before and after 10 treatment sessions during resting state, 2-back WM and AM tasks.\u003c/p\u003e\n\u003ch3\u003eWorking Memory Task\u003c/h3\u003e\n\u003cp\u003eWM refers to the cognitive system responsible for temporarily maintaining and manipulating information, and is considered a fundamental component of higher-order cognitive functions [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The task consisted of 130 trials, with target stimuli accounting for 25% of the trials. In each trial, a black letter was presented on a white background for 0.5 s, followed by a fixation cross displayed for 2000 ms (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Participants were instructed to press a button whenever the current letter matched the letter presented two trials earlier. Response accuracy and reaction time were recorded.\u003c/p\u003e\n\u003ch3\u003eAssociative Memory Task\u003c/h3\u003e\n\u003cp\u003eAM reflects the ability to form connections between unrelated items and store them as integrated representations, which is a core mechanism underlying episodic memory and plays a vital role in daily cognitive functioning [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In this study, AM was assessed using an object-word paired recall task, comprising encoding and retrieval phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). During encoding, participants were presented with 20 grayscale object-word pairs on printed cards, each shown for approximately 3 seconds with a 500 ms inter-stimulus interval. Participants were instructed to memorize each object-word association. During retrieval, two cards were presented: each contained the same object paired with a different word. Participants were asked to identify the correct pairing based on the encoding phase.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eData Preprocessing\u003c/h2\u003e\u003cp\u003eThe EEG data were preprocessed using EEGLAB and custom MATLAB scripts. The signals were first re-referenced to the bilateral mastoids (M1 and M2). Resting- and task-state EEG data were bandpass-filtered at 1\u0026ndash;40 Hz and 0.5\u0026ndash;45 Hz, respectively. All the data were then downsampled to 250 Hz. The resting-state data were segmented into 5-second epochs, while the task-state data were epoched relative to stimulus onset, spanning from 0 to 2000 ms. Artifacts were removed via independent component analysis (ICA).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePower Analysis\u003c/h3\u003e\n\u003cp\u003eSegmented EEG epochs were converted from the time domain to the frequency domain using Welch\u0026rsquo;s method. For each subject and electrode, spectral power within the theta band was summed for each epoch. Power values were then averaged across epochs for each electrode to enhance the signal-to-noise ratio. Subsequently, the power values were averaged for each subject and condition across electrodes within six regions of interest (ROIs): mid-frontal cortex (Fz, FCz), medial parietal cortex (CPz, Pz, POz), left prefrontal cortex (F1, F3, F5, AF3, FC3), right prefrontal cortex (F2, F4, F6, AF4, FC4), left parietal cortex (P1, P3, P5, CP5, PO5), and right parietal cortex (P2, P4, P6, CP4, PO4). These ROIs were selected based on established relevance to cognitive processes in prior literature [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003ePhase Synchronization\u003c/h3\u003e\n\u003cp\u003ePhase synchronization refers to the consistency of the phase angles between rhythmic neural signals originating from distinct cortical regions oscillating at a specific frequency. The Weighted PLI (wPLI) was used to quantify phase synchrony, accounting for non-zero phase lag interactions between signals while mitigating the effects of volume conduction [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In this study, resting- and task-state EEG data were filtered within the theta band (4\u0026ndash;8 Hz), and phase information was extracted using the Hilbert transform. The cross-spectrum between electrode pairs was calculated for each epoch, and the wPLI values were averaged across trials to enhance the reliability. Higher wPLI values indicate stronger phase consistency between brain regions. The fronto-parietal connectivity was assessed by averaging the wPLI values between the frontal and parietal ROIs.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ePhase-Amplitude Coupling\u003c/h2\u003e\u003cp\u003ePhase-amplitude coupling (PAC) between low-frequency phase and high-frequency amplitude was quantified using the mean vector length (MVL) method [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], which yields a modulation index (MI). This approach measures PAC by assessing the extent to which high-frequency amplitude is modulated by the phase of a lower frequency rhythm. The MVL is defined as:\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"187\" height=\"70\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\theta\\)\u003c/span\u003e\u003c/span\u003e is the total number of data points, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{t}\\)\u003c/span\u003e\u003c/span\u003e is a data point, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{a}}_{\\text{t}}\\)\u003c/span\u003e\u003c/span\u003e is the amplitude at data point, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{t}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\theta}_{\\text{t}}\\)\u003c/span\u003e\u003c/span\u003e is the phase angle at data point \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{t}\\)\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eThe theta-gamma PAC was computed at the electrode level by coupling the phase of theta activity (4\u0026ndash;8 Hz, in 2 Hz steps) with the amplitude of high-gamma activity (30\u0026ndash;45 Hz, in 2 Hz steps). The resulting values were aggregated across trials and time points for each subject and stimulation condition.