1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Thresholds of cutaneous afferents related to perceptual threshold across the human foot sole Authors: Nicholas D.J. Strzalkowski1, Robyn L. Mildren1, Leah R. Bent1 Affiliations: 1University of Guelph, Guelph ON, CANADA Running head: Cutaneous afferent firing and perceptual thresholds Corresponding author: Dr. Leah R. Bent Assistant Professor Department of Human Health and Nutritional Science Guelph, Ontario, N1G 2W1 E-mail: lbent@uoguelph.ca Phone: 519 824 4129 ext. 56442 Fax: 519-763-5902 Number of figures – 5 Number of tables - 2 23 24 25 26 27 28 29 30 31 32 33 Key words: foot sole, microneurography, perception, cutaneous afferent 34 35 Abstract Perceptual thresholds are known to vary across the foot sole, despite a reported 36 even distribution in cutaneous afferents. Skin mechanical properties have been proposed 37 to account for these differences, however, a direct relationship between foot sole afferent 38 firing, perceptual threshold and skin mechanical properties has not been previously 39 investigated. Using the technique of microneurography, we recorded the monofilament 40 firing thresholds of cutaneous afferents and associated perceptual thresholds across the 41 foot sole. In addition, receptive field hardness measurements were taken to investigate the 42 influence of skin hardness on these threshold measures. Afferents were identified as Fast 43 Adapting; FAI (n=48), FAII (n=13), or Slowly Adapting; SAI (n=21) or SAII (n=20), and 44 were grouped based on receptive field location (Heel, Arch, Metatarsals, Toes). Overall, 45 perceptual thresholds were found to most closely align with firing thresholds of FA 46 afferents. In contrast, SAI and SAII afferent firing thresholds were found to be 47 significantly higher than perceptual thresholds and are not thought to mediate 48 monofilament perceptual threshold across the foot sole. Perceptual thresholds and FAI 49 afferent firing thresholds were significantly lower in the Arch compared to other regions, 50 and skin hardness was found to positively correlate with both FAI and FAII afferent 51 firing and perceptual thresholds. These data support a perceptual influence of skin 52 hardness, which is likely the result of elevated FA afferent firing threshold at harder foot 53 sole sites. The close coupling between FA afferent firing and perceptual threshold across 54 foot sole indicates that small changes in FA afferent firing can influence perceptual 55 thresholds. 56 57 Introduction 58 It is well established that cutaneous feedback from the soles of the feet is 59 fundamental in the control of upright stance. Previous work has shown foot sole 60 cutaneous feedback to play a role in standing balance (Roll et al., 2002), gait (Perry et al., 61 2001; Eils et al., 2004), automatic postural adjustments (Inglis et al., 1994; Perry et al., 62 2000), as well as in the modulation of lower (Fallon et al., 2005) and upper limb (Bent 63 and Lowrey, 2013) muscle activity and vestibular reflexes (Muise et al., 2012). What 64 remains unclear is the capacity of individual types of foot sole cutaneous afferent classes 65 to transmit distinct tactile cues to the central nervous system (CNS) and what impact this 66 feedback has on balance control. 67 Tactile sensibility from the glabrous skin of the foot sole and hand arises from 68 four classes of low threshold cutaneous mechanoreceptors located in the dermal and 69 epidermal layers of the skin. Each class is sensitive to unique features of tactile stimuli 70 and demonstrate distinctive firing characteristics in response to indentation forces, skin 71 stretches, textures, and vibrations (Johansson et al., 1982; Johnson and Hsiao, 1992; 72 Aimonetti et al., 2007). Cutaneous afferent firing characteristics as well as receptive field 73 properties establish the classification of each subtype as fast adapting (FA) or slowly 74 adapting (SA), and type I (small, distinct borders) or type II (large, undefined borders). 75 The development of microneurography by Vallbo and Hagbarth in the 1960’s, allowed 76 for the direct comparison between primary afferent activity and perceptual experience 77 (Hagbarth and Vallbo, 1967). Pioneering work in the hand found light touch perceptual 78 threshold to most closely resemble the firing thresholds of FA afferents (Johansson and 79 Vallbo, 1979). In the most sensitive hand regions (fingers and lateral border), a small 80 amount of activity from FAI afferents, even single spikes, were capable of evoking a 81 percept. Further support for a one-to-one relationship between afferent firing has been 82 demonstrated through the electrical micro-stimulation of individual cutaneous afferents. 83 Using this technique, researchers have demonstrated that specific tactile sensations can be 84 evoked from the activity of single cutaneous afferents; e.g., flutter (FAI), vibration 85 (FAII), and pressure (SAI) (Ochoa and Torebjörk, 1983; Macefield et al., 1990). These 86 findings are in line with the lower envelope principle, in that perception can be set by 87 minimal activity in the most sensitive afferents (Parker and Newsome, 1998). 