Extensive mapping of somatosensory perception thresholds in the upper limb reveals an interaction between gender and stimulation position

Extensive mapping of somatosensory perception thresholds in the upper limb reveals an interaction between gender and stimulation position | 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 Extensive mapping of somatosensory perception thresholds in the upper limb reveals an interaction between gender and stimulation position Carolina Travassos, Alexandre Sayal, Bruno Direito, Paulo Fonte, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6043288/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Measuring perception thresholds in electrocutaneous stimulation offers valuable insights into sensory processing and supports the creation of personalized methods for diagnosing and treating somatosensory disorders. This study uses a custom non-invasive electrocutaneous stimulation device to test the impact of stimulation frequency, position along the upper limb, and participants’ gender on the perception thresholds. The device targeted 20 stimulation positions on the dorsal side of the right upper limb of 24 healthy participants. Perception thresholds for each participant and stimulation position were determined by a staircase procedure at two frequencies (30 Hz and 100 Hz). Our findings highlight the complex interplay between gender and stimulation position while suggesting that frequency does not significantly influence perception thresholds under these conditions. While males exhibited higher perception thresholds overall, the spatial pattern of perception thresholds along the upper limb thresholds were in general higher at the middle finger and hand compared to the forearm and upper arm. However, the interaction between gender and stimulation position indicates that the magnitude of these differences varies depending on the specific position. These results underscore the necessity of considering gender- and position-specific differences when analyzing somatosensory thresholds across the upper limb. Biological sciences/Neuroscience/Peripheral nervous system/Somatic system Biological sciences/Physiology/Neurophysiology Somatosensory cortex Perception thresholds Electrocutaneous stimulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Non-invasive electrocutaneous stimulation involves applying electrical pulses directly to the skin surface to stimulate the nerve endings, specifically mechanoreceptors, in the skin tissue 1 , 2 . Processing of non-invasive electrocutaneous stimulation in the human brain relies on the somatosensory information processing pathways, including sensory receptors, ascending pathways, and brain regions dedicated to interpreting and responding to these stimuli. Mechanoreceptors, specialized for processing the sense of touch, include Meissner’s Corpuscles (found exclusively in hairless/glabrous skin), Hair Units and Field Units (found only in hairy skin), Merkel’s Cells, Pacinian Corpuscles, and Ruffini Endings 1 – 4 . These receptors are located at specific depths in the skin tissue and exhibit distinct spatial and temporal resolutions, with each type of receptor being most responsive within specific frequency ranges 1 – 4 . Stimuli are then transmitted to the brain through the dorsal column pathway in the spinal cord, passing through the dorsal column nuclei and the ventral posterolateral nucleus of the thalamus, before reaching the primary somatosensory cortex (S1) 5 , 6 . Within S1, stimuli are processed in dedicated areas according to their topographic organization (somatotopy). Besides location information, S1 also processes simple features which are subsequently combined in higher-level areas to provide meaningful information 5 . Non-invasive electrocutaneous stimulation has been successfully applied in various fields, including the determination of somatosensory thresholds 7 – 12 , mapping the somatosensory cortex 6 , 13 – 17 , and developing electrotactile feedback systems, such as those used in prosthetic devices 1 , 18 . It is also a valid approach to explore the somatosensory hypersensitivity related to conditions like chronic pain (e.g., migraine) 19 , neurodevelopmental disorders (e.g., autism spectrum disorder and Tourette syndrome) 20 – 23 , and other conditions (e.g., obsessive-compulsive disorder) 24 , 25 . Electrocutaneous stimulation devices can create a range of sensations depending on the properties of the electrical signal (waveform, pulse frequency, amplitude, and width), the stimulation position (skin type, resistance, thickness, and hydration), and the electrodes (type, material, size, and shape), but also depending on the individual differences between participants (including factors like gender and age) 1 , 2 , 26 , 27 . Inadequate stimulation can lead to significant adverse effects for participants, including skin irritation, electric shock sensation, or even burns, making individualized calibration critical for safe application 1 , 2 . Most studies investigating somatosensory perception, attention, and intolerance/pain thresholds - defined as the levels at which stimuli induce noticeable sensations, capture the user's attention, or cause intolerable sensations, respectively - have focused on specific body areas, particularly the fingers and hands 7 – 12 . This limitation is often due to the low number of stimulation channels in commercially available stimulation devices and the heightened sensitivity of certain body regions that are represented in a larger somatosensory cortex area. According to the literature, the fingers and hands have the largest representation at the somatosensory cortex, making them a predominant area of study in human somatosensory research 6 . Consequently, research on the influence of a broader range of stimulation locations on electrocutaneous thresholds remains limited. For instance, a recent study by Dölker and colleagues utilized a commercially available stimulator with eight output channels positioned around the right upper arm to determine perception, attention, and intolerance thresholds 8 . They investigated how stimulation properties, such as pulse width, electrode size, and electrode position, affect these thresholds. Similarly, Geng and colleagues examined the impact of various stimulation parameters -including stimulation location, the number of simultaneously active electrodes, the number of stimulation pulses, and the time interval between stimulations at a pair of electrodes, on perception thresholds. This study used five electrodes positioned around the left forearm 7 . While the first study found no statistically significant differences in perception thresholds across stimulation positions around the upper arm, the second study reported statistically significant differences in perception thresholds across stimulation positions around the forearm. Another aspect that is reported to influence electrotactile thresholds is gender. However, studies that investigate gender-based differences in perception thresholds are also constrained by the limited number of stimulation channels. Seno and colleagues found higher perception thresholds in males compared to females by applying electrotactile stimulation on five electrodes on the forearm 10 . Furthermore, research on the effect of stimulation frequency on perception thresholds is also hindered by similar limitations in electrode configurations. A recent study, focusing on the perceptual characteristics of electrotactile feedback, investigated the influence of frequency on both perception and discomfort thresholds using only two electrodes on the finger 28 . Although they observed a trend toward decreasing thresholds as stimulation frequency increased from 10 Hz to 100 Hz, this trend was not statistically significant. On the other hand, they did find a significant association between the qualitative perception of the sensation reported and the increase in frequency 28 . The studies mentioned above highlight the influence of gender and stimulation position on perception threshold. However, they are limited in scope due to the focus on restricted body regions and the use of only a few stimulation channels. We aim to address this limitation by improving threshold mapping through increased spatial resolution and considering the entire upper limb. We follow the hypotheses that somatosensory perception thresholds depend on the stimulation position, subject gender, and stimulation frequency. To test this, we investigated how perception thresholds change across the dorsal side of the whole upper limb, accounting for sex differences and the stimulation frequency. Perception thresholds were determined following a staircase procedure using a custom stimulation device 29 . We also present a descriptive analysis of the participants’ perceptual responses ( subjective perceptions of electrical stimulation ) at the perception threshold intensity for each stimulation position and frequency. This threshold characterization is the first step for detailed and personalized human brain somatosensory mapping studies, using advanced neuroimaging techniques such as functional magnetic resonance imaging (fMRI). In summary, this comprehensive characterization of electrocutaneous perception thresholds across the dorsal side of the dominant upper limb in healthy participants highlighted the influence of gender and stimulation location, rather than frequency, on perception thresholds. Methods Participants Twenty-four healthy adult volunteers (12 females), aged between 21 and 39 years (mean age = 30.13 years, SD = 5.12) were included in this study. All participants were right-handed (mean laterality index: 85, SD = 10.72), as assessed through the Edinburgh inventory questionnaire 30 . All participants have no history of neuropsychological or psychiatric diseases, no deficits that prevent understanding and signing the informed consent, and no contraindications for performing electrocutaneous stimulation (e.g. skin lesions at the stimulation positions, suspected/diagnosed epilepsy and/or heart problems, and presence of active implantable medical devices). Additionally, participants were required to have upper limb dimensions sufficient for the application of stimulation electrodes (middle finger ≥ 6 cm, hand ≥ 8 cm, forearm ≥ 12 cm, and upper arm ≥ 14 cm). Inclusion criteria were verified in a screening interview previous to the stimulation session. Participants gave written informed consent so they could be included in the study, which was approved by the local Ethics Commission of the Faculty of Medicine of the University of Coimbra (CE-049/2021) and conducted following the Declaration of Helsinki. Experimental setup Stimulation setup Figure 1 depicts the schematic representation of the experimental setup implemented to determine the perception thresholds in the 20 electrodes placed at the dorsal side of the dominant upper limb using the custom-developed stimulation device. Electrode 1 is positioned at the tip of the middle finger, while electrode 20 is at the shoulder. The electrocutaneous stimulation device is a custom-built apparatus distinguished by its current-controlled, voltage-limited functionality and 20 independent stimulation channels 29 . It can generate positive rectangular stimulation signals with maximum energy per pulse of 6 mJ, within the range of 0 to 5 mA, a voltage up to 70 V, pulse widths between 0.2 and 5 ms, and frequencies up to 2.5 kHz). Previous work details its components, stimulus generation, operation procedures, and assessment 29 , 31 . Figure 1 - Schematic representation of the experimental setup implemented to determine somatosensory perception thresholds: electrical pulses, generated by the stimulation device according to the properties defined at the Experimental control computer, are delivered to each one of the 20 stimulation electrodes positioned on the dorsal side of the participant’s right upper limb: seven on the upper arm, six on the forearm, four on the hand, and three on the middle finger (distances between electrodes depend on the upper limb length of each participant). Participant preparation Before electrode placement at the dorsal side of the dominant upper limb, the participant's skin was carefully inspected for any injuries, as disruptions in the skin integrity could lead to unwanted pain during stimulation. Abrasive pads were used to remove non-conductive skin cells and to ensure low contact impedance at the electrodes’ attachment sites preparing the skin for stimulation 28 . Stimulation electrodes - Ag/AgCL laminated with a carbon composition contact area and a gel cavity (EL509, BIOPAC), were then placed at predetermined stimulation positions, spaced proportionately based on the length of each limb segment: middle finger, hand, forearm, and upper arm 34 . Anatomical landmarks were used to define each segment and