High-Definition Transcranial Direct Current Stimulation During Sleep

Summary

This protocol describes high-definition transcranial direct current stimulation during sleep with simultaneous electroencephalography monitoring to investigate causal relationships between targeted brain regions and sleep as well as its related processes.

Abstract

High-definition transcranial direct current stimulation (HD-tDCS) enables high spatial precision modulation of neural activity during sleep. This protocol presents a comprehensive methodology for applying HD-tDCS using a 4 x 1 ring electrode configuration while simultaneously recording high-definition electroencephalography (HD-EEG) to monitor sleep architecture and stimulation effects. The technique enables systematic tuning of multiple parameters, where each parameter selection produces distinct neurophysiological outcomes. This provides opportunities to target specific brain regions and oscillatory patterns specifically tailored to the research question. Successful implementation of this protocol requires attention to electrode placement, impedance management, and real-time sleep staging capability. The versatility of this methodology and its capacity to address causal relationships in sleep physiology invite a broad range of applications from basic neuroscience to clinical interventions based on sleep optimization. The standardized yet flexible framework described here establishes HD-tDCS as a robust platform for systematic investigations of sleep neurobiology as well as its clinical ramifications.

Introduction

Sleep is essential for brain health and cognition, and specific sleep oscillations have key supportive and restorative roles. The ability to modulate these sleep oscillations offers unique opportunities to establish causality between sleep, brain activity, and cognition. Various neuromodulation techniques have emerged in recent years, with transcranial direct current stimulation (tDCS) showing promise as a non-invasive approach to modulate neural activity during sleep^1,2,3,4.

Conventional tDCS applies direct electrical current through large pad electrodes, typically 25-35 cm², resulting in relatively diffuse stimulation⁵. The efficacy and specificity of tDCS during sleep depend on several key parameters that can be systematically manipulated. Electrode placement fundamentally determines which cortical regions receive stimulation; bifrontal configurations can either enhance slow oscillations and sleep spindles^6,7 or reduce total sleep time⁸, depending on the montage and polarity. Current intensity (typically 0.2-2.0 mA) exhibits dose-dependent effects on both the magnitude of neurophysiological responses and participant tolerability⁹. Temporal parameters, including total stimulation duration, ramp duration, and protocol structure (continuous vs. intermittent with specific inter-stimulation intervals), determine both immediate effects and after-effects; for example, intermittent protocols with stimulation-free intervals can enhance motor cortex excitability while reducing adaptation¹⁰. Additionally, the timing of stimulation relative to sleep architecture impacts outcomes; for instance, Stage 2 (N2) sleep stimulation primarily affects spindles and slow oscillations^6,7 while rapid eye movement (REM) sleep stimulation influences self-awareness during dreams¹¹. This diversity in outcomes from such subtleties in parameter selection exemplifies the exquisite sensitivity of sleep architecture-and related processes such as cognition-to targeted neuromodulation.

High-definition tDCS (HD-tDCS) employs smaller electrodes, such as the 4 x 1 ring montage (approximately 0.95 cm² surface area vs 2-35 cm² for conventional rubber electrodes), which allows for more focal targeting of brain regions with enhanced spatial precision-defined as the ability to concentrate electrical current within a specific target while minimizing spread to adjacent areas-compared to larger rubber electrodes used a priori¹². HD-tDCS also produces a more circumscribed electric field distribution, and the generated current is largely confined to cortical areas directly beneath the electrodes¹³. This advanced spatial specificity minimizes off-target effects and paves the way for high precision functional correlations between the stimulation sites and observed outcomes¹⁴. This selectivity allows for more accurate testing of brain region-specific hypotheses while reducing confounding influences from unintended stimulation of adjacent cortical areas^{5,12,13,14,15}. The improved focal precision and accuracy of HD-tDCS enable mechanistic interrogations of specific brain regions and their associations with distinct aspects of sleep physiology and cognition. The adaptability of HD-tDCS is amplified when coupled with continuous neurophysiological recordings, such as continuous high-definition electroencephalography (EEG). Hence, a platform with such translational promise is capable of advancing both basic neuroscience research and the development of clinical interventions for sleep and neuropsychiatric conditions⁴.

This protocol describes a comprehensive methodology for applying HD-tDCS during sleep using a 4 x 1 ring configuration while simultaneously recording EEG to monitor sleep architecture and stimulation effects. The approach is designed to be versatile, allowing researchers to customize stimulation parameters to address various research questions related to sleep neuroscience.