88 Previous work that has investigated tactile perception has focused almost 89 exclusively on cutaneous feedback from the hand. The fingers have been shown to have 90 lower perceptual thresholds compared to the palm, despite similar afferent firing 91 thresholds between these regions (Johansson and Vallbo, 1979). This led the authors to 92 postulate that cutaneous feedback is not weighted equally across the body, and that 93 central mechanisms may integrate input from the fingertips with more fidelity than the 94 palm of the hand. The higher density of afferents in the finger tips may increase the 95 probability of activating highly sensitive afferents leading to the disparity in perception 96 between these regions. However, Johansson and Vallbo (1979) argued this was not the 97 case since sub sensory stimuli at the palm still evoked firing in cutaneous afferents. Their 98 investigation suggests that perceptual threshold can be set by the firing capacity of the 99 most sensitive primary cutaneous afferents in some regions (e.g., in the fingers); while 100 additional factors may raise perceptual threshold in less sensitive skin regions (e.g., in the 101 palm). 102 The soles of the feet are not as sensitive as the hands, where in the feet, both 103 perceptual thresholds (Hennig and Sterzing, 2009) and cutaneous afferent firing 104 thresholds (Kennedy and Inglis, 2002) are reportedly higher. Perceptual threshold 105 differences have been reported across the foot sole (Kekoni et al., 1989; Hennig and 106 Sterzing, 2009; Strzalkowski et al., 2015); while mechanoreceptor density is thought to 107 be evenly distributed (Kennedy and Inglis, 2002). A direct comparison between foot sole 108 cutaneous afferent firing and perceptual sensitivity has not been made at the foot sole, 109 and the neural mechanisms underlying regional differences in perceptual threshold are 110 not well understood. 111 Mechanical properties of the skin have been shown to differ across the sole of the 112 foot (Strzalkowski et al., 2015) and between the foot sole and hand (Hoffmann et al., 113 1994). The ability of skin to deform and transmit force will presumably impact afferent 114 firing, and differences in skin properties have been proposed to account for disparities 115 between cutaneous afferent firing and perceptual thresholds between these regions 116 (Kekoni et al., 1989; Kowalzik et al., 1996; Kennedy and Inglis, 2002). While an attempt 117 has been made to link mechanical properties with afferent firing in the glabrous skin of 118 raccoons (Pubols & Pubols 1983), and with perceptual threshold in the foot (Strzalkowski 119 et al., 2015), the influence of skin mechanics on the actual firing of foot sole cutaneous 120 afferents has not been investigated. 121 The aim of the present study was to investigate the relationship between tactile 122 perceptual threshold and cutaneous afferent firing thresholds across the human foot sole. 123 Skin hardness within each afferent’s receptive field was also investigated to better 124 understand the potential influence of skin mechanics on afferent firing and perceptual 125 threshold. In following with previous work in the hand, FA afferents were expected to be 126 more sensitive to light touch (i.e., fire at lower forces) compared to SA afferents, and 127 have firing thresholds most similar to perceptual thresholds across the foot sole. Afferent 128 firing thresholds are expected to increase with skin hardness and, at least partially, 129 account for perceptual threshold differences across the foot sole. 130 Materials and Methods 131 Subjects 132 Fifty-nine recording sessions were performed on 21 healthy subjects (12 male 9 133 female, mean age 24, range 20-27). None of the participants had any known neurological 134 or musculoskeletal disorders. All subjects gave written informed consent to participate in 135 the experiment. The protocol was approved by the University of Guelph research ethics 136 board and complied with the declaration of Helsinki. 