measure its length, as illustrated in Fig. 2 . The length of the middle finger was measured from its tip to the base of the metacarpal bone. The hand was measured from the beginning of the carpal bones (at the base of the middle finger) to the midpoint of the wrist (at the level of the pisiform bone). The forearm was measured from the midpoint of the wrist to the midpoint of the elbow (at the level of the olecranon bone). The upper arm was measured from the midpoint of the elbow to the acromion bone. We utilized electrode conductor gel to improve conductivity 29 . The minimum distance between stimulation positions was determined based on literature regarding the two-point discrimination threshold for electrotactile sensation 35 . Therefore, the length of the upper limb served as an exclusion criteria. This criterion ensured that three electrodes could be appropriately positioned on the middle finger, four on the hand, six on the forearm, and seven on the upper arm, allowing for a minimum spacing of 1 cm between electrodes. Stimulation positions were registered using a navigation system (Localite), with an EEG PinPoint tool (Localite), which enabled us to digitalize the electrodes’ locations for each participant. After electrode placement, participants were instructed to sit in a comfortable position, relax their arm over the arm’s chair (all participants assumed the same position), and focus on perceiving the stimulation. Participants were instructed to strictly avoid contact between their left and right arms during stimulation, preventing a closed current loop through the heart. Upon concluding the experiment and removing all electrodes, the participants' skin was carefully examined for any signs of irritation, such as redness. Perception threshold determination - staircase procedure The determination of the perception thresholds wa s conducted using a staircase procedure 36 , 37 . Following a randomized stimulation sequence of electrodes, this interactive method identifies the threshold current (and corresponding voltage) for each stimulation position based on participants’ subjective judgments. The procedure started with current bursts of 2 mA, (lasting 4 s, with a pulse width of 0.2 ms; voltage depends on current) and adjusting them incrementally (either increasing or decreasing) based on the participant’s perception of the stimulation. If the participant reported feeling the stimulation, the current amplitude was decreased according to the step size; if the participant did not feel the stimulation, the current amplitude was increased. Step sizes for these adjustments were set at 1 mA, 0.5 mA, 0.25 mA, and 0.1 mA across successive stimulations. Participants could request repeated stimulation if they felt misjudged in their initial reports. Two frequencies were tested: 30 Hz and 100 Hz. These frequencies were chosen based on the literature(as frequencies around 20 Hz are typically most effective for sensory communication while avoiding discomfort) and the technical limitations of our stimulation device (which limited the maximum frequency to approximately 100 Hz with a pulse width of 0.2 ms) 4 , 7 , 29 . After determining the perception thresholds for all stimulation positions at a specific frequency, the frequency was changed, and the procedure was repeated for the other frequency. Half of the participants were randomly assigned to start with a frequency of 30 Hz, while the remaining started with 100 Hz. Perception thresholds determination, for all stimulation positions at each frequency, took about 15 minutes. Due to minor skin wounds, perception thresholds could not be determined for two participants at stimulation position 1 and for one participant at stimulation position 4. Additionally, it was also not feasible to determine the perception threshold for one participant at stimulation position 2 with a frequency of 30 Hz, as the participant did not perceive the stimulation even at the maximum intensity. Assessment of subjective perceptions at the perception threshold Once participants reached the perception thresholds for a given stimulation position, they were asked to describe their sensory experience, addressing quantitative, qualitative, spatial, and temporal dimensions 8 , 9 . In terms of quantitative perception , participants were asked to rate the intensity of the perceived sensation and pain on a visual analog scale (VAS) from 0 (no perceptible sensation/pain) to 10 (worst sensation/pain). Regarding qualitative perception , participants selected a single descriptor that best characterized the sensation perceived 8 : knocking, scratching, stinging, muscle twitch, tickling, itching, or pinching. These descriptors were selected based on the literature. Participants also had the option to use alternative adjectives if needed (termed as “Other” in the results). Spatial perception required participants to identify where the sensation was perceived, choosing from the following options: at a specific electrode, between electrodes, at two or more electrodes, extending beyond electrodes, or at other parts of the body. It was requested that the chosen electrode(s) or body part be pointed out. Lastly, regarding temporal perception , participants reported whether they could identify the start and end of the stimulation and whether it felt continuous or exhibited any discernible pattern (they could choose between intermittent at the same intensity, intermittent with intensity decreasing/increasing over time, and intermittent that initially increased/decreased then decreased/increased). Before the experiment began, participants were briefed on the questionnaire's assessment and the meaning of the perception threshold (but not the procedure to achieve it). Any inquiries from participants were addressed to ensure clarity. The overall experiment lasted approximately 90 minutes, including participant preparation (around 30 minutes), perception thresholds determination, and participants’ reports. Statistical Analysis To investigate how stimulation positions, stimulation frequency, and participants’ gender influenced the perception thresholds we used linear mixed-effect models (LMMs). LMMs offer a powerful statistical framework for analyzing cognitive neuroscience data since they accommodate both fixed and random effects, i.e. , effects where all levels of interest are included in the experiment (responsible for systematic variability in the data) and effects that have a non-systematic, idiosyncratic, unpredictable or random influence on the data, respectively 38 , 39 . In our case, stimulation position, stimulation frequency, and gender were considered fixed effects (or independent variables) and perception threshold was the dependent variable. Participants (participant ID) were considered as random effects, with a random intercept included for each participant to account for individual differences in baseline perception levels. This approach allowed us to model individual variability in perception thresholds that could not be explained by fixed effects, thereby minimizing the influence of such variability on the results 38 , 39 . To analyze the contributions of gender, stimulation positions, and stimulation frequency on perception thresholds, as well as possible interactions between these factors, we created two LMMs, each designed to capture different levels of spatial resolution in the analysis of stimulation positions: Model A - Stimulation positions were grouped according to segments of the upper limb (middle finger, hand, forearm, and upper arm), resulting in four levels for this variable (hereafter referred to as “Stimulation segment”). In this model, the individual contributions of the electrodes of each segment were averaged, allowing us to assess broader, segmental effects on perception thresholds. Model B - Each stimulation position was analyzed separately, with the variable “Stimulation positions” consisting of 20 levels, corresponding to the stimulation sites along the upper limb (0 for the tip of the middle finger and 20 for the last electrode at the shoulder). This model enabled a fine-grained analysis of spatial resolution and the influence of individual stimulation sites. By employing both models, we addressed our research question from complementary perspectives: Model A captured segmental-level statistical effects, while Model B provided position-specific insights into the role of spatial resolution in perception thresholds. In both models, we considered age as a covariate. When a significant effect was found, individual pairs of conditions were further compared using multiple pairwise comparisons with Bonferroni adjustment. The experimental results were analyzed using SPSS software (IBM, SPSS Statistics, version 28). Results Somatosensory Electrocutaneous Perception Thresholds Figure 3 - Distribution of the perception thresholds for each stimulation position (represented as electrodes 1 to 20) for 30 Hz (a) and 100 Hz (b) and female (orange) and male (blue) participants. Using Model A, we observed that both gender and stimulation segment have a significant impact on perception threshold (gender: F (1, 24.532) = 14.822, p = 7.460 \(\:\times\:{10}^{-4}\) , and stimulation segment: F (3, 929.097) = 237.795, p = 1.812 \(\:\times\:{10}^{-114}\) ). Additionally, a significant interaction between these factors was found ( F (3, 929.092) = 4.588, p = 0.003). Since frequency did not reach statistical significance ( F (1, 929.011) = 0.198, p = 0.656), it was removed from the model. After removing frequency and rerunning the model, both stimulation segments ( F (3, 929.092) = 237.106, p = 3.252 \(\:\times\:{10}^{-114}\) ), gender ( F (1, 24.528) = 14.802, p = 7.52 \(\:\times\:{10}^{-4}\) ) and their interaction ( F (3, 929.087) = 4.578, p = 0.003) remained significant. Given the significant interaction revealed by the mixed-effects analyses, we performed post hoc tests with Bonferroni correction for multiple comparisons. When fixing the stimulation segment, perception thresholds were significantly higher in males than females for electrodes at the middle finger ( p = 0.006), forearm ( p = 1.900 \(\:\times\:{10}^{-5}\) , and upper arm ( p = 4.690 \(\:\times\:{10}^{-4}\) ) - Fig. 4 (a). However, no significant gender difference was observed for the hand ( p = 0.050). For more details see Supplementary Table 1. Conversely, when fixing gender, post hoc tests revealed that perception thresholds were significantly higher at the middle finger and hand compared to the forearm and upper arm for both genders, as well as at the forearm compared to the upper arm for males - Fig. 4 (b). No statistical significance difference was found between the middle finger and hand for both genders. For more details see Supplementary Table 2. Considering Model B, we also found a significant impact of gender ( F (1, 24.005) = 8.289, p = 0.008) and stimulation position ( F (19, 872) = 61.504, p = 2.292 \(\:\times\:{10}^{-146}\) ), as well as a significant interaction between these factors ( F (19, 872) = 2.237, p = 0.002). Frequency did not reach statistical significance ( F (1, 872) = 0.027, p = 0.869), therefore it was removed from the model, which was then rerun. The results of the revised model were consistent with the previous one, with stimulation position, gender, and their interaction remaining statistically significant (stimulation position: F (19, 912) = 59.888, p = 1.014 \(\:\times\:{10}^{-145}\) ); gender ( F (1, 24.005) = 8.265, p = 0.008; interaction ( F (19, 912) = 2.189, p = 0.002). These results indicate that the effect of stimulation position and gender on perception thresholds cannot be interpreted isolatedly, since their effects interact. Post hoc analyses, corrected using Bonferroni for multiple comparisons, revealed that, when fixing the stimulation position, perception thresholds were significantly higher in males compared to females for all stimulation positions except one electrode at the middle finger (E2) and three electrodes at the hand (E4, E5, and E6) - Fig. 5 . Supplementary Table 3 details these results. On the other hand, when controlling for gender, post hoc tests revealed several significant differences between stimulation positions. The findings are consistent with those reported in model A. Detailed results can be found in Supplementary Table 4. Supplementary Fig. 1 shows the distribution of the amplitude of perception thresholds based on each stimulation site for both genders, regardless of frequency. Descriptive report of subjective perceptions at the perception threshold The assessment of subjective sensation intensity caused by stimulation at this level serves as a sanity check measure. The median sensation intensities reported are predominantly marked by low-intensity quantification (Supplementary Table 5). We also evaluated pain perception and found a median pain intensity of zero (independent of gender, frequency, and stimulation position). However, ten participants (five females) reported pain at one stimulation position (intensity of 1 on a 1–10 scale), regardless of the frequency. Additionally, four participants (one female) experienced pain at multiple positions, all rating their pain as 1 on a 10-point scale, except for one participant who rated it as 2. Regarding the qualitative aspects of subjects’ perception, we found that the most frequent descriptors were “Stinging”, closely followed by “Tickling”, and “Muscle twitch” (regardless of gender, stimulation position, and stimulation frequency). Supplementary Fig. 2 illustrates the distribution of qualitative descriptors for each stimulation position, gender, and stimulation frequency. Spatially, the most frequently reported spatial perception was “At the Active/Ground electrode", indicating that participants commonly felt the sensation at the electrode designated as the active or ground. These results were consistent across both genders and frequencies. Detailed results by gender, stimulation position, and stimulation frequency are available in Supplementary Fig. 3. When participants reported feeling the stimulation at a specific electrode, and this was the active or ground electrodes, we also examined at which one the sensation was felt - Supplementary Fig. 4. Participants successfully identified the onset and offset of stimulation in approximately 83% and 80% of cases, respectively. Most participants (75%) reported experiencing a continuous sensation when asked about the stimulation pattern. Other patterns identified included intermittent sensations (13%), sensations that decreased intensity over time (9%), sensations that increased intensity over time (2%), and sensations that initially increased and then decreased intensity (1%). Around 14% of participants did not respond to this question. Regarding the identification of the two different stimulation frequencies, only four participants (three of whom were male) were able to distinguish between them. Discussion This work is the first to explore the location-dependent variation of perception thresholds across the entire upper limb, using LMMs to capture both segmental and position-specific statistical effects. We investigated the impact of gender and stimulation frequency on these thresholds and found main effects of both factors and a significant interaction between them. The results were consistent across both statistical models, whether considering segmental (model A) or position-specific stimulation locations (model B). The first analysis model, which considered four segments of the upper limb (middle finger, hand, forearm, and upper arm), revealed the interaction between gender vs stimulation position at a regional level, providing a broader view of how perception thresholds differ by gender across major anatomical areas of the upper limb. The second analysis model, which considered individual stimulation positions, revealed where these gender differences were most pronounced within each segment, underscoring a more detailed sensitivity pattern that would be missed at the segment level. Both models consistently revealed significant effects of gender and stimulation position, as well as, their interaction. On the other hand, frequency did not have a statistically significant impact. In general, males have higher perception thresholds than females across most stimulation positions, except for the hand in Model A and select electrode positions in Model B, more specifically, at the second electrode on the middle finger and the first three electrodes on the hand. Regarding the overall pattern of perception thresholds through the upper limb, the trend was similar for both genders, with higher thresholds observed at the middle finger and hand (with no statistically significant difference between them) compared to the forearm and arm. Additionally, males have higher perception thresholds at the forearm than the arm. The results above could be attributed to both peripherical and central factors that differ between genders and across the upper limb. First, the epidermal thickness is different across the upper limb and between genders. According to a systematic review, thickness is lower at the dorsal forearm and much higher at the fingers 40 . Higher thickness results in increased skin impedance, indicative of greater resistance to the flow of an electric current 1 . Consequently, higher current intensities are needed to evoke perceived sensations. Our results corroborate this pattern, as we observed higher perception thresholds at the middle finger for both genders. Regarding differences between genders, it is known that women typically have thinner skin compared to men, resulting in lower skin impedance and, therefore, lower perception thresholds 1 , 7 , 41 . Previous research about morphological differences between genders suggests that the higher body fat percentage in females could facilitate the recruitment of nerve fibers, leading to lower perception thresholds 7 , 42 . The literature also reports a lower density of nerve fibers in men, which could also account for their higher perception thresholds 43 . Regarding nerve fiber distribution throughout the upper limb, the most significant discrepancies are observed at the fingertips and the palmar side of the hand compared to the upper arm, regardless of gender 3 , 6 . Therefore, we did not expect direct interference with perception thresholds determined at the dorsal side of the upper limb. Beyond these peripheral differences, central nervous system mechanisms also play a key role in shaping somatosensory perception. Somatosensory perception involves not only the somatosensory cortex but is also modulated by other brain areas, such as that involved in cognition (e.g., attention) and emotion (e.g. stress) 44 – 46 . These interconnected brain regions contribute to the integration, modulation, and interpretation of sensory inputs, ultimately shaping perceptual experience and behavioral responses. Neuroimaging research suggests that gender differences in somatosensory perception may be partly attributed to variations in cortical organization, connectivity, and structural differences in the size and thickness of the brain. Building on Penfield’s Homunculus, which represents a male figure, researchers have explored the possibility of creating a "hermunculus" to better explain the somatotopic organization of the female body 47 . However, current studies have predominantly focused on the genitalia and sexual function of women, leaving the broader somatosensory representation of the female body largely unexplored 47 . Regarding connectivity patterns, the literature highlights decreased functional connectivity in the parietal cortex of females, particularly within the sensorimotor network 48 – 51 . Zhang and colleagues further demonstrated that these lower functional connectivities in females were correlated with physical differences, such as body fat mass 48 . Concerning structural differences, a study performed on data from 5216 UK Biobank found that females generally exhibited greater cortical thickness in somatosensory regions, whereas males showed relatively higher volume and surface area in these regions 50 . This study also noted that gender differences in functional connectivity persisted even after controlling for brain size, especially in sensory and association areas 50 . These findings suggest that males and females may process somatosensory information in distinct ways. However, to the best of our knowledge, there is no evidence linking these structural and functional differences to perceptual outcomes in somatosensory stimulation. Future neuroimaging research exploring gender differences in the neuronal pathways of somatosensory processing could provide important insights into the mechanisms underlying the perceptual differences observed in this study. Additionally, several other factors reported in the literature have implications for the electrode-skin interface and may contribute to variations in perception thresholds. These include skin moisture content (or hydration level), as the higher water content in the skin enhances its ability to conduct electrical currents 10 ; and exposure to external factors, which can lead to variations in sensitivity. For example, fingers and hands, often unprotected and frequently used throughout the day, may develop tougher, less sensitive skin than the arms, typically protected by clothes 52 . However, this study did not incorporate additional measures to correlate these factors with our findings directly. Lastly, stimulation frequency did not impact the somatosensory perception thresholds recorded, aligning with previous findings that frequency primarily influences the quality of perceived sensations (due to the targeted mechanoreceptors) rather than threshold intensity 1 , 2 . Participants' subjective perceptions showed low sensation intensity, as expected, with some minor discomfort reported (1–2 on a 0–10 scale), likely due to individual sensitivity differences. The most common qualitative perceptions were stinging, ticking, and muscle twitch. This is primarily influenced by the type of mechanoreceptors activated, but also by other factors such as stimulation parameters, electrodes, and physiological variables, making a direct comparison with existing literature difficult 1 , 2 . Spatially, perception remained mostly localized to the active or ground electrode sites, with minimal mislocalization, particularly in the forearm and upper arm, where larger receptive fields may explain the effect 53 . This finding aligns with expectations and highlights the high spatial acuity of the evoked sensations. Regarding temporal perception, good onset and offset identification of the stimulation was achieved. However, only four participants could differentiate between the two stimulation frequencies tested. Despite the contributions here provided, some experimental steps contributed to limitations in our conclusions. For example, while the method of constant stimuli is generally regarded as the most reliable approach for estimating perception thresholds due to its ability to minimize biases from participant anticipation, it is also notably time-consuming 54 . Given the large number of electrodes and the risk of reduced participant attention during prolonged experiments, this method was impractical for our study. Instead, we opted for the staircase method, carefully selecting the step size. Larger step sizes risk overestimating the threshold, while smaller step sizes could underestimate it. Drawing on previous research, we chose a smaller step size of 0.1 mA 7 , 42 . This approach allowed us to determine the perception thresholds for 20 electrodes in approximately 15 minutes, yielding results consistent with existing literature. Additionally, although VAS is very intuitive, the results obtained are subject-specific therefore the overall conclusion may be misleading, as two subjects may rate identical stimuli with different VAS scores. Lastly, we did not assess the skin sensation deficits of our participants, so we can not discard any influence from this. Future studies should aim to integrate the findings from this work to develop optimized somatosensory mapping protocols of the cortical representation of the upper limb at S1. Additionally, these studies should investigate potential processing differences in sensory-dedicated brain regions, focusing on how these differences may affect somatosensory perception. It would also be valuable to investigate somatosensory perception thresholds in clinical populations with known impaired somatosensory processing, such as psychiatric, neurodevelopmental, and/or chronic pain disorders, to shed light on the associated somatosensory processing alterations 22 , 23 , 25 . Investigating somatosensory processing in clinical populations may also provide crucial insights into how these findings can be translated into therapeutic interventions, ultimately improving treatment strategies for individuals with sensory processing deficits. Conclusions This study provides valuable insights into how perception thresholds are influenced by stimulation location and gender across the entire upper limb in healthy participants considering broader (based on upper limb segments) and fine-tuned (based on individual stimulation positions) approaches. Our findings revealed that perception thresholds vary significantly along the dorsal side of the dominant upper limb depending on gender, highlighting the need for customized current intensities to accommodate such differences. In contrast, stimulation frequency had no significant effect on these thresholds. These results enhance our understanding of the factors affecting electrocutaneous stimulation and contribute to the growing knowledge of how to effectively implement electrocutaneous stimulation across various fields. Declarations Data Availability The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. Acknowledgments The authors would like to thank all participants who took part in this study. Author contributions CT, AS, BD, TS, and MC-B conceived and designed the study. CT conducted the experiments, analyzed the data, and wrote the manuscript. CT, AS, and PF designed and developed the somatosensory electrical stimulator used in this study. CT and AS developed the code for the threshold definition staircase approach. All authors reviewed the manuscript and approved the final version. Funding This research was funded by FCT (UIDB/04950/2020, UIDP/04950/2020). CT and AS were funded by Siemens Healthineers Portugal and the FCT PhD fellowship 2023.04160.BD (CT) and 2023.04365.BD (AS). BD is funded by FCT (CEECINST/00117/2021/CP2784/CT0002). TS is funded by 2021.01469.CEECIND. Competing interests The authors declare no competing interests. References Kourtesis, P., Argelaguet, F., Vizcay, S., Marchal, M. & Pacchierotti, C. Electrotactile Feedback Applications for Hand and Arm Interactions: A Systematic Review, Meta-Analysis, and Future Directions. IEEE Trans. Haptics 15 , 479–496 (2022). Zhou, Z. et al. Electrotactile Perception Properties and Its Applications: A Review. IEEE Trans. Haptics 15 , 464–478 (2022). 