Combined Regulatory Effects of 40 Hz eTNS and Multisensory Stimulation on Brain Networks

Physical neuromodulation at 40 Hz has emerged as a promising therapeutic approach, with visual and auditory stimulation demonstrating the capacity to entrain gamma oscillations and confer cognitive benefits. Analogous effects have also been observed with electrical approaches, including external trigeminal nerve stimulation (eTNS). While the prevailing assumption in the field suggests that combining multiple sensory modalities will more comprehensively recruit distributed brain circuitry and yield greater therapeutic effects, the mechanistic foundations of this additive hypothesis remain contested. Furthermore, recent evidence indicates that network-level reorganization, rather than localized gamma entrainment, may represent a more causally relevant substrate for these cognitive improvements.

This study is designed to systematically characterize the independent and interactive effects of 40Hz auditory, visual, and eTNS stimulation on brain state dynamics, functional connectivity, and graph-theoretic network properties. The trial employs a fully crossed 2×2×2 within-subject design. A total of 23 participants will undergo all eight possible combinations of 40Hz stimulation modalities: sham (no stimulation), auditory only, visual only, eTNS only, auditory + visual, auditory + eTNS, visual + eTNS, and the three-way combination of auditory + visual + eTNS.

During the experimental session, the eTNS will specifically target the maxillary branch of the trigeminal nerve. The eight stimulation conditions will be administered in a randomized order for each participant. Each stimulation block will last for 5 minutes, separated by 1-minute inter-stimulus intervals to prevent carry-over effects.

Continuous electroencephalography (EEG) will be recorded throughout the session using a 32-channel eego™ mylab system (ANT Neuro, Netherlands) to capture real-time cortical responses. Following data acquisition, standard preprocessing pipelines will be applied, including notch filtering, 0.5-45Hz bandpass filtering, average re-referencing, ICA-based artifact rejection, and bad channel interpolation.

• 40 Hz External Trigeminal Nerve Stimulation (Device): Electrical stimulation targeting the maxillary branch of the trigeminal nerve at a frequency of 40 Hz
• 40 Hz Auditory Stimulation (Device): Auditory sensory stimulation delivered at 40 Hz for 5-minute durations
• 40 Hz Visual Stimulation (Device): Visual sensory stimulation delivered at a frequency of 40 Hz for 5-minute durations
• Sham Stimulation (Other): Inactive control stimulation designed to mimic the sensory experience of the active modalities without delivering actual 40 Hz therapeutic stimulation

Primary outcomes

• Whole-Brain Functional Connectivity (Phase-Locking Value) [Day1 (during the single experimental session)]

Secondary outcomes

• EEG Microstate Dynamics (Mean Duration and Segment Density) [Up to 1 hour (during the single experimental session)]: Brain state dynamics evaluated via microstate analysis of global field power (GFP) peaks. Primary metrics extracted include the mean duration and segment density (occurrence rate per minute) of identified microstate classes to assess modality-specific regulation.
• EEG Phase-Locking Value (PLV)-Derived Global Efficiency [Day1 (during the single experimental session)]: Continuous electroencephalography (EEG) data will be recorded. The specific parameter extracted from the EEG is the Phase-Locking Value (PLV) across theta (4-7Hz), alpha (8-12Hz), beta (13-30Hz), and gamma (31-45Hz) frequency bands. These PLV functional connectivity matrices will be used to construct weighted brain networks across a sparsity threshold range of 0.1 to 0.5. Global efficiency, a graph-theoretic metric representing network-wide communication integration, will then be calculated and reported based on these PLV networks.
• EEG Phase-Locking Value (PLV)-Derived Small-World Propensity [Day 1 (during the single experimental session)]: Continuous electroencephalography (EEG) data will be recorded. The specific parameter extracted from the EEG is the Phase-Locking Value (PLV) across theta (4-7Hz), alpha (8-12Hz), beta (13-30Hz), and gamma (31-45Hz) frequency bands. These PLV functional connectivity matrices will be used to construct weighted brain networks across a sparsity threshold range of 0.1 to 0.5. Small-world propensity, a graph-theoretic metric evaluating the balance between local network specialization and global integration, will then be calculated and reported based on these PLV networks.

Sex: All, ages: 18 Years to any, accepts healthy volunteers

Inclusion Criteria:

* Healthy adults aged ≥18 years old.
* Normal or corrected-to-normal vision and hearing (essential for 40Hz visual and auditory stimulation).
* Right-handedness.
* Willing to participate in the study and capable of providing written informed consent.

Exclusion Criteria:

* Personal or family history of epilepsy or seizures (crucial safety criteria for 40Hz flicker and electrical stimulation).
* History of any neurological or psychiatric disorders.
* Presence of metallic implants in the head, cardiac pacemakers, or other implanted electronic medical devices.
* Skin lesions, infections, or hypersensitivity at the eTNS stimulation site (maxillary branch of the trigeminal nerve).
* Current use of psychoactive medications or substances that may significantly alter EEG signals or brain state dynamics.
* Pregnant or lactating individuals.
* Poor sleep quality or irregular sleep-wake cycles prior to the experiment.

• Xue-Juan Yang, Principal investigator, Xidian University