This protocol introduces a scalp acupuncture-based method for localizing the primary motor cortex (M1) hotspot for the flexor digitorum superficialis (FDS) muscle and validates its effectiveness against the conventional C3 site.
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Method Article
This protocol introduces a scalp acupuncture-based method for localizing the primary motor cortex (M1) hotspot for the flexor digitorum superficialis (FDS) muscle and validates its effectiveness against the conventional C3 site.
The precise localization of the primary motor cortex (M1) corresponding to specific muscles is critical for enhancing the efficacy of neuromodulation. When navigated transcranial magnetic stimulation (nTMS) or other mapping tools are unavailable, stimulation is typically applied at the C3 site of the standard 10-20 EEG system. However, emerging evidence indicates that C3 may not be the optimal site for targeting hand muscles. Therefore, the goal of this study is to propose an innovative neuroanatomy-based localization method to identify the M1 hotspot for the hand muscle and to validate its effectiveness compared with the C3 site. A step-by-step protocol was developed to identify the M1 hotspot for the flexor digitorum superficialis (FDS) muscle using an acupuncture-based approach. The identified hotspot was then compared with the conventional C3 site by evaluating baseline and post-neuromodulation motor evoked potentials (MEPs). Representative data demonstrated that the proposed M1 hotspot yielded significantly higher MEP amplitudes than the C3 site, both at the baseline and following neuromodulation. The proposed approach provides a validated and practical method for localizing the M1 hotspot for the FDS. This technique has the potential to improve the effectiveness of neuromodulation in clinical practice when advanced neuroimaging tools are not accessible.
Precisely localizing the cortical hotspot in the primary motor cortex (M1) for neuromodulation targeting specific muscles is critical for optimizing motor rehabilitation outcomes1. Navigated transcranial magnetic stimulation (nTMS) has been adopted as a non-invasive reliable methodology for motor mapping compared to direct cortical stimulation2. However, in the absence of such tools, C3 (left hemisphere) and C4 (right hemisphere), defined by the standard 10-20 EEG system, are commonly used as M1 targeting for delivering neuromodulation3. However, studies have indicated that C3 may not represent the actual M1 hotspot4,5, and may not be the optimal site for delivering neuromodulation, as it yields lower motor evoked potential (MEP) amplitudes compared to alternative sites in the surrounding region6. In addition, high-definition transcranial electrical stimulation (HD-tES) has the potential to enhance its modulatory effects7,8,9,10; however, it requires more precise localization of the targeted stimulation area compared to conventional montages11,12. Therefore, there is a need for a method to accurately identify the M1 hotspot when advanced neuroimaging tools are not accessible.
Chinese scalp acupuncture is a contemporary acupuncture technique integrating traditional Chinese needling methods with modern neuroanatomy of representative areas of the cerebral cortex13. In contrast to traditional acupuncture, scalp acupuncture involves subcutaneous needle insertion across defined zones that align with cortical regions responsible for central nervous system functions13. Several scalp acupuncture systems have been developed and utilized for the treatment of motor impairments, including those established by Jiao, Lin, Jin, and Tang, as well as the internationally standardized China scalp-point protocol14. Among these approaches, Jiao's scalp acupuncture integrates modern neuroanatomy and neurophysiological principles with traditional Chinese acupuncture techniques, creating an innovative method for modulating central nervous system function15,16. However, whether its concepts can be applied and validated in upper limb neuromodulation studies remains unknown.
Therefore, this protocol aims to explore a new approach for localizing the M1 hotspot of the flexor digitorum superficialis (FDS) muscle using scalp acupuncture landmarks (AC) and to validate this approach by comparing corticospinal excitability with that of the conventional C3 site before and after HD-tES intervention. However, the current approach has only been validated in healthy participants with clearly identifiable scalp anatomical landmarks and for the FDS muscle specifically. Whether this method can be generalized to other upper limb muscles or applied to individuals with neurological impairments remains uncertain.
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Participants were right-handed healthy adults with no history of neurological or musculoskeletal disorders affecting the upper limbs. The research protocol was reviewed and approved by the Committee for the Protection of Human Subjects (CPHS) at The University of Texas Health Science Center at Houston. Written informed consent was obtained from all participants prior to participation.
1. Preparation
2. Localize the upper motor point (UMP)
NOTE: The Upper Motor Point (UMP) is defined as the point located 0.5 cm posterior to the midpoint of the anterior-posterior line, which extends from the glabella (the midpoint between the eyebrows) to the inion.