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Perception thresholds and qualitative perceptions for electrocutaneous stimulation. Sci. Rep. 12 , 7335 (2022). Dölker, E.-M. et al. Sensation thresholds in electrocutaneous stimulation. Curr. Dir. Biomed. Eng. 6 , 372–375 (2020). Seno, S., Shimazu, H., Kogure, E., Watanabe, A. & Kobayashi, H. Factors Affecting and Adjustments for Sex Differences in Current Perception Threshold With Transcutaneous Electrical Stimulation in Healthy Subjects. Neuromodulation Technol. Neural Interface 22 , 573–579 (2019). Geng, B., Yoshida, K., Petrini, L. & Jensen, W. Evaluation of sensation evoked by electrocutaneous stimulation on forearm in nondisabled subjects. J. Rehabil. Res. Dev. 49 , 297 (2012). Zhou, Z., Wang, X., Yang, Y., Zeng, J. & Liu, H. Exploring Perceptual Intensity Properties Using Electrotactile Stimulation on Fingertips. IEEE Trans. Haptics 16 , 805–815 (2023). Kurth, R. et al. fMRI shows multiple somatotopic digit representations in human primary somatosensory cortex: NeuroReport 11 , 1487–1491 (2000). Deuchert, M. et al. Event-related fMRI of the somatosensory system using electrical ¢nger stimulation. 5. Blankenburg, F. Evidence for a Rostral-to-Caudal Somatotopic Organization in Human Primary Somatosensory Cortex with Mirror-reversal in Areas 3b and 1. Cereb. Cortex 13 , 987–993 (2003). Hartwig, V. et al. A Compatible Electrocutaneous Display for functional Magnetic Resonance Imaging application. in 2006 International Conference of the IEEE Engineering in Medicine and Biology Society 1021–1024 (IEEE, New York, NY, 2006). doi:10.1109/IEMBS.2006.260279. Roux, F.-E., Djidjeli, I., Durand, J.-B., Taylor, J. & Carson, R. Functional architecture of the somatosensory homunculus detected by electrostimulation. J Physiol 16 (2017). Malešević, N. & Antfolk, C. Sensory feedback in upper limb prosthetics: advances and challenges. Nat. Rev. Neurol. 20 , 449–450 (2024). Hodkinson, D. J. et al. Primary Somatosensory Cortices Contain Altered Patterns of Regional Cerebral Blood Flow in the Interictal Phase of Migraine. PLOS ONE 10 , e0137971 (2015). Isaacs, D. & Riordan, H. Sensory hypersensitivity in Tourette syndrome: A review. Brain Dev. 42 , 627–638 (2020). Baum, S. H., Stevenson, R. A. & Wallace, M. T. Behavioral, perceptual, and neural alterations in sensory and multisensory function in autism spectrum disorder. Prog. Neurobiol. 134 , 140–160 (2015). Balasco, L., Provenzano, G. & Bozzi, Y. Sensory Abnormalities in Autism Spectrum Disorders: A Focus on the Tactile Domain, From Genetic Mouse Models to the Clinic. Front. Psychiatry 10 , 1016 (2020). Robertson, C. E. & Baron-Cohen, S. Sensory perception in autism. Nat. Rev. Neurosci. 18 , 671–684 (2017). Isaacs, D. et al. Sensory Hypersensitivity Severity and Association with Obsessive-Compulsive Symptoms in Adults with Tic Disorder. Neuropsychiatr. Dis. Treat. Volume 16 , 2591–2601 (2020). Van Den Boogert, F. et al. Sensory processing difficulties in psychiatric disorders: A meta-analysis. J. Psychiatr. Res. 151 , 173–180 (2022). Szeto, A. Y. J. & Saunders, F. A. Electrocutaneous Stimulation for Sensory Communication in Rehabilitation Engineering. IEEE Trans. Biomed. Eng. BME-29 , 300–308 (1982). Pamungkas, D. S. & Caesarendra, W. Overview Electrotactile Feedback for Enhancing Human Computer Interface. J. Phys. Conf. Ser. 1007 , 012001 (2018). Manoharan, S. & Park, H. Characterization of perception by transcutaneous electrical Stimulation in terms of tingling intensity and temporal dynamics. Biomed. Eng. Lett. 14 , 35–44 (2024). Travassos, C. et al. Development and assessment of a new multichannel electrocutaneous device for non-invasive somatosensory stimulation for magnetic resonance applications. Preprint at https://doi.org/10.1101/2024.05.27.595320 (2024). Oldfield, R. C. The assessment and analysis of handedness: The Edinburgh inventory. Neuropsychologia 9 , 97–113 (1971). Travassos, C. et al. Assessing MR-compatibility of somatosensory stimulation devices: A systematic review on testing methodologies. Front. Neurosci. 17 , 1071749 (2023). BIOPAC Systems, Inc. ELPAD - Abrasive Pads 10/PK. https://www.biopac.com/product/abrasive-pads-10pk/. BIOPAC Systems, Inc. Gel101A - Electrode Gel, Isotonic, 114g. https://www.biopac.com/product/electrode-gel-isotonic-114-g/. BIOPAC Systems, Inc. EL509 - Disposable RT Dry Electrodes. https://www.biopac.com/product/disp-rt-dry-electrode-100pk/. Solomonow, M., Lyman, J. & Freedy, A. Electrotactile two-point discrimination as a function of frequency, body site, laterality, and stimulation codes. Ann. Biomed. Eng. 5 , 47–60 (1977). Cornsweet, T. N. The Staircase-Method in Psychophysics. Am. J. Psychol. 75 , 485 (1962). Doll, R. J., Buitenweg, J. R., Meijer, H. G. E. & Veltink, P. H. Tracking of nociceptive thresholds using adaptive psychophysical methods. Behav. Res. Methods 46 , 55–66 (2014). Müller, S., Scealy, J. L. & Welsh, A. H. Model Selection in Linear Mixed Models. Stat. Sci. 28 , (2013). Scandola, M. & Tidoni, E. Reliability and Feasibility of Linear Mixed Models in Fully Crossed Experimental Designs. Adv. Methods Pract. Psychol. Sci. 7 , 25152459231214454 (2024). Lintzeri, D. A., Karimian, N., Blume‐Peytavi, U. & Kottner, J. Epidermal thickness in healthy humans: a systematic review and meta‐analysis. J. Eur. Acad. Dermatol. Venereol. 36 , 1191–1200 (2022). Sandby-Møller, J., Poulsen, T. & Wulf, H. C. Epidermal Thickness at Different Body Sites: Relationship to Age, Gender, Pigmentation, Blood Content, Skin Type and Smoking Habits. Acta Derm. Venereol. 83 , 410–413 (2003). Maffiuletti, N. A., Herrero, A. J., Jubeau, M., Impellizzeri, F. M. & Bizzini, M. Differences in electrical stimulation thresholds between men and women. Ann. Neurol. 63 , 507–512 (2008). Gøransson, L. G., Mellgren, S. I., Lindal, S. & Omdal, R. The effect of age and gender on epidermal nerve fiber density. Neurology 62 , 774–777 (2004). Wang, L.-H., Ding, W.-Q. & Sun, Y.-G. Spinal ascending pathways for somatosensory information processing. Trends Neurosci. 45 , 594–607 (2022). Gomez-Ramirez, M., Hysaj, K. & Niebur, E. Neural mechanisms of selective attention in the somatosensory system. J. Neurophysiol. 116 , 1218–1231 (2016). Kragel, P. A. & LaBar, K. S. Somatosensory Representations Link the Perception of Emotional Expressions and Sensory Experience. eneuro 3 , ENEURO.0090-15.2016 (2016). Di Noto, P. M., Newman, L., Wall, S. & Einstein, G. The Hermunculus: What Is Known about the Representation of the Female Body in the Brain? Cereb. Cortex 23 , 1005–1013 (2013). Zhang, R., Rolls, E. T., Cheng, W. & Feng, J. Different cortical connectivities in human females and males relate to differences in strength and body composition, reward and emotional systems, and memory. Brain Struct. Funct. 229 , 47–61 (2023). Zhang, C. et al. Sex and Age Effects of Functional Connectivity in Early Adulthood. Brain Connect. 6 , 700–713 (2016). Ritchie, S. J. et al. Sex Differences in the Adult Human Brain: Evidence from 5216 UK Biobank Participants. Cereb. Cortex 28 , 2959–2975 (2018). Serio, B. et al. Sex differences in functional cortical organization reflect differences in network topology rather than cortical morphometry. Nat. Commun. 15 , 7714 (2024). Samain-Aupic, L., Dione, M., Ribot-Ciscar, E., Ackerley, R. & Aimonetti, J.-M. Relations between tactile sensitivity of the finger, arm, and cheek skin over the lifespan showing decline only on the finger. Front. Aging Neurosci. 16 , 1387136 (2024). Vallbo, A. B., Olausson, H., Wessberg, J. & Kakuda, N. Receptive field characteristics of tactile units with myelinated afferents in hairy skin of human subjects. J. Physiol. 483 , 783–795 (1995). Ehrenstein, W. H. & Ehrenstein, A. Psychophysical methods. in Modern techniques in neuroscience research 1211–1241 (Springer, Berlin, 1999). Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformationv5.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 07 Nov, 2025 Reviews received at journal 05 Apr, 2025 Reviews received at journal 24 Mar, 2025 Reviewers agreed at journal 20 Mar, 2025 Reviewers agreed at journal 20 Mar, 2025 Reviewers agreed at journal 20 Mar, 2025 Reviewers invited by journal 09 Mar, 2025 Editor assigned by journal 28 Feb, 2025 Editor invited by journal 20 Feb, 2025 Submission checks completed at journal 19 Feb, 2025 First submitted to journal 16 Feb, 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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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6043288","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":418551287,"identity":"daf9271f-314a-4caf-980a-149bc67c26bf","order_by":0,"name":"Carolina Travassos","email":"","orcid":"","institution":"University of Coimbra (UC)","correspondingAuthor":false,"prefix":"","firstName":"Carolina","middleName":"","lastName":"Travassos","suffix":""},{"id":418551288,"identity":"e38b8262-b57c-44b8-a633-2b818073fd42","order_by":1,"name":"Alexandre Sayal","email":"","orcid":"","institution":"University of Coimbra (UC)","correspondingAuthor":false,"prefix":"","firstName":"Alexandre","middleName":"","lastName":"Sayal","suffix":""},{"id":418551289,"identity":"ef620ef9-19d6-47fe-a5aa-a9200ef8aeeb","order_by":2,"name":"Bruno Direito","email":"","orcid":"","institution":"University of Coimbra (UC)","correspondingAuthor":false,"prefix":"","firstName":"Bruno","middleName":"","lastName":"Direito","suffix":""},{"id":418551290,"identity":"c488e61a-7fa1-47b6-b1e3-ac7f42f93b39","order_by":3,"name":"Paulo Fonte","email":"","orcid":"","institution":"University of Coimbra (UC)","correspondingAuthor":false,"prefix":"","firstName":"Paulo","middleName":"","lastName":"Fonte","suffix":""},{"id":418551291,"identity":"5d02c0b8-800c-47dd-b52f-630faa6fce11","order_by":4,"name":"Teresa Sousa","email":"","orcid":"","institution":"University of Coimbra (UC)","correspondingAuthor":false,"prefix":"","firstName":"Teresa","middleName":"","lastName":"Sousa","suffix":""},{"id":418551292,"identity":"749b9f6c-d6c1-4b04-8740-0ba06d441582","order_by":5,"name":"Miguel Castelo-Branco","email":"data:image/png;base64,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","orcid":"","institution":"University of Coimbra (UC)","correspondingAuthor":true,"prefix":"","firstName":"Miguel","middleName":"","lastName":"Castelo-Branco","suffix":""}],"badges":[],"createdAt":"2025-02-16 23:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6043288/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6043288/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77102124,"identity":"0662d30b-c3ec-4169-9337-321bc439eea4","added_by":"auto","created_at":"2025-02-25 07:30:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":248010,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the experimental setup implemented to determine somatosensory perception thresholds: electrical pulses, generated by the stimulation device according to the properties defined at the Experimental control computer, are delivered to each one of the 20 stimulation electrodes positioned on the dorsal side of the participant’s right upper limb: seven on the upper arm, six on the forearm, four on the hand, and three on the middle finger (distances between electrodes depend on the upper limb length of each participant).