137 Microneurography 138 Microneurography was used to identify and record the firing patterns of single 139 cutaneous afferents from the right tibial nerve. Subjects lay prone on an adjustable table 140 with both legs extended, and supported with Versa Form positioning pillows. The path of 141 the tibial nerve and microelectrode insertion sites were located at the level of the popliteal 142 fossa using transdermal electrical stimulation (1-ms square wave pulse, 1Hz 0-10mA, 143 Grass S48, SIU-Isolation Unit, Grass Instruments). A low impedance reference electrode 144 (uninsulated, tungsten, 200µm diameter; FHC Inc. Bowdoinham, ME, USA) was inserted 145 percutaneously to a depth of 0.5cm, 2cm medial to the predetermined recording site. A 146 recording electrode (insulated 10MΩ, tungsten, 200µm diameter, 1-2 µm recording tip, 147 55mm length; FHC Inc.) was then inserted at the recording site and manipulated by hand 148 to penetrate the nerve and to isolate single units. Electrode manipulations were guided by 149 subject sensations as well as audio feedback of the neural activity initiated by mechanical 150 activation (light tapping, stroking and stretching) of the foot sole skin. Neural recordings 151 were amplified and band-pass filtered (gain 104, bandwidth 300Hz-3kHz, model ISO- 152 180; World Precision Instruments, Sarasota, FL), digitally sampled (40kHz), and stored 153 for analysis (CED 1401 and Spike2 version 6; Cambridge Electronic Design). Spike 154 morphology was used to generate templates for the visual classification of single units. 155 The sample of cutaneous afferents through microneurographic recordings is thought to be 156 random, and the ratio of afferent classes and distribution of receptive fields in the present 157 study are thought to reflect a representative sample of the cutaneous population in the 158 foot sole. 159 Cutaneous mechanoreceptor identification 160 Single afferents were classified as fast adapting type I (FAI) or II (FAII), and 161 slowly adapting type I (SAI) or II (SAII) based on previously described criteria 162 (Johansson, 1978; Kennedy and Inglis, 2002). Briefly, FA afferents adapt quickly to 163 sustained indentations and are highly sensitivity to dynamic events. In contrast, SA 164 afferents respond throughout sustained indentations, and demonstrate a firing rate 165 proportional to the magnitude of skin displacement. Type I afferents typically have small 166 receptive fields with distinct borders and multiple hotspots, while type II afferents have 167 large receptive fields with less well defined borders and a single hotspot. 168 Afferent firing and perceptual threshold testing 169 After a single afferent was isolated, Semmes-Weinstein monofilaments (Touch 170 Test®, North Coast Medical Inc, Gilroy, California) were used to measure afferent firing 171 thresholds (AFT), perceptual threshold, and to measure receptive field location and size. 172 AFT was defined as the minimum monofilament force (mN), which reliably (100% 173 confidence of unit identification) evoked an afferent discharge in at least three of four 174 applications. AFT was determined at the most sensitive receptive field location (hotspot) 175 for each identified cutaneous afferent. Perceptual threshold was also measured at each 176 afferent’s receptive field hotspot following single unit recordings. The AFT test site was 177 marked with a pen to ensure perceptual threshold was measured at the same location. A 178 modified 4-2-1 search method was employed (Dyck et al., 1993), and subjects were 179 instructed that there would be multiple catch trials in which no monofilaments would be 180 applied. Subjects were instructed to answer with a simple yes/no response when they 181 were at least 90% confident that they perceived the tactile stimulus. Perceptual thresholds 182 were determined to be the lowest monofilament force (mN) correctly perceived on at 183 least 75% of applications. It is notable that perceptual threshold is the perception of force 184 within an identified region (RF). Given the nature of microneurography, where we are 185 recording from one single afferent, it may be possible for perception threshold to be 186 lower than AFT when we are not recording from the most sensitive afferent. 187 Receptive field characteristics 188 Afferent receptive fields were measured with monofilaments that applied a force 189 4-5 times greater than AFT, and were drawn on the skin using a fine tip pen (Figure 1). 190 Receptive fields were always oval or circular in shape, and the major and minor axes 191 were used to calculate receptive field area (mm2) (Table 1). Efforts were made to identify 192 and map all isolated single afferents, however searching was focused to the foot sole, and 193 only afferents with their receptive field in the plantar surface were included in AFT and 194 perceptual threshold analyses. 195 Hardness measurements were taken at the receptive fields of each identified 196 cutaneous afferent using a handheld durometer (Type 1600-OO, Rex Gauge, Brampton, 197 Ontario, CAN). The durometer had a 2mm diameter column-shaped indenter, which is 198 ideally suited for skin measurements (Kissin et al., 2006). Durometers provide hardness 199 measurements in arbitrary units (au) between 1 (softest) and 100 (hardest), based on the 200 penetration depth of the indenter. Two measurements of hardness were taken at each 201 receptive field and averaged. Hardness measurements were not taken at some toe sites 202 (10 of 30) due to the receptive field being too close to the nail, or an inability for the 203 durometer to fit on the toe. 204 Data analysis 205 The dependent variable assessed for both afferent firing threshold