Protocol

The study was approved and conducted following our institute's ethical guidelines. Informed consent with full explanations about the procedures, risks, and benefits was obtained from the participants of the study.

1. Contraindications and safety considerations

1. Prior to experimental set-up, confirm that the participant does not have any contraindications for HD-tDCS based on current safety guidelines^16,17 (Table 1).
2. Inspect the participant's scalp thoroughly for skin lesions. Avoid stimulating areas with damage or inflammation.
3. Review the participant's medications, as central nervous system-acting drugs might alter the effects of stimulation.
4. Procure informed consent with full explanations about the procedures, risks, and benefits.
5. Screen baseline symptoms¹⁶ for pre-existing symptoms that might be confused with stimulation-related effects^16,17. These include: headache, scalp discomfort, tingling, itching, burning sensation, skin redness, sleepiness, trouble concentrating, and mood changes.

2. Equipment preparation

NOTE: This protocol describes the use of separate, independent devices for HD-tDCS and EEG recording (Figure 1). Some commercially available systems integrate both stimulation and recording capabilities within a single device. Users of such integrated systems should consult manufacturer-specific instructions.

1. Gather all necessary equipment and materials (Table 2, Figure 2, and Figure 3).
2. Ensure the conventional tDCS device has an adequate power supply.
   1. Turn on both devices and check the power status according to the manufacturer's specifications.
   2. For battery-powered devices: If low battery indicators illuminate, replace batteries (for user-replaceable systems) or recharge the device (for integrated battery systems).
   3. For software-controlled devices: Verify power status through the control interface.
   4. For mains-powered devices: Ensure proper connection to the power outlet with appropriate medical-grade isolation.
3. Inspect all electrodes for signs of unusual wear or damage.
   1. For Ag/AgCl sintered ring electrodes: Check for deposits of electrolysis products on the electrode surface.
   2. For carbon rubber electrodes: Check for cracks, brittleness, or uneven wear.
   3. For disposable electrodes: Verify they have not exceeded single-use or recommended usage limits.
   4. Replace any damaged or excessively used electrodes following the manufacturer's guidelines for replacement frequency.
4. Connect the cables of the Ag/AgCl sintered ring electrodes to the matching receivers on the 4 x 1 adapter output cable.
   1. Connect the center electrode lead to the center receiver plug.
   2. Connect the remaining electrodes to the surrounding plugs.

3. EEG cap and HD-tDCS electrode preparation (Figure 2 and Figure 3)

1. Select the appropriate size EEG cap for the participant based on the head circumference. Refer to the manufacturer's manual for the EEG cap size selection.
2. Determine HD-tDCS electrode polarity configuration based on study objectives. For anodal stimulation, the center electrode serves as the anode (positive) with the surrounding electrodes as cathodes (negative). For cathodal stimulation, the center electrode serves as the cathode (negative) with the surrounding electrodes as anodes (positive). Common placements (and expected brain targets) include:
   1. Use the bilateral frontolateral (F3/F4) placement to target the prefrontal cortex⁶.
   2. Use the central (Cz) placement to target sensorimotor areas¹⁸.
   3. Use the occipital (Oz) placement to target the visual cortex¹⁹.
   4. Use the parietal (P3/P4) to target association cortices²⁰.
   5. Optional) Use computational field modeling to visualize and optimize current distribution ( Figure 4):
      1. Obtain the participant's structural MRI if available, or use a standard head model.
      2. Use field modeling software (e.g., SIMNIBS, ROAST, or manufacturer-specific tools like HD-Explore) to simulate current flow^21,22.
      3. Input electrode positions and stimulation parameters (current intensity, polarity).
      4. Evaluate the predicted field distribution to ensure adequate targeting of the intended region of interest.
      5. Adjust the electrode positions as needed to optimize field focality and intensity at the target.
3. Remove the EEG recording electrodes at the chosen stimulation location from the EEG cap. This allows the HD-tDCS stimulation electrodes to occupy these positions so that they have direct scalp contact through the same openings in the cap. The removed EEG electrodes cannot record meaningful data from these locations during stimulation due to saturation from the stimulation current.
4. Insert the HD-tDCS electrodes or electrode holders into the cap (Figure 2).
   ​NOTE: Some HD-tDCS systems use electrodes that are directly inserted into the cap without separate holders. Follow manufacturer-specific instructions.
   1. For systems with electrode holders: Position and secure the holders so that the side containing a cap faces the EEG cap's exterior and the opposite side (lacking a cap) faces the EEG cap's interior, such that it will contact the participant's scalp.
   2. For systems with direct-insertion electrodes: Follow manufacturer guidelines for proper electrode orientation and securing mechanism.
5. Apply adhesive conductive paste (high viscosity, chloride-free; Ten-20) paste to prepare the EEG cap, as recommended by the manufacturer.
   1. Reverse the EEG cap inside-out and place it on a foam head.
   2. Flatten any wrinkles in the fabric of the cap.
   3. Using a tongue depressor, cover the EEG electrodes with paste. Do not use paste on HD-tDCS stimulation electrodes.
   4. Shape the paste into domes. Apply less paste in the front where there is less hair and more at the crown and back of the head.
   5. Avoid applying too much paste to each electrode site and getting paste on the fabric, as excessive spillage can cause bridging.