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6043288/v1/234b5d43e2dcd724e1920ff0.png"},{"id":77100817,"identity":"517b842e-c531-4baf-9633-233b325807bd","added_by":"auto","created_at":"2025-02-25 07:22:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":434926,"visible":true,"origin":"","legend":"\u003cp\u003eAnatomical landmarks used for defining and measuring the upper limb segments (middle finger, hand, forearm, and upper arm) to enable accurate electrode placement.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6043288/v1/c6ca44c3d2e89aa271a4c959.png"},{"id":77100495,"identity":"93f081f2-f121-479a-ac49-401b34294a4b","added_by":"auto","created_at":"2025-02-25 07:14:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":164576,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of the perception thresholds for each stimulation position (represented as electrodes 1 to 20) for 30 Hz (a) and 100 Hz (b) and female (orange) and male (blue) participants.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6043288/v1/e8e19a94dd964daa9a5f4a5f.png"},{"id":77100490,"identity":"9bff21cd-9701-4322-8da7-78bc991e1f8c","added_by":"auto","created_at":"2025-02-25 07:14:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":132263,"visible":true,"origin":"","legend":"\u003cp\u003eEstimated marginal means of perception thresholds for females (orange) and males (blue) over the stimulation segments (middle finger, hand, forearm, and upper arm). Error bars represented the standard error of the mean (SEM). Significant differences between genders at each electrode (a) and between stimulation segments for each gender (b) are signalized with asterisks (**\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001), being corrected for multiple comparisons using the Bonferroni method.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6043288/v1/c125340d948668e6ba0e441a.png"},{"id":77102527,"identity":"a45e333c-c700-45a6-937e-8d27be6714cf","added_by":"auto","created_at":"2025-02-25 07:38:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":131327,"visible":true,"origin":"","legend":"\u003cp\u003eEstimated marginal means of perception thresholds for females (orange) and males (blue) over stimulation positions (represented as electrodes E1 to E20). Error bars represented the standard error of the mean (SEM). Significant differences between genders at each stimulation position are signalized with asterisks (*\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001), being corrected for multiple comparisons using the Bonferroni method.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6043288/v1/79519a0d00508ce29ef5a389.png"},{"id":77103525,"identity":"d660ad93-0202-4799-8bb5-50da6c521f62","added_by":"auto","created_at":"2025-02-25 07:46:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1934231,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6043288/v1/44460062-906f-4d98-b605-3a8c495ee65e.pdf"},{"id":77100488,"identity":"0992b187-6ab0-4745-9d9c-e33424263979","added_by":"auto","created_at":"2025-02-25 07:14:13","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":992111,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationv5.docx","url":"https://assets-eu.researchsquare.com/files/rs-6043288/v1/db29831460999170668b5ad8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Extensive mapping of somatosensory perception thresholds in the upper limb reveals an interaction between gender and stimulation position","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNon-invasive electrocutaneous stimulation involves applying electrical pulses directly to the skin surface to stimulate the nerve endings, specifically mechanoreceptors, in the skin tissue\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Processing of non-invasive electrocutaneous stimulation in the human brain relies on the somatosensory information processing pathways, including sensory receptors, ascending pathways, and brain regions dedicated to interpreting and responding to these stimuli. Mechanoreceptors, specialized for processing the sense of touch, include Meissner\u0026rsquo;s Corpuscles (found exclusively in hairless/glabrous skin), Hair Units and Field Units (found only in hairy skin), Merkel\u0026rsquo;s Cells, Pacinian Corpuscles, and Ruffini Endings \u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. These receptors are located at specific depths in the skin tissue and exhibit distinct spatial and temporal resolutions, with each type of receptor being most responsive within specific frequency ranges\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Stimuli are then transmitted to the brain through the dorsal column pathway in the spinal cord, passing through the dorsal column nuclei and the ventral posterolateral nucleus of the thalamus, before reaching the primary somatosensory cortex (S1)\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Within S1, stimuli are processed in dedicated areas according to their topographic organization (somatotopy). Besides location information, S1 also processes simple features which are subsequently combined in higher-level areas to provide meaningful information\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNon-invasive electrocutaneous stimulation has been successfully applied in various fields, including the determination of somatosensory thresholds\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, mapping the somatosensory cortex \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, and developing electrotactile feedback systems, such as those used in prosthetic devices\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. It is also a valid approach to explore the somatosensory hypersensitivity related to conditions like chronic pain (e.g., migraine)\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, neurodevelopmental disorders (e.g., autism spectrum disorder and Tourette syndrome)\u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, and other conditions (e.g., obsessive-compulsive disorder)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eElectrocutaneous stimulation devices can create a range of sensations depending on the properties of the electrical signal (waveform, pulse frequency, amplitude, and width), the stimulation position (skin type, resistance, thickness, and hydration), and the electrodes (type, material, size, and shape), but also depending on the individual differences between participants (including factors like gender and age)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Inadequate stimulation can lead to significant adverse effects for participants, including skin irritation, electric shock sensation, or even burns, making individualized calibration critical for safe application\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Most studies investigating somatosensory perception, attention, and intolerance/pain thresholds - defined as the levels at which stimuli induce noticeable sensations, capture the user's attention, or cause intolerable sensations, respectively - have focused on specific body areas, particularly the fingers and hands\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. This limitation is often due to the low number of stimulation channels in commercially available stimulation devices and the heightened sensitivity of certain body regions that are represented in a larger somatosensory cortex area. According to the literature, the fingers and hands have the largest representation at the somatosensory cortex, making them a predominant area of study in human somatosensory research\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Consequently, research on the influence of a broader range of stimulation locations on electrocutaneous thresholds remains limited. For instance, a recent study by D\u0026ouml;lker and colleagues utilized a commercially available stimulator with eight output channels positioned around the right upper arm to determine perception, attention, and intolerance thresholds\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. They investigated how stimulation properties, such as pulse width, electrode size, and electrode position, affect these thresholds. Similarly, Geng and colleagues examined the impact of various stimulation parameters -including stimulation location, the number of simultaneously active electrodes, the number of stimulation pulses, and the time interval between stimulations at a pair of electrodes, on perception thresholds. This study used five electrodes positioned around the left forearm\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. While the first study found no statistically significant differences in perception thresholds across stimulation positions around the upper arm, the second study reported statistically significant differences in perception thresholds across stimulation positions around the forearm. Another aspect that is reported to influence electrotactile thresholds is gender. However, studies that investigate gender-based differences in perception thresholds are also constrained by the limited number of stimulation channels. Seno and colleagues found higher perception thresholds in males compared to females by applying electrotactile stimulation on five electrodes on the forearm\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Furthermore, research on the effect of stimulation frequency on perception thresholds is also hindered by similar limitations in electrode configurations. A recent study, focusing on the perceptual characteristics of electrotactile feedback, investigated the influence of frequency on both perception and discomfort thresholds using only two electrodes on the finger\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Although they observed a trend toward decreasing thresholds as stimulation frequency increased from 10 Hz to 100 Hz, this trend was not statistically significant. On the other hand, they did find a significant association between the qualitative perception of the sensation reported and the increase in frequency\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe studies mentioned above highlight the influence of gender and stimulation position on perception threshold. However, they are limited in scope due to the focus on restricted body regions and the use of only a few stimulation channels. We aim to address this limitation by improving threshold mapping through increased spatial resolution and considering the entire upper limb. We follow the hypotheses that somatosensory perception thresholds depend on the stimulation position, subject gender, and stimulation frequency. To test this, we investigated how perception thresholds change across the dorsal side of the whole upper limb, accounting for sex differences and the stimulation frequency. Perception thresholds were determined following a staircase procedure using a custom stimulation device\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. We also present a descriptive analysis of the participants\u0026rsquo; perceptual responses \u003cb\u003e(\u003c/b\u003esubjective perceptions of electrical stimulation\u003cb\u003e)\u003c/b\u003e at the perception threshold intensity for each stimulation position and frequency. This threshold characterization is the first step for detailed and personalized human brain somatosensory mapping studies, using advanced neuroimaging techniques such as functional magnetic resonance imaging (fMRI).\u003c/p\u003e \u003cp\u003eIn summary, this comprehensive characterization of electrocutaneous perception thresholds across the dorsal side of the dominant upper limb in healthy participants highlighted the influence of gender and stimulation location, rather than frequency, on perception thresholds.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eParticipants\u003c/h2\u003e \u003cp\u003eTwenty-four healthy adult volunteers (12 females), aged between 21 and 39 years (mean age\u0026thinsp;=\u0026thinsp;30.13 years, SD\u0026thinsp;=\u0026thinsp;5.12) were included in this study. All participants were right-handed (mean laterality index: 85, SD\u0026thinsp;=\u0026thinsp;10.72), as assessed through the Edinburgh inventory questionnaire\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. All participants have no history of neuropsychological or psychiatric diseases, no deficits that prevent understanding and signing the informed consent, and no contraindications for performing electrocutaneous stimulation (e.g. skin lesions at the stimulation positions, suspected/diagnosed epilepsy and/or heart problems, and presence of active implantable medical devices). Additionally, participants were required to have upper limb dimensions sufficient for the application of stimulation electrodes (middle finger\u0026thinsp;\u0026ge;\u0026thinsp;6 cm, hand\u0026thinsp;\u0026ge;\u0026thinsp;8 cm, forearm\u0026thinsp;\u0026ge;\u0026thinsp;12 cm, and upper arm\u0026thinsp;\u0026ge;\u0026thinsp;14 cm). Inclusion criteria were verified in a screening interview previous to the stimulation session. Participants gave written informed consent so they could be included in the study, which was approved by the local Ethics Commission of the Faculty of Medicine of the University of Coimbra (CE-049/2021) and conducted following the Declaration of Helsinki.