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eAssessments and Outcomes\u003c/h2\u003e\u003cp\u003eThe MoCA was used to evaluate global cognition, supplemented by a battery of neuropsychological tests to assess specific cognitive domains, including the Trail Making Test (TMT) Parts A and B to assess processing speed, attention, and cognitive flexibility, the Coding test to assess executive function, and the Stroop test to measure response inhibition.\u003c/p\u003e\u003cp\u003eDuring the Stroop-Word test, participants were presented with color words printed in incongruent inks and asked to read the word (target) while ignoring the color (distractor). The total time taken to name all words was recorded. Conversely, in the Stroop-Color test, participants were required to name the ink color (target) while ignoring the word itself (distractor). The number of correct responses made within 90 seconds was recorded.\u003c/p\u003e\u003cp\u003eAt the end of the treatment, adverse events related to electrical stimulation\u0026mdash;such as dizziness, headache, and scalp discomfort\u0026mdash;were recorded. Participants were invited to complete a follow-up assessment of MoCA at four weeks after the intervention.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were conducted using SPSS (version 25.0; IBM Corp.). Demographic and clinical data were compared between groups: continuous variables were analyzed using independent-sample t-tests if normally distributed, or the Mann\u0026ndash;Whitney U test otherwise; and categorical variables were compared using Fisher\u0026rsquo;s exact test.\u003c/p\u003e\u003cp\u003eThe effects of tACS on behavioral and neuroelectrophysiological measures were evaluated with simple contrasts (pre versus post stimulation) within each group using paired t tests or Wilcoxon signed-rank tests. To control for time-related confounds, we also performed double contrasts to compare the differences in pre- to post-intervention changes between the two groups using independent-sample t-tests or Mann\u0026ndash;Whitney U tests, as appropriate. Pearson correlation analysis was then performed to examine the relationships between changes in EEG-derived metrics and improvements in behavioral and electrophysiological measures.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eParticipants\u003c/h2\u003e\u003cp\u003eAmong the 40 patients initially recruited, 30 met the eligibility criteria and were enrolled into the study. One participant from each group discontinued the intervention due to adverse effects (head tension or sleep disturbance). One patient in the dual-node group and four in the single-node group withdrew due to loss of interest or COVID-19-related lockdown restrictions. Finally, a total of 13 patients in the dual-node group and 10 in the single-node group completed all the tACS sessions. Seventeen participants (10 in the dual-node group and 5 in the single-node group) were lost to the 4-week follow-up due to pandemic-related constraints. Given the substantial attrition during follow-up, only pre- and post-treatment data were included.\u003c/p\u003e\u003cp\u003eAmong the 23 patients who completed the treatment, valid resting-state EEG data were available for 9 in the dual-node group and 8 in the single-node group. For the WM task, usable EEG data were obtained from 8 participants per group. For the AM task, data from 8 participants per group were included in the analysis. The two groups were balanced in demographic or clinical characteristics (Table\u0026nbsp;1).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eTreatment effects on cognitive function and memory performance\u003c/h2\u003e\u003cp\u003eThere were no significant differences between the dual-node and single-node groups on baseline clinical or neuropsychological measures (Table\u0026nbsp;1). Regarding global cognition, the MoCA scores were increased in the dual-node (versus single-node) group from pre- to post-stimulation (t\u0026thinsp;=\u0026thinsp;2.482, p\u0026thinsp;=\u0026thinsp;0.022). Specifically, the dual-node group increased significantly in the MoCA scores after tACS (t\u0026thinsp;=\u0026thinsp;5.148, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while no change was observed in the single-node group (t\u0026thinsp;=\u0026thinsp;2.236, p\u0026thinsp;=\u0026thinsp;0.052) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). For specific cognitive domains, the dual-node group improved in the Stroop-Word test (Stroop-Word: T\u0026thinsp;=\u0026thinsp;12, p\u0026thinsp;=\u0026thinsp;0.034), Stroop-Color test (Stroop-Color: t\u0026thinsp;=\u0026thinsp;3.504, p\u0026thinsp;=\u0026thinsp;0.004), and number completed in the Coding test (t\u0026thinsp;=\u0026thinsp;2.198, p\u0026thinsp;=\u0026thinsp;0.048), which, however, failed to survive the double contrast (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe two groups did not differ at baseline behavioral performance for the WM task (accuracy: p\u0026thinsp;=\u0026thinsp;0.535; reaction time: p\u0026thinsp;=\u0026thinsp;0.623) and the AM task (recall accuracy: p\u0026thinsp;=\u0026thinsp;0.072; reaction time: p\u0026thinsp;=\u0026thinsp;0.385). Simple contrasts indicated improved WM accuracy after dual-node stimulation (T\u0026thinsp;=\u0026thinsp;5, p\u0026thinsp;=\u0026thinsp;0.005) but no change in the single-node group (t\u0026thinsp;=\u0026thinsp;0.054, p\u0026thinsp;=\u0026thinsp;0.958), which, however, failed to survive the double contrast (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The dual-node group also exhibited an increase in recall accuracy during the AM task (T\u0026thinsp;=\u0026thinsp;0, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), which was again absent in the single-node group (t\u0026thinsp;=\u0026thinsp;0, p\u0026thinsp;=\u0026thinsp;1.000). A double contrast confirmed that this increase was specific to the dual-node group (t\u0026thinsp;=\u0026thinsp;3.822, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003cb\u003eTreatment effects on resting-state theta power and connectivity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTopographic distributions of theta power before and after tACS treatment are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. Dual-node group demonstrated increased theta power over the right DLPFC electrodes after tACS (T\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;=\u0026thinsp;0.028), while the single-node group showed no changes (T\u0026thinsp;=\u0026thinsp;12, p\u0026thinsp;=\u0026thinsp;0.401). A double contrast confirmed the specific increase in the dual-node (versus single-node) group (t\u0026thinsp;=\u0026thinsp;2.452, p\u0026thinsp;=\u0026thinsp;0.027) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The theta power in the mid-frontal cortex showed an increased trend after dual-node stimulation (T\u0026thinsp;=\u0026thinsp;8, p\u0026thinsp;=\u0026thinsp;0.086), which was again absent in the single-node group (T\u0026thinsp;=\u0026thinsp;14, p\u0026thinsp;=\u0026thinsp;0.575); this increase was specific to the dual-node group (t\u0026thinsp;=\u0026thinsp;2.277, p\u0026thinsp;=\u0026thinsp;0.038) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The dual-node group increased in the right frontoparietal wPLI (T\u0026thinsp;=\u0026thinsp;5, p\u0026thinsp;=\u0026thinsp;0.022) but not the single-node group (t\u0026thinsp;=\u0026thinsp;0.749, p\u0026thinsp;=\u0026thinsp;0.478), which, however, failed to survive the double contrast (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTreatment effects on theta activity during the WM task\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe dual-node group (t\u0026thinsp;=\u0026thinsp;2.595, p\u0026thinsp;=\u0026thinsp;0.036), but not the single-node group (t=-0.226, p\u0026thinsp;=\u0026thinsp;0.827), increased in the wPLI values for right frontal-to-parietal ROI connectivity during the WM task, which, however, failed to survive the double contrast (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). For cross-frequency coupling, the dual-node group demonstrated increased theta-gamma PAC in the right DLPFC electrodes after tACS (t\u0026thinsp;=\u0026thinsp;2.759, p\u0026thinsp;=\u0026thinsp;0.028), which was absent in the single-node group (T\u0026thinsp;=\u0026thinsp;17, p\u0026thinsp;=\u0026thinsp;0.889); the double contrast confirmed a specific increase in the dual-node group (t\u0026thinsp;=\u0026thinsp;2.784, p\u0026thinsp;=\u0026thinsp;0.015) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTreatment effects on theta activity during the AM task\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDuring the AM task, the wPLI values for right frontoparietal connectivity was increased in the dual-node group (T\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;=\u0026thinsp;0.049) but not in the single-node group (t=-1.046, p\u0026thinsp;=\u0026thinsp;0.330), which survived the double contrast (t\u0026thinsp;=\u0026thinsp;2.784, p\u0026thinsp;=\u0026thinsp;0.015) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The theta-gamma PAC (MVL-MI) in right DLPFC electrodes was elevated in the dual-node group (t\u0026thinsp;=\u0026thinsp;3.683, p\u0026thinsp;=\u0026thinsp;0.008) but remained unchanged in the single-node group (T\u0026thinsp;=\u0026thinsp;11, p\u0026thinsp;=\u0026thinsp;0.327), which, however, failed to survive the double contrast (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eBehavioral-neurophysiological correlations\u003c/h2\u003e\u003cp\u003eAlthough within-group analyses did not reveal significant correlations between changes in behavioral measures and electrophysiological indicators, several correlations