4. Participant set-up

1. Seat the participant comfortably in a chair.
2. Apply the facial and ground reference electrodes with adhesive conductive paste using gold cup electrodes. Secure the electrodes with medical-grade tape.
   1. Clean sites with an alcohol swab, exfoliate with mildly abrasive preparation gel (e.g., Nuprep) on a cotton swab (e.g., Q-tip), and remove all fluids with a dry gauze square. Electrode sites follow standard polysomnography montage requirements for sleep staging according to American Academy of Sleep Medicine (AASM) criteria²³ and include:
      Reference (forehead); serve as reference electrodes
      A1/A2 (mastoids, bone behind ears); serve as reference electrodes.
      LOC/ROC (right and left outer canthi near eyes, right raised, left lowered); record eye movements.
      EMGs (2 on chin); record muscle tone for distinguishing sleep stages.
      ​Ground (clavicle/collar bone); stabilizes the amplifier and reduces common-mode noise.
   2. Fill electrodes with adhesive conductive paste and apply to the site while avoiding exposing the paste on the back of the electrode.
   3. For the HD-tDCS return electrode, place the relevant stimulation electrode on the collar bone and secure it with tape. Apply saline solution or gel per the manufacturer's specifications.
3. Locate and mark the central point for EEG cap placement.
   1. First, localize the vertex (Cz).
      1. Measure the distance from the nasion (junction of the forehead and nasal bones) to the inion (most prominent point of the occipital bone). Mark the halfway point with a line.
      2. Measure the distance between the left and right pre-auricular points. Mark the halfway point with a line.
      3. Cz lies at the intersection of these lines. Lightly spray the hair with water using a spray bottle ( Figure 3) to facilitate EEG cap placement.
4. Invert the prepared EEG cap snugly and comfortably on the participant's head, targeting the Cz electrode with the marked Cz site. Secure the EEG cap on the participant's head with the chin straps.
5. Using a syringe, fill the electrode sites with conductive gel (e.g., e-gel).
   1. Pull up the electrode slightly and insert the large-gauge, blunt-tip needle tip into the center of the electrode. Gently wiggle the needle to move hair out of the way and apply conductive gel (~0.5 mL). Gently push the electrode down to ensure a strong connection between the gel, adhesive conductive paste, and scalp.
   2. Avoid spreading gel beyond the circumference of the electrode site to prevent shunting of current.
   3. Apply more conductive gel on the crown and back of the head to compensate for the higher density of hair.
   4. Press each electrode down against the scalp.
6. Position the Ag/AgCl sintered ring electrodes in each HD-tDCS electrode holder ( Figure 2, right panel).
   1. Place with the rough surface facing down and the smooth, rounded surface facing up.
   2. Lower the ring electrode until it rests on the base of the holder.
   3. Add more high-viscosity conductive gel (e.g., HD-gel) to cover the electrode.
   4. Use the caps provided to lock the electrodes in place.