and perceptual 206 threshold was the applied monofilament force level (mN) necessary to evoke an afferent 207 discharge or a percept, respectively. Analysis of variance (ANOVA) procedures were 208 conducted on log-transformed AFT and perceptual threshold data to correct for violations 209 of normality and homogeneity. A one-way ANOVA was used to determine if AFTs 210 differed between afferent classes (FAI, FAII, SAI, SAII). Significant effects were 211 followed up with a Gabriel post hoc analysis. Additionally, a mixed design ANOVA was 212 performed to determine if there were differences between afferent class firing threshold 213 and associated perceptual thresholds (within factor), and if these differences were present 214 at different foot sole locations (between factor). Significant effects were followed up with 215 one-way ANOVAs and a Gabriel post hoc test. 216 Pearson’s product-moment coefficients were calculated to measure the 217 relationship between afferent class firing thresholds and associated perceptual thresholds. 218 Relationships between receptive field hardness and AFT as well as receptive field 219 hardness and perceptual threshold were also explored using Pearson’s correlations. 220 The cumulative probabilities of afferent firing and the generation of a percept 221 were calculated across monofilament force levels. These data demonstrate the proportion 222 of afferents within each class that reached threshold, as well as the proportion of percepts 223 evoked, at a given monofilament force application. 224 Results 225 One hundred and two afferents were successfully identified with receptive fields 226 in the plantar surface of the foot sole. These included 48 FAI (47%), 13 FAII (13%), 21 227 SAI (20%) and 20 SAII (20%) (Figure 1). An additional 9 units were identified in the nail 228 bed, dorsum and back of the ankle (nail bed: 2 SAII, dorsum: 1 SAII, ankle: 1 FAI, 2 229 FAII, 1 SAI, 2 SAII), however all non-foot sole units were excluded from analysis. 230 Cutaneous afferent class characteristics are presented in Table 1. 231 Afferent class firing threshold 232 One-way repeated measures ANOVA revealed that there was a significant 233 difference in afferent firing threshold between afferent classes (p<0.001) (Figure 2). Post 234 hoc analysis indicated that AFT did not significantly differ between FAI (mean 13.2mN) 235 and FAII (mean 12.0mN) afferents (p=0.498), and that both FAI and FAII afferents had 236 significantly lower thresholds compared to SAI (mean 49.6mN) and SAII (222.5mN) 237 afferents (all p-values <0.001). In addition, SAI AFT was significantly lower than SAII 238 AFT (p=0.001). 239 Across foot sole locations, FAI AFTs were found to be significantly different 240 (p=0.005), while location differences were not found for FAII (p=0.174), SAI (p=0.143), 241 or SAII (p=0.964) afferent classes (evaluated using one-way repeated measures 242 ANOVAs). It should be noted that FAI afferents were the most abundant (n=48), thus the 243 relatively lower sample size of the other classes may have contributed to the absence of 244 observed differences in AFT across foot sole locations. Post hoc analysis revealed FAI 245 AFTs to be significantly lower at the Arch compared to the Heel (p=0.019) and Toes 246 (p=0.043), and there was a trend toward a lower threshold at the Arch in comparison to 247 the Met (p=0.073) (Figure 3.A). 248 Perceptual thresholds 249 Perceptual thresholds significantly differed across foot sole locations (p<0.001; 250 One-way repeated measures ANOVA). Similar to FAI AFT, the Arch displayed the 251 lowest perceptual thresholds; post hoc analysis revealed that perceptual threshold at the 252 Arch was significantly lower in comparison to the Heel (p<0.001), Met (p=0.003) and 253 Toes (p=0.007) (Figure 3.B). 254 Relationship between afferent firing threshold and perceptual threshold 255 Overall, perceptual threshold (mean 14.63mN) was found to be most similar to 256 both FAI (mean 13.2mN) and FAII (mean 12.0mN) AFTs (Figure 2). Two-way mixed 257 ANOVA results indicated that across the foot sole, there were no significant differences 258 between perceptual threshold and FAI or FAII AFT (p>0.05) (Figure 4). In contrast, SAI 259 and SAII AFTs were found to be significantly higher than perceptual threshold (SAI 260 p=0.004, SAII p=0.001), (Figure 2). Post hoc analysis showed that SAI AFT at the Toes 261 was significantly higher compared to perceptual threshold (p=0.011), with a similar trend 262 at the arch (p=0.073), and an opposite trend of lower SAI AFT compared to perceptual 263 threshold at the Heel (p=0.053) (Figure 4). SAII AFTs were significantly higher than 264 perceptual threshold at the Arch and Met (p<0.001) (Figure 4). Minimum, maximum and 265 median threshold values across foot sole sites are represented in Table 2. The small 266 sample sizes at some locations (one FAII and SAII at the Heel, and 1 SAII at the toes) 267 limited the comparisons that could be made. 268 The cumulative probability of afferent firing and perceptual threshold across 269 monofilament forces (mN) is presented in Figure 5. These data demonstrate differences 270 in the proportion of afferents recruited in each class across monofilament force levels. 