showed statistical significance when data from both groups were combined. Specifically, increased theta-gamma PAC in the right prefrontal electrodes was positively correlated with improved accuracy on the WM task (R\u0026thinsp;=\u0026thinsp;0.59, p\u0026thinsp;=\u0026thinsp;0.016) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Increased wPLI values for right frontoparietal theta-phase synchronization were positively correlated with improvements in MoCA scores (R\u0026thinsp;=\u0026thinsp;0.80, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and AM task accuracy (R\u0026thinsp;=\u0026thinsp;0.55, p\u0026thinsp;=\u0026thinsp;0.028), but was negatively correlated with Trail Making Test-Part B (TMT-B) completion time (R=-0.68, p\u0026thinsp;=\u0026thinsp;0.0034) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-D).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study investigated the therapeutic effects of dual-node network-targeted tACS versus single-node intervention in patients with aMCI. The results demonstrate that dual-node tACS targeting the right DLPFC and PPC significantly improved global cognition compared with single-node stimulation applied solely over the right DLPFC. These behavioral benefits were accompanied by improvements in neurophysiological measures, including resting-state theta power, frontoparietal connectivity, and theta-gamma PAC during memory tasks.\u003c/p\u003e\u003cp\u003e Dual-node stimulation led to a greater improvement in global cognition relative to single-node stimulation, as measured by the MoCA. This finding aligns with the established role of the FPN in supporting executive control and memory functions [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Prior studies applying rTMS or tDCS to individual nodes within the prefrontal or parietal cortex [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] have demonstrated limited efficacy in aMCI patients. Neuroimaging evidence showed the FPN disconnection in AD and aMCI [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], making network-targeted stimulation a more scientifically grounded approach for cognitive enhancement than the single-node intervention. Although research in clinical populations remains vacant, the superiority of dual-node network-targeted to single-node interventions has been confirmed in improving memory performance among healthy individuals [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Of note, in this study, the group differences in clinical improvements was statistically significant in global cognition rather than in isolated cognitive domains, suggesting the global cognition a more sensitive indicator of stimulation effects.\u003c/p\u003e\u003cp\u003eIn terms of neurophysiological outcomes, the dual-node group exhibited a more significant increase after tACS in resting-state theta power over the right DLPFC and the mid-frontal cortex compared with the single-node group. These results indicate that, at the dimension of resting-state spectral power, the FPN-targeted intervention primarily affects the PFC functioning. The PFC is involved in a range of cognitive processes including memory and executive control, with dysfunction implicated in various cognition-related disorders [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Theta oscillations originate from the mid-frontal cortex and reflect a common computational mechanism underlying cognitive control [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Theta-tACS applied over the mid-frontal cortex has enhanced memory consolidation [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], and in-phase theta stimulation between the mid-frontal cortex and the DLPFC increased inter-regional phase synchronization and cognitive control ability in healthy volunteers [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Therefore, we posit that that dual-node stimulation applied over the FPN may exert effects not only on the targeted prefrontal regions but also propagate to upstream cortical areas involved in cognitive control.\u003c/p\u003e\u003cp\u003eDuring the WM task, we observed a preferential increase in theta-gamma PAC at the right prefrontal electrodes areas following dual-node stimulation. The increase in theta-gamma PAC suggests that modulating slower rhythmic activity within the FPN can regulate faster, local oscillatory processes associated with memory function. The PAC reflects the supervisory influence of large-scale network oscillations operating at lower frequencies over localized high-frequency activity [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Theta-gamma PAC is a fundamental mechanism for integrating localized neural information into a coherent stream that supports complex memory processes [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Impairments in this coupling have been documented in memory disorders including AD and aMCI [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], indicating its potential as an intervention target. In healthy volunteers, in-phase theta-tACS applied over the DLPFC-PPC [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] or frontotemporal [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] pathways preferentially enhanced theta-gamma PAC compared with anti-phase dual-node or single-node stimulation. The positive correlation between increased PAC and improved WM accuracy further supports its role as a quantitative indicator of WM enhancement. Notably, increased theta-gamma PAC were observed specifically during task performance but not at rest, suggesting that the stimulation effects are state-dependent and may become more evident when the cognitive resources are engaged.