5. HD-EEG system setup

1. Prepare the appropriate software for the recording computer.
2. If a high-density headbox (or similar hardware) is involved, ensure all electrodes are connected to the appropriate amplifiers. This stage may involve using a fiber-optic connector cable, and variations may exist in the set-up of the recording hardware.
3. Do not plug in the stimulation electrodes at this stage.
4. Turn on the amplifier and launch the EEG recording software.
   1. After creating a new study file, select the profile option appropriate for a high-density montage.
   2. Verify the destination for file storage and filters appropriate for the montage.
5. Check impedances.
   1. Set the impedance limit to 25 kΩ and aim for impedances below 10 kΩ.
   2. For electrodes with high impedance, consider the following adjustments:
      1. If all or most are high, check if all the electrodes are properly connected. Check the reference electrode and, if necessary, redo the reference electrodes.
      2. If all or most are unusually low, check that the ground is connected.
      3. If half are completely red, check the snugness of the cap and amplifier.
      4. If an individual electrode is high, press down on the electrode. If the impedance remains high, add more conductive gel with the syringe, gently scrape the scalp with the syringe tip for the scalp electrode, or redo the facial electrode. If the problem persists or excess impedance is detected, check the connection between the cap, the connector, and the amplifier.
6. Begin recording EEG.
7. Perform biocalibrations.
   1. Ask the participant to open and close their eyes for ~10 s. Observe for alpha activity in most participants. Expect larger amplitudes at posterior channels than at anterior channels. Monitor for amplitude variations across the scalp.
   2. Ask the participant to look left, right, up, and down. Subsequently, ask the participant to clench their teeth. Observe movements in the eye and chin electrode recordings.
   3. Ask the participant to blink a few times.
      ​NOTE: Blink artifacts should appear as large deflections in the frontal electrodes (e.g., Fp1, Fp2), as these are closest to the eyes. Electrodes further from the eyes may show smaller deflections.
   4. Add additional biocalibrations as dictated by study protocol (e.g., respiration, limb movement, etc.)

6. HD-tDCS stimulator set-up

1. Prepare the HD-tDCS stimulation electrodes and stimulator. Connect the electrodes to the stimulator only after the following have been completed first.
   1. Ensure that the stimulator is connected to a power outlet, or its batteries are fully charged and properly installed.
   2. Switch on the stimulator.
2. After the device is on, plug the electrode cable into the back of the device.
   NOTE: Failure to turn the device on before plugging could lead to a mild shock. A long or specially ordered electrode cable may be necessary so that the stimulation can be triggered from the control room or location outside the sleeping space to decrease the likelihood of disrupting the participant's sleep.
3. Set stimulation parameters based on study design. Settings include the intensity, waveform (this will be DC for tDCS protocols), duration, and ramp times.
   NOTE: These parameters will vary depending on the intended target and physiology within the brain. Most devices also possess settings to separate stimulation and sham protocols. At this stage, the current distribution for the stimulation montage should be configured. For a typical 4 x 1 configuration, the center electrode receives 100% of the programmed current intensity, while this current is distributed across the four return electrodes (25% each).
4. Set active stimulation or sham mode as per the study protocol. If the study involves blinding, appropriate steps are warranted to prevent participants or researchers from knowing which mode is being used. Some stimulators have built-in stimulation/sham functions to help with this.
5. If the HD-tDCS electrodes exhibit high impedance (e.g., greater than 600 kΩ), deliver a brief test stimulation (5-10 s at programmed intensity) under the SHAM setting to check the connectivity. Ideally, complete this prior to the participant falling asleep.
   1. Under the SHAM setting, deliver the pulse. Expect to see the impedance drop 3-5 s after delivering the pulse. Once impedance reduction is confirmed, immediately abort the stimulation (total test duration should not exceed 10 s).
   2. Add more high-viscosity conductive gel and/or check the connections until the impedance falls to less than 20-25 kΩ when a sham pulse is applied.

7. Sleep monitoring and stimulation protocol

1. Prepare the sleep environment.
   1. Minimize ambient light and noise.
   2. Ensure the participant's reported comfort.
   3. Offer the participant earplugs.
   4. Mark the time in the EEG recording when the lights are turned off.
2. Monitor the EEG for sleep onset and specific stages to initiate the stimulation protocol based on the study design. Decisions regarding the stimulation protocol and its point of initiation should consider the duration/continuity of stimulation (i.e., continuous vs intermittent) as well as the targeted physiology/sleep architecture.
3. Mark the start of stimulation in the EEG recording with an appropriate notation.
4. If the participant awakens during stimulation:
   1. Abort the stimulator.
   2. Mark the time point with an appropriate notation on the EEG recording.
   3. Monitor for the participant to return to the sleep stage of interest per study design and stimulation protocol.
   4. Resume the stimulation protocol as appropriate. If using an intermittent stimulation design, the aborted stimulation period is typically considered over, and the next stimulation would be the next one in the schedule (e.g., if Stimulation #3 is aborted, the protocol picks up with Stimulation #4 once resumed).
   5. Complete the stimulation protocol.
      1. Once the final stimulation period ends, unplug the stimulation cord (connecting the HD-tDCS electrodes) from the stimulator before turning off the stimulator.
         ​NOTE: Again, failure to unplug before turning the device off could result in a minor shock, potentially waking the participant.
      2. Allow the participant to continue sleeping (as appropriate), undisturbed, depending on the study design.