271 FAII afferents were shown to be the most sensitive, exhibiting a higher percentage of 272 recruitment at lower forces compared to the other classes. By 1mN of force, 40% of FAII 273 afferents reached threshold whereas 20% of FAI, and 0% of SAI and SAII afferents were 274 firing. The proportion of trials perceived, increased with larger monofilament force and 275 most closely related to the recruitment of FA afferents. Perceptual threshold was reached 276 in 10% of trials before any SAI or SAII afferents reached firing threshold. At 277 approximately 6mN of force, 50% of monofilament applications were perceived, while 278 only 14% of SAI and 0% of SAII afferents reached threshold. In contrast 56% of FAI and 279 63% FAII were recruited by 6mN of force. These data demonstrate that FAI and FAII 280 afferent firing thresholds are lower than perceptual threshold in some instances, and that 281 perception threshold is likely reached in the absence of SAI and SAII firing. Furthermore, 282 a significant correlation was found between FAI AFT and perceptual threshold (r=0.489, 283 p=<0.001). In contrast, significant correlations were not found between perceptual 284 threshold and AFT for any other afferent classes (Table 3). 285 286 Receptive field hardness influences FA afferent firing threshold and perceptual threshold 287 Receptive field hardness was found to significantly correlate with perceptual 288 threshold (r=0.433, p=<0.001). Similarly, receptive field hardness was found to 289 significantly correlate with FAI AFT (r=0.357, p=0.018), as well as FAII AFT (r=0.758, 290 p=0.007) (Table 3). No significant correlations were found between SAI or SAII 291 receptive field hardness and AFT, although a trend was found for SAII afferents (SAI: 292 r=-0.109, p=0.678, SAII: r=0.422, p=0.064). These data suggest that the effects of skin 293 hardness on perceptual threshold parallel the effects of skin hardness on FA afferent 294 firing. 295 Discussion 296 The present study examined the relationship between cutaneous afferent firing 297 thresholds and perceptual thresholds across the human foot sole. We have demonstrated 298 that monofilament perceptual threshold is mediated by the activity of fast adapting 299 afferents, and, in turn, that both fast adapting afferent firing and perceptual thresholds 300 may be influenced by skin hardness. Across all foot sole locations, perceptual thresholds 301 did not significantly differ from the firing thresholds of FAI and FAII afferents. The Arch 302 was perceptually the most sensitive region and also contained the most sensitive FAI 303 afferents. In contrast, SAI and SAII afferents were significantly less sensitive than 304 perceptual threshold across the foot sole and are thus not thought to mediate 305 monofilament perceptual threshold. 306 Psychophysical Detection 307 Cutaneous afferents are the fundamental units that convey tactile feedback to the 308 central nervous system. The lower envelope principle postulates that perceptual 309 thresholds are set by the most sensitive afferents, and predicts that perceptual variability 310 can be accounted for in the variability of individual afferent firing (Parker and Newsome, 311 1998). Alternatively, afferent temporal or spatial summation may be required for tactile 312 stimuli to have perceptual significance. In such pooling-models, the relationship between 313 perception and afferent firing thresholds is expected to be small, as fluctuations in the 314 activity of single neurons would have a minimal impact on whether cutaneous activity is 315 perceived (Parker and Newsome, 1998). Microneurography provides a tool to obtain 316 single unit recordings from awake human subjects, and thus permits the relationship 317 between cutaneous afferent firing and perception to be directly examined. This is the first 318 study to link the activity of single cutaneous afferents to perceptual threshold across the 319 foot sole. 320 Afferent and perceptual thresholds across the foot sole 321 Previous reports of foot sole cutaneous afferent firing thresholds exhibit a range in 322 median values, which are similar to the threshold ranges measured in the present study. In 323 all cases FAII afferents were found to have the lowest monofilament thresholds, with 324 median values reported from 0.73-4mN (3.9mN in the present study). In most cases FAI 325 afferents had the second lowest thresholds (3.84-11.8mN, 5.9mN present study), 326 followed by SAI (4.08-35.6mN, 39.2mN present study) and SAII afferents (1.42 – 327 115.3mN, 122.6mN present study) (Kennedy and Inglis, 2002; Fallon et al., 2005; Bent 328 and Lowrey, 2013; Lowrey et al., 2013). Collectively, these