Figure 1: Localizing the Upper Motor Point. (A) The midpoint between the eyebrows (Glabella). (B) The external occipital protuberance located at the midline of the occipital bone (Inion). (C) The left preauricular point, located anterior to the tragus of the ear. (D) Located 0.5 cm posterior to the intersection of the mid-sagittal line and the transverse line connecting the left and right preauricular points; this point is defined as the Upper Motor Point (UMP). Please click here to view a larger version of this figure.
3. Localize the lower motor point (LMP)
NOTE: Lower Motor Point (LMP) is defined as the intersection of the line extending from the lateral end of the eyebrow to the inion with the anterior hairline.
4. Localize the M1 FDS hotspot (AC)
NOTE: The M1 FDS hotspot is defined as the point located at two-fifths of the distance from the Upper Point (Up) to the Lower Motor Point (LMP) along the motor area line (Figure 2C).

Figure 2: Localizing the Lower Motor Point and the FDS hotspot. (A) The intersection of the line extending from the lateral end of the eyebrow to the inion with the anterior hairline. (B) The Lower Motor Point (LMP). (C) The Upper Motor Point (UMP), the FDS hotspot (AC), and the LMP. Please click here to view a larger version of this figure.
5. M1 FDS Hotspot (AC) validation
NOTE: Instruct the participants to sit in a chair adjusted to a comfortable height, put their right hands on a soft pillow, and instruct them to relax during the stimulation session.

Figure 3: EMG electrode and TMS coil placement. (A) The bipolar EMG electrode placement at FDS and (B) the placement of single-pulse TMS at M1 FDS hotspot. Please click here to view a larger version of this figure.
6. HD-tES intervention

Figure 4: The setup of HD-tES. The center electrode is targeting at C3 or AC with 4 reference electrodes located at the surrounding channels. Please click here to view a larger version of this figure.
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The results revealed three key findings: i) The proposed method (AC), based on neuroanatomy-guided scalp acupuncture, successfully identified the M1 hotspot for the FDS muscle, as it overlapped with the TMS-guided hotspot. ii) The identified M1 FDS hotspot did not overlap with the conventionally targeted C3 site; moreover, the AC site exhibited higher baseline MEP amplitudes compared to C3. iii) HD-tACS intervention at the AC site significantly increased MEP amplitudes, whereas no significant change was observed followin...
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In this study, an innovative approach was developed to localize the M1 hotspot for the FDS muscle based on scalp acupuncture landmarks. The proposed AC site showed strong potential as the M1 hotspot for the FDS muscle, with higher baseline and post-modulation MEP amplitudes compared to the conventionally used C3 site.
The corticospinal tract is one of the most critical descending pathways originating from the motor cortex for voluntary motor behavior20,
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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
The authors are grateful to Dr. Yingchun Zhang, Professor in the Department of Biomedical Engineering at the University of Miami, for providing valuable guidance and expertise in conducting the EMG analysis.
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Electrode Gel | Soterix Medical Inc., New York, USA | https://soterixmedical.com/research/hd-tdcs/accessories/hd-gel | |
| EMG | TMSi, Oldenzaal, The Netherlands | https://www.tmsi.artinis.com/product-overview | |
| High-definition transcranial electrical stimulation (HD-tES) | Soterix Medical Inc., New York, USA | https://soterixmedical.com/research/hd-tdcs/4x1 | |
| TMS (Magstim Bistim² 200) | Magstim Company Ltd., Whitland, Wales, UK | 3231-00 |
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