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental setup\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStimulation setup\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;1 depicts the schematic representation of the experimental setup implemented to determine the perception thresholds in the 20 electrodes placed at the dorsal side of the dominant upper limb using the custom-developed stimulation device. Electrode 1 is positioned at the tip of the middle finger, while electrode 20 is at the shoulder.\u003c/p\u003e \u003cp\u003eThe electrocutaneous stimulation device is a custom-built apparatus distinguished by its current-controlled, voltage-limited functionality and 20 independent stimulation channels\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. It can generate positive rectangular stimulation signals with maximum energy per pulse of 6 mJ, within the range of 0 to 5 mA, a voltage up to 70 V, pulse widths between 0.2 and 5 ms, and frequencies up to 2.5 kHz). Previous work details its components, stimulus generation, operation procedures, and assessment\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;1 - Schematic representation of the experimental setup implemented to determine somatosensory perception thresholds: electrical pulses, generated by the stimulation device according to the properties defined at the Experimental control computer, are delivered to each one of the 20 stimulation electrodes positioned on the dorsal side of the participant\u0026rsquo;s right upper limb: seven on the upper arm, six on the forearm, four on the hand, and three on the middle finger (distances between electrodes depend on the upper limb length of each participant).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eParticipant preparation\u003c/h3\u003e\n\u003cp\u003eBefore electrode placement at the dorsal side of the dominant upper limb, the participant's skin was carefully inspected for any injuries, as disruptions in the skin integrity could lead to unwanted pain during stimulation. Abrasive pads were used to remove non-conductive skin cells and to ensure low contact impedance at the electrodes\u0026rsquo; attachment sites preparing the skin for stimulation\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Stimulation electrodes - Ag/AgCL laminated with a carbon composition contact area and a gel cavity (EL509, BIOPAC), were then placed at predetermined stimulation positions, spaced proportionately based on the length of each limb segment: middle finger, hand, forearm, and upper arm \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Anatomical landmarks were used to define each segment and measure its length, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The length of the middle finger was measured from its tip to the base of the metacarpal bone. The hand was measured from the beginning of the carpal bones (at the base of the middle finger) to the midpoint of the wrist (at the level of the pisiform bone). The forearm was measured from the midpoint of the wrist to the midpoint of the elbow (at the level of the olecranon bone). The upper arm was measured from the midpoint of the elbow to the acromion bone. We utilized electrode conductor gel to improve conductivity\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe minimum distance between stimulation positions was determined based on literature regarding the two-point discrimination threshold for electrotactile sensation \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Therefore, the length of the upper limb served as an exclusion criteria. This criterion ensured that three electrodes could be appropriately positioned on the middle finger, four on the hand, six on the forearm, and seven on the upper arm, allowing for a minimum spacing of 1 cm between electrodes.\u003c/p\u003e \u003cp\u003e Stimulation positions were registered using a navigation system (Localite), with an EEG PinPoint tool (Localite), which enabled us to digitalize the electrodes\u0026rsquo; locations for each participant.\u003c/p\u003e \u003cp\u003e After electrode placement, participants were instructed to sit in a comfortable position, relax their arm over the arm\u0026rsquo;s chair (all participants assumed the same position), and focus on perceiving the stimulation. Participants were instructed to strictly avoid contact between their left and right arms during stimulation, preventing a closed current loop through the heart.\u003c/p\u003e \u003cp\u003eUpon concluding the experiment and removing all electrodes, the participants' skin was carefully examined for any signs of irritation, such as redness.\u003c/p\u003e\n\u003ch3\u003ePerception threshold determination - staircase procedure\u003c/h3\u003e\n\u003cp\u003eThe determination of the perception thresholds wa\u003cb\u003es\u003c/b\u003e conducted using a staircase procedure\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Following a randomized stimulation sequence of electrodes, this interactive method identifies the threshold current (and corresponding voltage) for each stimulation position based on participants\u0026rsquo; subjective judgments. The procedure started with current bursts of 2 mA, (lasting 4 s, with a pulse width of 0.2 ms; voltage depends on current) and adjusting them incrementally (either increasing or decreasing) based on the participant\u0026rsquo;s perception of the stimulation. If the participant reported feeling the stimulation, the current amplitude was decreased according to the step size; if the participant did not feel the stimulation, the current amplitude was increased. Step sizes for these adjustments were set at 1 mA, 0.5 mA, 0.25 mA, and 0.1 mA across successive stimulations. Participants could request repeated stimulation if they felt misjudged in their initial reports. Two frequencies were tested: 30 Hz and 100 Hz. These frequencies were chosen based on the literature(as frequencies around 20 Hz are typically most effective for sensory communication while avoiding discomfort) and the technical limitations of our stimulation device (which limited the maximum frequency to approximately 100 Hz with a pulse width of 0.2 ms)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAfter determining the perception thresholds for all stimulation positions at a specific frequency, the frequency was changed, and the procedure was repeated for the other frequency. Half of the participants were randomly assigned to start with a frequency of 30 Hz, while the remaining started with 100 Hz. Perception thresholds determination, for all stimulation positions at each frequency, took about 15 minutes.\u003c/p\u003e \u003cp\u003e Due to minor skin wounds, perception thresholds could not be determined for two participants at stimulation position 1 and for one participant at stimulation position 4. Additionally, it was also not feasible to determine the perception threshold for one participant at stimulation position 2 with a frequency of 30 Hz, as the participant did not perceive the stimulation even at the maximum intensity.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of subjective perceptions at the perception threshold\u003c/h2\u003e \u003cp\u003eOnce participants reached the perception thresholds for a given stimulation position, they were asked to describe their sensory experience, addressing quantitative, qualitative, spatial, and temporal dimensions\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn terms of \u003cb\u003equantitative perception\u003c/b\u003e, participants were asked to rate the intensity of the perceived sensation and pain on a visual analog scale (VAS) from 0 (no perceptible sensation/pain) to 10 (worst sensation/pain). Regarding \u003cb\u003equalitative perception\u003c/b\u003e, participants selected a single descriptor that best characterized the sensation perceived\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e: knocking, scratching, stinging, muscle twitch, tickling, itching, or pinching. These descriptors were selected based on the literature. Participants also had the option to use alternative adjectives if needed (termed as \u0026ldquo;Other\u0026rdquo; in the results). \u003cb\u003eSpatial perception\u003c/b\u003e required participants to identify where the sensation was perceived, choosing from the following options: at a specific electrode, between electrodes, at two or more electrodes, extending beyond electrodes, or at other parts of the body. It was requested that the chosen electrode(s) or body part be pointed out. Lastly, regarding \u003cb\u003etemporal perception\u003c/b\u003e, participants reported whether they could identify the start and end of the stimulation and whether it felt \u003cb\u003econtinuous or exhibited any discernible pattern\u003c/b\u003e (they could choose between intermittent at the same intensity, intermittent with intensity decreasing/increasing over time, and intermittent that initially increased/decreased then decreased/increased).\u003c/p\u003e \u003cp\u003eBefore the experiment began, participants were briefed on the questionnaire's assessment and the meaning of the perception threshold (but not the procedure to achieve it). Any inquiries from participants were addressed to ensure clarity. The overall experiment lasted approximately 90 minutes, including participant preparation (around 30 minutes), perception thresholds determination, and participants\u0026rsquo; reports.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eTo investigate how stimulation positions, stimulation frequency, and participants\u0026rsquo; gender influenced the perception thresholds we used linear mixed-effect models (LMMs). LMMs offer a powerful statistical framework for analyzing cognitive neuroscience data since they accommodate both fixed and random effects, \u003cem\u003ei.e.\u003c/em\u003e, effects where all levels of interest are included in the experiment (responsible for systematic variability in the data) and effects that have a non-systematic, idiosyncratic, unpredictable or random influence on the data, respectively\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In our case, stimulation position, stimulation frequency, and gender were considered fixed effects (or independent variables) and perception threshold was the dependent variable. Participants (participant ID) were considered as random effects, with a random intercept included for each participant to account for individual differences in baseline perception levels. This approach allowed us to model individual variability in perception thresholds that could not be explained by fixed effects, thereby minimizing the influence of such variability on the results\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo analyze the contributions of gender, stimulation positions, and stimulation frequency on perception thresholds, as well as possible interactions between these factors, we created two LMMs, each designed to capture different levels of spatial resolution in the analysis of stimulation positions:\u003c/p\u003e \u003cp\u003eModel A - Stimulation positions were grouped according to segments of the upper limb (middle finger, hand, forearm, and upper arm), resulting in four levels for this variable (hereafter referred to as \u0026ldquo;Stimulation segment\u0026rdquo;). In this model, the individual contributions of the electrodes of each segment were averaged, allowing us to assess broader, segmental effects on perception thresholds.\u003c/p\u003e \u003cp\u003eModel B - Each stimulation position was analyzed separately, with the variable \u0026ldquo;Stimulation positions\u0026rdquo; consisting of 20 levels, corresponding to the stimulation sites along the upper limb (0 for the tip of the middle finger and 20 for the last electrode at the shoulder). This model enabled a fine-grained analysis of spatial resolution and the influence of individual stimulation sites.\u003c/p\u003e \u003cp\u003eBy employing both models, we addressed our research question from complementary perspectives: Model A captured segmental-level statistical effects, while Model B provided position-specific insights into the role of spatial resolution in perception thresholds.\u003c/p\u003e \u003cp\u003eIn both models, we considered age as a covariate. When a significant effect was found, individual pairs of conditions were further compared using multiple pairwise comparisons with Bonferroni adjustment. The experimental results were analyzed using SPSS software (IBM, SPSS Statistics, version 28).