median values demonstrate 329 that large ranges in afferent firing thresholds may exist within classes, however, these 330 afferent firing thresholds are averaged across the foot sole and not distinct by region. We 331 made the link between afferent location and firing threshold because it is an important 332 measure to identify factors contributing to both AFT and perceptual threshold. FAI AFT 333 was found to be significantly lower at the arch compared to the Heel and Toes, while SA 334 afferents did not show significant differences in threshold across the foot sole. 335 Interestingly, perceptual threshold was also found to be lowest in the Arch region 336 compared to the Heel, Met and Toes, which is in agreement with previous work (Nurse 337 and Nigg, 1999; Eils et al., 2002; Hennig and Sterzing, 2009; Zhang and Li, 2012). In 338 general, we found that across the foot sole, regional perceptual threshold differences 339 closely mirrored the firing thresholds of FAI and FAII afferents; the force required to 340 activate these fast adapting afferents was not significantly different from those required to 341 reach perceptual threshold. Although the relative perceptual contributions between FAI 342 and FAII afferents cannot be determined from the present data, our results provide strong 343 evidence that only FA, and not SA, afferent firing contributes to monofilament perceptual 344 thresholds across the foot sole. 345 Regional differences: Receptive field hardness 346 The ability of cutaneous afferents to fire is set by the capacity of the skin and 347 surrounding tissue to deform and transmit force to the mechanoreceptor endings. 348 Mechanical property differences between the hands and feet and across the foot sole have 349 been suggested to account for perceptual and afferent firing differences between these 350 regions, however, this relationship has not been previously investigated (Kekoni et al., 351 1989; Trulsson, 2001; Kennedy and Inglis, 2002). Significant differences in hardness 352 were found across the foot sole regions investigated in the current study. Additionally, we 353 found significant correlations between both FAI and FAII AFT with receptive field 354 hardness, which supports an influence of skin hardness on FA AFT. Perceptual thresholds 355 were also found to correlate with receptive field hardness. As a whole, these correlational 356 data suggest that across the foot sole, higher FA AFTs may be the result of harder skin, 357 and as a consequence, perceptual thresholds are increased. These data cannot make this 358 link unequivocally, but when considered along with the significant regional differences 359 observed in FAI AFT and perceptual thresholds, it appears that indeed, receptive field 360 hardness has an influence on these measures. These data suggest that regional differences 361 in foot sole hardness may partially explain the consistent regional differences in foot sole 362 monofilament thresholds reported in the literature. 363 Afferent characteristics between the hands and feet 364 The hands and feet purportedly contain the same classes of mechanoreceptive 365 afferents, despite serving distinct functional roles. Tactile feedback from the feet aids in 366 the control of posture and upright stance by providing information about sway and weight 367 distribution under the feet (Kavounoudias et al., 1998). In contrast, the hands are 368 commonly used to manipulate objects and require high tactile acuity. It is therefore not 369 surprising that firing thresholds of afferents in the hands are reported to be lower than 370 those in the feet (Johansson et al., 1980; Kennedy and Inglis, 2002). Median 371 monofilament afferent firing thresholds of RA (FAI), FAII, SAI and SAII afferents in the 372 hand have been reported to be 0.58, 0.54, 1.3 and 7.5mN respectively (Johansson et al., 373 1980); these are 7-30 times more sensitive than the median afferent class thresholds 374 found in the present study and in other studies examining cutaneous receptors in the feet 375 (Kennedy and Inglis, 2002; Fallon et al., 2005; Bent and Lowrey, 2013; Lowrey et al., 376 2013). Elevated thresholds across the foot sole may reflect a peripheral adaptation of foot 377 sole afferents that enables them to optimally function under loaded conditions. Despite 378 these observations of overall elevated thresholds in the foot sole, the relative thresholds 379 between afferent classes appear to be preserved in the feet; in both the hands and feet, 380 FAII afferents are typically the most sensitive to perpendicular light touch followed by 381 FAI and SAI afferents, while SAIIs characteristically are the least sensitive (Johansson et 382 al., 1980; Kennedy and Inglis, 2002). 