\u003c/p\u003e\u003cp\u003eDual-node stimulation was superior to single-site stimulation in improving AM performance and right frontal-to-parietal theta-phase synchronization during the AM task. Moreover, the increase in theta phase synchronization was positively correlated with improvements in AM recall accuracy and MoCA scores, but negatively correlated with shorter completion times on the TMT-B. AM decline is a recognized marker of cognitive impairment and an early sign of AD [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Age-related cognitive decline has been linked to reduced cortical information transmission, exemplified by diminished theta phase synchronization [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. While studies of transcranial electrical stimulation (tES) targeted the prefrontal or temporal regions to improve AM in older healthy or cognition-impaired adults, results have been inconsistent in treatment efficacy [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In contrast, although fewer studies applied parietal stimulation, most of them report beneficial effects on AM [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], which may suggest a stronger memory enhancement effect for the parietal stimulation than those of the frontotemporal cortex. Successful associative retrieval recruits the PPC [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Recent work applying high-frequency rTMS to the left PPC that showed strongest functional connectivity with the hippocampus demonstrated enhanced AM, hippocampal network connectivity, and parietal activity during retrieval [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Based on the importance of the PPC in AM, we propose that synchronous stimulation over the FPN may produce reinforcement effects on the network dynamics than single-node stimulation by leveraging the functional role of the parietal cortex. The correlations between increases in right frontal-parietal theta phase synchronization and performance in cognitive function (MoCA, AM accuracy, and TMT-B) may suggest that a better synchronization within the FPN leads to more cognitive benefits, further indicating the importance of network-level intervention.\u003c/p\u003e\u003cp\u003eSeveral issues should be considered in future research. Larger sample sizes and the inclusion of a sham control group will be helpful. An ideal control might incorporate anti-phase stimulation; however, ethical considerations arise when applying such protocols to cognitively impaired populations. Longitudinal follow-ups with regular assessments on cognitive function were required to determine the long-term duration of treatment effects. Furthermore, neuromodulation may be more effective when is personalized to entertain intrinsic brain rhythms and ongoing brain states. Future research should account for inter-individual variability in brain physiology and anatomy. The development of individualized tACS protocols is expected to improve the efficacy of dual-site interventions.\u003c/p\u003e\u003cp\u003eIn conclusion, this study demonstrates that modulating the FPN functioning by theta-band in-phase stimulation rather than a local region is more effective for cognitive disorders. These findings support the potential of network-targeted dual-node tACS as a non-invasive therapeutic approach for cognitive impairment.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAD, Alzheimer’s dementia; tACS, transcranial alternating current stimulation; rTMS, repetitive transcranial magnetic stimulation; tDCS, transcranial direct current stimulation; DLPFC, dorsolateral prefrontal cortex; WM, working memory; FPN, fronroparietal network; AM, associative memory; MMSE, Mini Mental State Examination; MoCA, Montreal Cognitive Assessment; CDR, Clinical Dementia Rating Scale; ICA, independent component analysis; wPLI, weighted phase lag index; MVL, mean vector length; MI, modulation index; PAC, phase-amplitude coupling; TMT, Trail Making Test.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the participants in this trial.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLi Wang: Conceptualization, Investigation, Methodology, Project administration, Funding acquisition, Supervision, Writing original draft, Writing-review \u0026amp; editing. Zhixin Piao, Hongfang Su: Project administration. Yiwen Jiang: Data curation, Methodology. Anshun Kang, Fenglin Zhu: Methodology. Tianyi Yan: Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the 2030 Major Project of the Ministry of Science and Technology (2022ZD0208500), the Beijing Institute of Technology Research Fund Program for Young Scholars (3160012222109), and the National Natural Science Foundation of China (U20A20191).