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSomatosensory Electrocutaneous Perception Thresholds\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e - Distribution of the perception thresholds for each stimulation position (represented as electrodes 1 to 20) for 30 Hz (a) and 100 Hz (b) and female (orange) and male (blue) participants.\u003c/p\u003e \u003cp\u003eUsing Model A, we observed that both gender and stimulation segment have a significant impact on perception threshold (gender: \u003cem\u003eF\u003c/em\u003e(1, 24.532)\u0026thinsp;=\u0026thinsp;14.822, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.460\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:{10}^{-4}\\)\u003c/span\u003e\u003c/span\u003e, and stimulation segment: \u003cem\u003eF\u003c/em\u003e(3, 929.097)\u0026thinsp;=\u0026thinsp;237.795, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.812\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:{10}^{-114}\\)\u003c/span\u003e\u003c/span\u003e). Additionally, a significant interaction between these factors was found (\u003cem\u003eF\u003c/em\u003e(3, 929.092)\u0026thinsp;=\u0026thinsp;4.588, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003). Since frequency did not reach statistical significance (\u003cem\u003eF\u003c/em\u003e(1, 929.011)\u0026thinsp;=\u0026thinsp;0.198, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.656), it was removed from the model. After removing frequency and rerunning the model, both stimulation segments (\u003cem\u003eF\u003c/em\u003e(3, 929.092)\u0026thinsp;=\u0026thinsp;237.106, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.252\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:{10}^{-114}\\)\u003c/span\u003e\u003c/span\u003e), gender (\u003cem\u003eF\u003c/em\u003e(1, 24.528)\u0026thinsp;=\u0026thinsp;14.802, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.52\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:{10}^{-4}\\)\u003c/span\u003e\u003c/span\u003e) and their interaction (\u003cem\u003eF\u003c/em\u003e(3, 929.087)\u0026thinsp;=\u0026thinsp;4.578, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003) remained significant. Given the significant interaction revealed by the mixed-effects analyses, we performed post hoc tests with Bonferroni correction for multiple comparisons. When fixing the stimulation segment, perception thresholds were significantly higher in males than females for electrodes at the middle finger (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006), forearm (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.900 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:{10}^{-5}\\)\u003c/span\u003e\u003c/span\u003e, and upper arm (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.690 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:{10}^{-4}\\)\u003c/span\u003e\u003c/span\u003e) \u003cb\u003e-\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e(a). However, no significant gender difference was observed for the hand (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.050). For more details see Supplementary Table\u0026nbsp;1. Conversely, when fixing gender, post hoc tests revealed that perception thresholds were significantly higher at the middle finger and hand compared to the forearm and upper arm for both genders, as well as at the forearm compared to the upper arm for males - Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e(b). No statistical significance difference was found between the middle finger and hand for both genders. For more details see Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsidering Model B, we also found a significant impact of gender (\u003cem\u003eF\u003c/em\u003e(1, 24.005)\u0026thinsp;=\u0026thinsp;8.289, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008) and stimulation position (\u003cem\u003eF\u003c/em\u003e(19, 872)\u0026thinsp;=\u0026thinsp;61.504, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.292 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:{10}^{-146}\\)\u003c/span\u003e\u003c/span\u003e), as well as a significant interaction between these factors (\u003cem\u003eF\u003c/em\u003e(19, 872)\u0026thinsp;=\u0026thinsp;2.237, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002). Frequency did not reach statistical significance (\u003cem\u003eF\u003c/em\u003e(1, 872)\u0026thinsp;=\u0026thinsp;0.027, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.869), therefore it was removed from the model, which was then rerun. The results of the revised model were consistent with the previous one, with stimulation position, gender, and their interaction remaining statistically significant (stimulation position: \u003cem\u003eF\u003c/em\u003e(19, 912)\u0026thinsp;=\u0026thinsp;59.888, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.014\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:{10}^{-145}\\)\u003c/span\u003e\u003c/span\u003e); gender (\u003cem\u003eF\u003c/em\u003e(1, 24.005)\u0026thinsp;=\u0026thinsp;8.265, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008; interaction (\u003cem\u003eF\u003c/em\u003e(19, 912)\u0026thinsp;=\u0026thinsp;2.189, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002). These results indicate that the effect of stimulation position and gender on perception thresholds cannot be interpreted isolatedly, since their effects interact. Post hoc analyses, corrected using Bonferroni for multiple comparisons, revealed that, when fixing the stimulation position, perception thresholds were significantly higher in males compared to females for all stimulation positions except one electrode at the middle finger (E2) and three electrodes at the hand (E4, E5, and E6) - Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Supplementary Table\u0026nbsp;3 details these results. On the other hand, when controlling for gender, post hoc tests revealed several significant differences between stimulation positions. The findings are consistent with those reported in model A. Detailed results can be found in Supplementary Table\u0026nbsp;4.\u003c/p\u003e \u003cp\u003eSupplementary Fig.\u0026nbsp;1 shows the distribution of the amplitude of perception thresholds based on each stimulation site for both genders, regardless of frequency.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDescriptive report of subjective perceptions at the perception threshold\u003c/h2\u003e \u003cp\u003eThe assessment of subjective sensation intensity caused by stimulation at this level serves as a sanity check measure. The median sensation intensities reported are predominantly marked by low-intensity quantification (Supplementary Table\u0026nbsp;5).\u003c/p\u003e \u003cp\u003eWe also evaluated pain perception and found a median pain intensity of zero (independent of gender, frequency, and stimulation position). However, ten participants (five females) reported pain at one stimulation position (intensity of 1 on a 1\u0026ndash;10 scale), regardless of the frequency. Additionally, four participants (one female) experienced pain at multiple positions, all rating their pain as 1 on a 10-point scale, except for one participant who rated it as 2.\u003c/p\u003e \u003cp\u003e Regarding the qualitative aspects of subjects\u0026rsquo; perception, we found that the most frequent descriptors were \u0026ldquo;Stinging\u0026rdquo;, closely followed by \u0026ldquo;Tickling\u0026rdquo;, and \u0026ldquo;Muscle twitch\u0026rdquo; (regardless of gender, stimulation position, and stimulation frequency). Supplementary Fig.\u0026nbsp;2 illustrates the distribution of qualitative descriptors for each stimulation position, gender, and stimulation frequency.\u003c/p\u003e \u003cp\u003e Spatially, the most frequently reported spatial perception was \u0026ldquo;At the Active/Ground electrode\", indicating that participants commonly felt the sensation at the electrode designated as the active or ground. These results were consistent across both genders and frequencies. Detailed results by gender, stimulation position, and stimulation frequency are available in Supplementary Fig.\u0026nbsp;3. When participants reported feeling the stimulation at a specific electrode, and this was the active or ground electrodes, we also examined at which one the sensation was felt - Supplementary Fig.\u0026nbsp;4.\u003c/p\u003e \u003cp\u003eParticipants successfully identified the onset and offset of stimulation in approximately 83% and 80% of cases, respectively. Most participants (75%) reported experiencing a continuous sensation when asked about the stimulation pattern. Other patterns identified included intermittent sensations (13%), sensations that decreased intensity over time (9%), sensations that increased intensity over time (2%), and sensations that initially increased and then decreased intensity (1%). Around 14% of participants did not respond to this question. Regarding the identification of the two different stimulation frequencies, only four participants (three of whom were male) were able to distinguish between them.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis work is the first to explore the location-dependent variation of perception thresholds across the entire upper limb, using LMMs to capture both segmental and position-specific statistical effects. We investigated the impact of gender and stimulation frequency on these thresholds and found main effects of both factors and a significant interaction between them.\u003c/p\u003e \u003cp\u003eThe results were consistent across both statistical models, whether considering segmental (model A) or position-specific stimulation locations (model B). The first analysis model, which considered four segments of the upper limb (middle finger, hand, forearm, and upper arm), revealed the interaction between gender vs stimulation position at a regional level, providing a broader view of how perception thresholds differ by gender across major anatomical areas of the upper limb. The second analysis model, which considered individual stimulation positions, revealed where these gender differences were most pronounced within each segment, underscoring a more detailed sensitivity pattern that would be missed at the segment level. Both models consistently revealed significant effects of gender and stimulation position, as well as, their interaction. On the other hand, frequency did not have a statistically significant impact. In general, males have higher perception thresholds than females across most stimulation positions, except for the hand in Model A and select electrode positions in Model B, more specifically, at the second electrode on the middle finger and the first three electrodes on the hand. Regarding the overall pattern of perception thresholds through the upper limb, the trend was similar for both genders, with higher thresholds observed at the middle finger and hand (with no statistically significant difference between them) compared to the forearm and arm. Additionally, males have higher perception thresholds at the forearm than the arm.\u003c/p\u003e \u003cp\u003eThe results above could be attributed to both peripherical and central factors that differ between genders and across the upper limb. First, the epidermal thickness is different across the upper limb and between genders. According to a systematic review, thickness is lower at the dorsal forearm and much higher at the fingers\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Higher thickness results in increased skin impedance, indicative of greater resistance to the flow of an electric current\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Consequently, higher current intensities are needed to evoke perceived sensations. Our results corroborate this pattern, as we observed higher perception thresholds at the middle finger for both genders. Regarding differences between genders, it is known that women typically have thinner skin compared to men, resulting in lower skin impedance and, therefore, lower perception thresholds\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Previous research about morphological differences between genders suggests that the higher body fat percentage in females could facilitate the recruitment of nerve fibers, leading to lower perception thresholds\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The literature also reports a lower density of nerve fibers in men, which could also account for their higher perception thresholds\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Regarding nerve fiber distribution throughout the upper limb, the most significant discrepancies are observed at the fingertips and the palmar side of the hand compared to the upper arm, regardless of gender\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Therefore, we did not expect direct interference with perception thresholds determined at the dorsal side of the upper limb.