383 Previous seminal work in the hand investigated the mechanisms behind the 384 perception of light touch in the glabrous skin of the palm and fingers (Johansson and 385 Vallbo, 1979). These authors found FAI and FAII afferent firing thresholds to mirror 386 perceptual thresholds in the fingers and lateral boarder of the hand; which is similar to the 387 relationship we found in the foot sole. However, while we found FA AFT and perceptual 388 threshold to correlate across the entire foot sole, they found a discrepancy in the palm of 389 the hand, where FA afferent firing thresholds were considerably lower than perceptual 390 thresholds. This disparity suggests that perception at the palm of the hand may be limited 391 by noise or processing inefficiencies within the central nervous system. Such a 392 discrepancy between AFT and perceptual threshold was not found in any regions of the 393 foot sole. The alignment of FA afferent firing thresholds with perceptual thresholds in the 394 most sensitive regions of the hands (fingers and lateral boarder) are consistent with the 395 lower envelope principle and with the present observations across the foot sole whereby 396 minimal input from a few afferents is able to generate a percept. 397 Functional implications 398 This study extends the large body of work that has investigated cutaneous afferent 399 firing and sensory perception in the hand and the foot sole. Considering the importance of 400 detailed tactile feedback from the fingers, it makes functional sense that minimal afferent 401 input from the fingers would have a significant impact on perception (Johansson and 402 Vallbo, 1979). It may then be a surprise that a similar relationship, albeit at elevated 403 thresholds, is present in the foot sole where high tactile discrimination may not be 404 necessary for the control of standing balance. Research has identified a large proportion 405 of FAI afferents in the foot sole, which highlights skin’s important role in dynamic 406 balance (Kennedy and Inglis 2002, Fallon et al 2005). The transmission of FAI afferent 407 information, with minimal firing and low signal noise, would ensure the fidelity of 408 cutaneous dynamic input for balance and locomotor tasks. In the present study, the FA 409 afferent-perceptual correspondence supports that small changes in FA afferent firing 410 thresholds can have a significant impact on perceptual threshold, and potentially on 411 balance control. In support of this concept, low amplitude white noise vibration applied 412 to the foot sole has been shown to improve balance control in stroke and diabetic patients 413 (Priplata et al., 2005). These vibrations are thought to increase the detection of weak 414 cutaneous signals from the soles of the feet. Therefore, small changes in FA afferent 415 firing are thought to impact both tactile perception, and balance control. 416 While the current study was conducted in a young healthy population, these data 417 can help inform clinical assessments of tactile sensitivity. Diabetic neuropathy, which is 418 present in 80% of both type 1 and 2 diabetics, is commonly diagnosed and assessed with 419 monofilament testing (Valk et al., 1997; Collins et al., 2010). In these patients, the 420 standard which is used to diagnose sensory neuropathy is a threshold of 10g (98mN) or 421 higher, typically in the plantar surface of the great toe (Kumar et al., 1991; Lambert et al., 422 2009). The current data suggests that monofilament thresholds in the foot sole are 423 mediated by the activity of FA afferents, and monofilament testing does not provide a 424 measure of SA afferent function. Clinically, monofilaments remain a simple tool to assess 425 tactile sensibility, however, other techniques, such as vibration, grating orientation tasks 426 and temperature thresholds are needed to understand the function of the complete 427 peripheral sensory system. 428 Limitations 429 Microneurography is a powerful technique in that it provides a comparison 430 between afferent activity and perception in human subjects. A limitation of studying 431 single neurons is the inability to measure population behaviour at different levels within 432 the nervous system. The number of afferents responding to each monofilament 433 application is unknown, but almost certainly includes more than the individual afferent 434 being recorded. Consequently, the influence of spatial summation on these monofilament 435 threshold outcomes remains unknown. Additionally, foot sole location and afferent class 436 comparisons would be strengthened with large sample sizes, however microneurography 437 does not permit the selection of skin units based on class or foot sole location. Perceptual 438 threshold is a relatively simple psychophysical measure, and may only be mediated by 439 FA afferents. Understanding the perceptual contributions of SAI and SAII afferents could 440 be achieved with different tactile stimuli and associated psychophysical tasks; such as 441 stimulus intensity threshold, location, and texture perception (Johnson and Hsiao, 1992). 