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used during the current study are available from the corresponding or first author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval was received from Beijing Institute of Technology (BIT-EC-H-2021112). 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Stimulation of the Posterior Cortical-Hippocampal Network Enhances Precision of Memory Recollection. \u003cem\u003eCurr Biol\u003c/em\u003e 2017; \u003cstrong\u003e27\u003c/strong\u003e: 465\u0026ndash;470.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"translational-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"tp","sideBox":"Learn more about [Translational Psychiatry](http://www.nature.com/tp/)","snPcode":"41398","submissionUrl":"https://mts-tp.nature.com/cgi-bin/main.plex","title":"Translational Psychiatry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Alzheimer’s disease, Theta oscillations, Transcranial alternating current stimulation, Mild cognitive impairment, Frontoparietal network","lastPublishedDoi":"10.21203/rs.3.rs-7785019/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7785019/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe efficacy of current transcranial stimulation in cognitive disorders is limited by single-node intervention. Recent evidence indicates that amnestic mild cognitive impairment (aMCI) is associated with dysconnectivity in the frontoparietal network (FPN) and theta oscillations; modulating the FPN with theta-frequency stimulation represents a promising intervention for aMCI.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe developed a noninvasive transcranial alternating current stimulation (tACS) protocol for modulating long-range theta interactions within the FPN in aMCI patients. Thirty patients with aMCI were randomly assigned to 2 mA, 6 Hz, 25 min, and 10 sessions of dual-node tACS applied over right FPN (i.e., the DLPFC and the posterior parietal cortex) or single-node tACS over right DLPFC, followed by clinical visits at 4 weeks after treatment. Participants also undergone EEG recordings during resting state, 2-back working memory, and associative memory task before and after intervention.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eCompared with single-node stimulation, dual-node stimulation produced more sgnificant improvements in global cognition, as measured by Montreal Cognitive Assessment. Dual-node stimulation enhanced resting-state theta power in dorsolateral and midline prefrontal cortices. Furthermore, dual-node stimulation was also superior to single-site stimulation in improving memory performance and network dynamics, including theta-gamma phase-amplitude coupling in right dorsolateral prefrontal cortex during the working memory task and right frontal-to-parietal theta-phase synchronization during the associative memory task.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThis study demonstrates behavioural benefits and neural mechanisms of dual-node stimulation in ameliorating cognitive impairment, providing a promising approach for achieving network-level intervention in cognitive disorders.\u003c/p\u003e\u003ch2\u003eTrial registration\u003c/h2\u003e\u003cp\u003eChinese Clinical Trial Registry, ChiCTR2200058652 Registration Date\u003c/p\u003e","manuscriptTitle":"Comparative Efficacy of Dual- vs. Single-Node tACS in Amnestic Mild Cognitive Impairment: Behavioral and EEG Evidence","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-29 03:57:48","doi":"10.21203/rs.3.rs-7785019/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-12-16T08:47:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-11-01T19:58:42+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-10-30T13:21:48+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-24T20:07:33+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-18T06:59:58+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-10-13T15:17:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-08T11:13:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-08T11:13:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Translational Psychiatry","date":"2025-10-08T01:44:43+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2025-10-07T14:48:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"translational-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"tp","sideBox":"Learn more about [Translational Psychiatry](http://www.nature.com/tp/)","snPcode":"41398","submissionUrl":"https://mts-tp.nature.com/cgi-bin/main.plex","title":"Translational Psychiatry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"149f2e59-a94d-49e5-aaa8-2b95f747e10c","owner":[],"postedDate":"October 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":56216000,"name":"Biological sciences/Neuroscience/Learning and memory"},{"id":56216001,"name":"Health sciences/Diseases"}],"tags":[],"updatedAt":"2026-03-24T10:35:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-29 03:57:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7785019","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7785019","identity":"rs-7785019","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}