\u003c/p\u003e \u003cp\u003eBeyond these peripheral differences, central nervous system mechanisms also play a key role in shaping somatosensory perception. Somatosensory perception involves not only the somatosensory cortex but is also modulated by other brain areas, such as that involved in cognition (e.g., attention) and emotion (e.g. stress)\u003csup\u003e\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. These interconnected brain regions contribute to the integration, modulation, and interpretation of sensory inputs, ultimately shaping perceptual experience and behavioral responses. Neuroimaging research suggests that gender differences in somatosensory perception may be partly attributed to variations in cortical organization, connectivity, and structural differences in the size and thickness of the brain. Building on Penfield\u0026rsquo;s Homunculus, which represents a male figure, researchers have explored the possibility of creating a \"hermunculus\" to better explain the somatotopic organization of the female body\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. However, current studies have predominantly focused on the genitalia and sexual function of women, leaving the broader somatosensory representation of the female body largely unexplored\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Regarding connectivity patterns, the literature highlights decreased functional connectivity in the parietal cortex of females, particularly within the sensorimotor network\u003csup\u003e\u003cspan additionalcitationids=\"CR49 CR50\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Zhang and colleagues further demonstrated that these lower functional connectivities in females were correlated with physical differences, such as body fat mass\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Concerning structural differences, a study performed on data from 5216 UK Biobank found that females generally exhibited greater cortical thickness in somatosensory regions, whereas males showed relatively higher volume and surface area in these regions\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. This study also noted that gender differences in functional connectivity persisted even after controlling for brain size, especially in sensory and association areas\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. These findings suggest that males and females may process somatosensory information in distinct ways. However, to the best of our knowledge, there is no evidence linking these structural and functional differences to perceptual outcomes in somatosensory stimulation. Future neuroimaging research exploring gender differences in the neuronal pathways of somatosensory processing could provide important insights into the mechanisms underlying the perceptual differences observed in this study.\u003c/p\u003e \u003cp\u003eAdditionally, several other factors reported in the literature have implications for the electrode-skin interface and may contribute to variations in perception thresholds. These include skin moisture content (or hydration level), as the higher water content in the skin enhances its ability to conduct electrical currents\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e; and exposure to external factors, which can lead to variations in sensitivity. For example, fingers and hands, often unprotected and frequently used throughout the day, may develop tougher, less sensitive skin than the arms, typically protected by clothes\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. However, this study did not incorporate additional measures to correlate these factors with our findings directly.\u003c/p\u003e \u003cp\u003eLastly, stimulation frequency did not impact the somatosensory perception thresholds recorded, aligning with previous findings that frequency primarily influences the quality of perceived sensations (due to the targeted mechanoreceptors) rather than threshold intensity\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eParticipants' subjective perceptions showed low sensation intensity, as expected, with some minor discomfort reported (1\u0026ndash;2 on a 0\u0026ndash;10 scale), likely due to individual sensitivity differences. The most common qualitative perceptions were stinging, ticking, and muscle twitch. This is primarily influenced by the type of mechanoreceptors activated, but also by other factors such as stimulation parameters, electrodes, and physiological variables, making a direct comparison with existing literature difficult\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Spatially, perception remained mostly localized to the active or ground electrode sites, with minimal mislocalization, particularly in the forearm and upper arm, where larger receptive fields may explain the effect\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. This finding aligns with expectations and highlights the high spatial acuity of the evoked sensations. Regarding temporal perception, good onset and offset identification of the stimulation was achieved. However, only four participants could differentiate between the two stimulation frequencies tested.\u003c/p\u003e \u003cp\u003eDespite the contributions here provided, some experimental steps contributed to limitations in our conclusions. For example, while the method of constant stimuli is generally regarded as the most reliable approach for estimating perception thresholds due to its ability to minimize biases from participant anticipation, it is also notably time-consuming\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Given the large number of electrodes and the risk of reduced participant attention during prolonged experiments, this method was impractical for our study. Instead, we opted for the staircase method, carefully selecting the step size. Larger step sizes risk overestimating the threshold, while smaller step sizes could underestimate it. Drawing on previous research, we chose a smaller step size of 0.1 mA\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. This approach allowed us to determine the perception thresholds for 20 electrodes in approximately 15 minutes, yielding results consistent with existing literature. Additionally, although VAS is very intuitive, the results obtained are subject-specific therefore the overall conclusion may be misleading, as two subjects may rate identical stimuli with different VAS scores. Lastly, we did not assess the skin sensation deficits of our participants, so we can not discard any influence from this.\u003c/p\u003e \u003cp\u003eFuture studies should aim to integrate the findings from this work to develop optimized somatosensory mapping protocols of the cortical representation of the upper limb at S1. Additionally, these studies should investigate potential processing differences in sensory-dedicated brain regions, focusing on how these differences may affect somatosensory perception. It would also be valuable to investigate somatosensory perception thresholds in clinical populations with known impaired somatosensory processing, such as psychiatric, neurodevelopmental, and/or chronic pain disorders, to shed light on the associated somatosensory processing alterations\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Investigating somatosensory processing in clinical populations may also provide crucial insights into how these findings can be translated into therapeutic interventions, ultimately improving treatment strategies for individuals with sensory processing deficits.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study provides valuable insights into how perception thresholds are influenced by stimulation location and gender across the entire upper limb in healthy participants considering broader (based on upper limb segments) and fine-tuned (based on individual stimulation positions) approaches. Our findings revealed that perception thresholds vary significantly along the dorsal side of the dominant upper limb depending on gender, highlighting the need for customized current intensities to accommodate such differences. In contrast, stimulation frequency had no significant effect on these thresholds. These results enhance our understanding of the factors affecting electrocutaneous stimulation and contribute to the growing knowledge of how to effectively implement electrocutaneous stimulation across various fields.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank all participants who took part in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCT, AS, BD, TS, and MC-B conceived and designed the study. CT conducted the experiments, analyzed the data, and wrote the manuscript. CT, AS, and PF designed and developed the somatosensory electrical stimulator used in this study. CT and AS developed the code for the threshold definition staircase approach. All authors reviewed the manuscript and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by FCT (UIDB/04950/2020, UIDP/04950/2020). CT and AS were funded by Siemens Healthineers Portugal and the FCT PhD fellowship 2023.04160.BD (CT) and 2023.04365.BD (AS). BD is funded by FCT (CEECINST/00117/2021/CP2784/CT0002). TS is funded by 2021.01469.CEECIND.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKourtesis, P., Argelaguet, F., Vizcay, S., Marchal, M. \u0026amp; Pacchierotti, C. 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Psychophysical methods. in \u003cem\u003eModern techniques in neuroscience research\u003c/em\u003e 1211\u0026ndash;1241 (Springer, Berlin, 1999).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Somatosensory cortex, Perception thresholds, Electrocutaneous stimulation","lastPublishedDoi":"10.21203/rs.3.rs-6043288/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6043288/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMeasuring perception thresholds in electrocutaneous stimulation offers valuable insights into sensory processing and supports the creation of personalized methods for diagnosing and treating somatosensory disorders. This study uses a custom non-invasive electrocutaneous stimulation device to test the impact of stimulation frequency, position along the upper limb, and participants\u0026rsquo; gender on the perception thresholds. The device targeted 20 stimulation positions on the dorsal side of the right upper limb of 24 healthy participants. Perception thresholds for each participant and stimulation position were determined by a staircase procedure at two frequencies (30 Hz and 100 Hz). Our findings highlight the complex interplay between gender and stimulation position while suggesting that frequency does not significantly influence perception thresholds under these conditions. While males exhibited higher perception thresholds overall, the spatial pattern of perception thresholds along the upper limb thresholds were in general higher at the middle finger and hand compared to the forearm and upper arm. However, the interaction between gender and stimulation position indicates that the magnitude of these differences varies depending on the specific position. These results underscore the necessity of considering gender- and position-specific differences when analyzing somatosensory thresholds across the upper limb.\u003c/p\u003e","manuscriptTitle":"Extensive mapping of somatosensory perception thresholds in the upper limb reveals an interaction between gender and stimulation position","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-25 07:14:08","doi":"10.21203/rs.3.rs-6043288/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-07T07:51:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-05T11:57:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-24T12:34:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62788531201931749137732697368101898377","date":"2025-03-20T21:14:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"121524760386658745145448686911380263603","date":"2025-03-20T15:59:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"183154325554451971559724646443577973006","date":"2025-03-20T06:27:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-09T13:20:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-28T16:29:01+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-02-20T12:02:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-02-19T09:58:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-02-16T23:15:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9073be25-d619-4590-8d79-82adb31f158f","owner":[],"postedDate":"February 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":44626494,"name":"Biological sciences/Neuroscience/Peripheral nervous system/Somatic system"},{"id":44626495,"name":"Biological sciences/Physiology/Neurophysiology"}],"tags":[],"updatedAt":"2026-01-06T07:53:44+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-25 07:14:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6043288","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6043288","identity":"rs-6043288","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}