442 Conclusions 443 The current findings indicate that minimal FA afferent input from the foot sole 444 can give rise to tactile percepts. These findings are in agreement with the lower envelope 445 principle in that perception is set by the activity of the most sensitive FA afferents. SAI 446 and SAII afferents were found to have elevated firing thresholds compared to FA 447 afferents, and their firing did not contribute to foot sole light touch perceptual thresholds. 448 Additionally, regional differences in receptive field hardness appear to relate to, and 449 influence, the firing thresholds of FA afferents; this is thought to contribute to regional 450 differences in perception across the foot sole. 451 452 Grants: This work was supported by funding from the Natural Science and Engineering 453 Research Council (NSERC) of Canada Postsecondary Graduate Scholarship (Doctoral) to 454 N.D.J. Strzalkowski, NSERC Canada Graduate Scholarship (Masters) to R.L. Mildren, 455 and NSERC Discovery Grant to L.R. Bent. 456 Disclosure statement: The authors declare that there are no conflicts of interest, financial 457 or otherwise. 458 Author Contributions: N.D.J.S. and L.R.B. conception and design of research; N.D.J.S., 459 R.L.M. and L.R.B. performed experiments; N.D.J.S. analyzed data; N.D.J.S., R.L.M. and 460 L.R.B interpreted results; N.D.J.S. drafted manuscript; N.D.J.S., R.L.M. and L.R.B 461 edited, revised and approved the final version of manuscript. 462 References 463 464 465 Aimonetti J-MJ, Hospod VV, Roll J-PJ, Ribot-Ciscar EE. Cutaneous afferents provide a neuronal population vector that encodes the orientation of human ankle movements. J. Physiol. (Lond.) 580: 649–658, 2007. 466 467 468 Bent LR, Lowrey CR. Single low-threshold afferents innervating the skin of the human foot modulate ongoing muscle activity in the upper limbs. J. Neurophysiol. 109: 1614– 1625, 2013. 469 470 471 Collins S, Visscher P, De Vet HC, Zuurmond WWA, Perez RSGM. 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Gait & Posture (October 11, 2012). doi: 10.1016/j.gaitpost.2012.09.012. 565 566 567 568 Table Captions 569 570 571 572 573 574 575 576 577 578 579 Table 1: The number and percent of each afferent class identified as well as the monofilament threshold and receptive field area (mean and range) 580 581 Table 2: Afferent firing threshold (AFT) values across foot sole locations (mN). Data represented are minimum (min), maximum (max) and Median values. Table 3: Correlation data for comparisons between afferent firing threshold and perceptual threshold, afferent firing threshold and receptive field hardness, and perceptual threshold and receptive field hardness. 582 Figure Captions 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 Figure 1: Afferent class receptive field distribution. Grey ovals indicate the relative size and location of cutaneous afferent receptive fields identified across the foot sole. FAI (fast adapting type I), FAII (fast adapting type II), SAI (slowly adapting type I), SAII (slowly adapting type II). These represent the receptive fields for all afferents included in the current study. Figure 2: Mean (±SD) monofilament perceptual threshold (hashed bar) and afferent class firing thresholds (black bars). FAI and FAII afferent firing thresholds were significantly lower than SAI and SAII (all p-values <0.001) but were not different than perceptual threshold (p>0.05). SAI afferent firing threshold was significantly lower than SAII (p=0.001) and both SAI and SAII afferent firing thresholds were significantly higher than perceptual threshold (SAI p=0.004; SAII p<0.001). The letters a, b and c identify threshold categories that significantly differ from each other. Figure 3: (A) Mean (±SD) FAI afferent firing thresholds at the Heel, Arch, Met and Toes. FAI AFTs were significantly lower at the Arch compared to the Heel (p=0.019) and Toes (p=0.043). (B) Mean (±SD) perceptual thresholds at each foot region. Perceptual thresholds were lowest in the Arch compared to all other sites (p<0.05). Figure 4: Mean (±SD) afferent firing and perceptual threshold at the Heel, Arch, Met and Toes for each afferent class (FAI, FAII, SAI, SAII). There were no significant differences between FAI or FAII afferent firing (AFT) and perceptual threshold at any foot sole location. SAI AFTs were significantly higher than perceptual threshold at the Toes (p=0.011) and SAII AFTs were significantly higher than perceptual threshold at the Arch and Met (p-values<0.001). Figure 5: Cumulative probability of afferent class firing and perceptual threshold. This demonstrates the proportion of percepts evoked and afferent firing thresholds reached at a given monofilament force level. Lines represent FAI (single black line), FAII (double black line), SAI (single grey line), SAII (double grey line) and perception (dotted line). These data demonstrate that some FAI and FAII AFTs were lower than perceptual threshold, and perceptual threshold was reached in 60% of trials in the absence of substantial SAI and SAII contributions.