8. Session completion and equipment removal

1. Wake the participant at the appropriate time.
   1. Mark the time in the EEG recording when the lights are turned on.
   2. Gently wake the participant.
2. Stop the EEG recording.
3. Disconnect the equipment.
   1. Remove the caps from the HD-tDCS electrode holders.
   2. Gently remove the Ag/AgCl ring electrodes, using a blunt tool if necessary to avoid pulling on cables.
   3. Clean the electrodes with a damp paper towel and dry them for storage.
4. Remove the EEG cap and clean the electrode sites to remove the gels.
5. Offer the participant cleaning products to remove gel from their hair and skin.
6. Evaluate post-stimulation adverse event assessment (similar to the screen in step 1.5). Document severity (e.g., mild, moderate, or severe) and probable relationship to stimulation to compare responses to baseline screening.
   NOTE: Most HD-tDCS adverse events are mild and transient, typically resolving within hours following stimulation^16,17. If this is not the case, institutional protocols should be invoked for serious adverse events reporting.

Results

When the protocol is implemented correctly, HD-tDCS during sleep produces measurable and reproducible changes in sleep architecture and physiology that vary systematically with the chosen stimulation parameters. The focal distribution from bilateral frontolateral stimulation in Figure 4-one possible montage-exemplifies the spatial precision of 4 x 1 ring electrodes. Successful protocol implementation is confirmed through several key indicators. Optimal electr...

Discussion

This protocol provides a standardized yet flexible methodological framework for delivering HD-tDCS during sleep with simultaneous EEG monitoring. The technique's primary strength lies in its focal, parameter-controlled neuromodulation and the ability to directly monitor its neurophysiological effects. This allows for tests of causality between targeted brain activity and sleep-related processes.

Successful and reliable execution of HD-tDCS during sleep requires attention to certain paramet...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ag/AgCl sintered ring electrodes	Soterix Medical	HD-1AGE-12	For stimulation set-up
Alcohol swabs	NA	NA	For EEG set-up
Ear plugs	NA	NA	For helping participant to fall asleep
EC2	Natus	CR-003	For EEG set-up
EEG recording cap (with attached electrodes)	Natus	21114	For EEG set-up
Electro-gel	Electro-Cap International, Inc.	E11	For EEG set-up
Gauze squares	NA	NA	For EEG set-up
Gold cup electrodes	Natus	F-E5GH-48	For electrooculography (EOG) and electromyography (EMG) collection
HD-EEG system with 64-channel capability	Natus	Brain Monitor EEG Amplifier	For EEG set-up
HD-gel	Soterix Medical	HD-1GEL	For stimulation set-up
HD-tDCS electrode holders	Soterix Medical	HD-1A.2	For stimulation set-up
HD-tDCS stimulation adapter	Soterix Medical 4x1 Multichannel Stimulation Adapter	Model 4X1-C3A	For stimulation set-up
HD-tDCS stimulation system	Soterix Medical M×N HD Transcranial Stimulation System	Model 9002	For stimulation set-up
Head model for securing and preparing gel on EEG recording cap (if needed)		For EEG set-up
Nuprep	Weaver and Company	10-30	For EEG set-up
Q-tips	Q-tips	https://www.qtips.com/	For EEG set-up
Scissors	NA	NA	For EEG set-up
Specialized connector cable/cord	Soterix Medical	CUS_MxN_CBL	For stimulation electrodes
Spray bottle (and cup) with water	NA	NA	For EEG set-up
Tape	NA	NA	For EEG set-up
Ten cc syringe with 16-gauge blunt tip	NA	NA	For EEG/stimulation set-up
Ten-20	Weaver and Company	10-20-8	For EEG set-up
Tongue depressor	NA	NA	For EEG set-up
Towels	NA	NA	For EEG set-up