Cutaneous inputs from perineal region facilitates and modulates spinal locomotor activity and reduces cutaneous reflexes from the foot in spinal cats

It is well known that mechanically stimulating the perineal region potently facilitates hindlimb locomotion and weight support in mammals with a spinal transection (spinal mammals). However, how perineal stimulation mediates this excitatory effect is poorly understood. We evaluated the effect of mechanically stimulating (vibration or pinch) the perineal region on ipsilateral (9-14 ms onset) and contralateral (14-18 ms onset) short-latency cutaneous reflex responses evoked by electrically stimulating the superficial peroneal or distal tibial nerve in seven adult spinal cats where hindlimb movement was restrained. Cutaneous reflexes were evoked before, during, and after mechanical stimulation of the perineal region. We found that vibration or pinch of the perineal region effectively triggered rhythmic activity, unilateral and bilateral to nerve stimulation. When electrically stimulating nerves, adding perineal stimulation modulated rhythmic activity by decreasing cycle and burst durations and by increasing the amplitude of flexors and extensors. Perineal stimulation also disrupted the timing of the ipsilateral rhythm, which had been entrained by nerve stimulation. Mechanically stimulating the perineal region decreased ipsilateral and contralateral short-latency reflex responses evoked by cutaneous inputs, a phenomenon we observed in muscles crossing different joints and located in different limbs. The results suggest that the excitatory effect of perineal stimulation on locomotion and weight support is not mediated by increasing cutaneous reflex gain and instead points to an excitation of central pattern-generating circuitry. Our results are consistent with a state-dependent modulation of reflexes by spinal interneuronal circuits. Significance Statement Mechanically stimulating the skin of the perineal region strongly facilitates hindlimb locomotion in mammals following a complete spinal cord injury (SCI). Despite its remarkable effectiveness in promoting hindlimb locomotion in spinal cord-injured mammals, we do not know how this is mediated. The present study provides data on how inputs from the perineal region interact with neuronal circuits that generate locomotor-like activity and reflexes from the foot. A better understanding of how inputs from the perineal region interact with neuronal circuits of the spinal cord could lead to non-invasive approaches to restore walking in people with SCI.

is not mediated by increasing cutaneous reflex gain and instead points to an excitation of 48 central pattern-generating circuitry. Our results are consistent with a state-dependent 49 modulation of reflexes by spinal interneuronal circuits.

Introduction
To record the electrical activity of muscles (EMG, electromyography), we directed pairs 151 of Teflon-insulated multistrain fine wires (AS633; Cooner Wire) subcutaneously from two 152 head-mounted 34-pin connectors (Omnetics) that were sewn into the belly of selected 153 hindlimb muscles. To verify electrode placement during surgery, we electrically stimulated 154 each muscle through the appropriate head connector channel to assess the biomechanically 155 desired muscle contraction. The current data set includes EMG from the following muscles: a National Instruments card (NI 6032E), acquired with custom-made acquisition software, 162 and stored on a computer. 163 For bipolar nerve stimulation, pairs of Teflon-insulated multistrain fine wires (AS633; 164 Cooner Wire) were passed through a silicon tubing. A horizontal slit was made in the tubing 165 and wires within the tubing were stripped of their insulation. The ends protruding through 166 the cuff were knotted to hold the wires in place and glued. The ends of the wires away from 167 the cuff were inserted into four-pin connectors (Hirose or Samtec) for bipolar nerve 168 stimulation. Cuff electrodes were directed subcutaneously from head-mounted connectors 169 and placed around the left and right superficial peroneal (SP) and tibial (Tib) nerves at ankle 170 level. At this level, the SP nerve is purely cutaneous whereas the Tib nerve is mixed, 171 primarily innervating the plantar skin but also intrinsic foot muscles (Bernard et al., 2007). The spinal cord was completely transected (spinalization) between the 12th and 13 th 175 thoracic vertebrae. An incision of the skin was made over the last thoracic vertebrae. After 176 carefully setting aside muscle and connective tissue, a small laminectomy of the dorsal bone 177 was made to expose the spinal cord. Lidocaine (xylocaine) was applied topically and two to 178 three injections were made within the spinal cord, which was then transected with surgical defined as the voltage required to elicit a small consistent short-latency (8-10 ms) excitatory 210 response in an ipsilateral flexor, such as St. Each stimulation consisted of a train of three 0.2 211 ms pulses delivered at 300 Hz. In each condition, we delivered ~45 stimuli for 90 s at 0.5 Hz

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(1 train every 2 s), i.e. ~15 stimulations were given for each period (Fig. 1B, top). The 213 vibration was delivered using a custom device with a 120-Hz frequency and an amplitude of  EMG analysis and quantification. We measured some EMG parameters in each period of 226 the trial in selected muscles. Only trials where rhythmic bursts of the selected muscles 227 observed before, during and after the perineal stimulation were selected (n = 9-14 trials). To 228 quantify EMG bursts, we selected a flexor (IP) and an extensor (LG) muscle. An 229 experimenter (Merlet) determined burst onsets and offsets of selected muscles by visual 230 inspection from the raw EMG waveforms using a custom-made program. Cycle duration was 231 measured as the time between two successive bursts and burst duration was determined as 232 the time from onset to offset (Fig. 1B, bottom). We characterized mean EMG amplitude by 233 integrating the full-wave EMG burst from onset to offset and dividing it by its burst duration. 234 We also measured the interval of time between flexor burst onset and the first electrical 235 stimulus of the train normalized to cycle duration, termed the phasing (Fig. 1B, bottom). We 236 also measured the phasing between ipsilateral and contralateral flexor burst onsets. 237 Reflex analysis and quantification. The step-by-step procedure for quantifying reflex 238 responses is illustrated in Fig. 1C. Reflex responses were sorted into 3 periods: before, during  Based on the terminology introduced by Duysens and Loeb (1980), we defined short-latency 249 (⁓8-10 ms) excitatory (P1) or inhibitory responses (N1) and mid-latency (⁓20-25 ms) 250 excitatory responses (P2). We also classified responses in contralateral muscles as P1 or N1 251 because they had onsets <18 ms, which is the minimal latency for reflexes with a relay in the 252 brainstem in cats (Shimamura & Livingston, 1963 analyses. Therefore, we analyzed ipsilateral and contralateral short-latency responses with 257 onset latencies of ⁓9-14 ms and ⁓14-18 ms, respectively.

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The window for the short-latency excitatory (P1) or inhibitory (N1) responses started 259 when the averaged EMG was greater than the blEMG for ≥ 3 ms using 95% confidence 260 intervals and with a minimal latency of 7 ms. Onset latencies had to be adjusted because 261 response onset differed slightly depending on the muscle and between cats. The window 262 ended when the reflex response returned to blEMG for ≥ 3 ms or at a maximal latency of ~40 263 ms for some muscles. The onset and offset of the windows were the same for each period of 264 the trial. When we observed the appearance of N1 or P1 responses during perineal stimulation 265 with no response observed before or after, we placed a window with identical onsets and 266 offsets for comparison. When we observed a switch from a P1 response to an N1 response 267 within a trial, we adjusted the onset and the offset of the window at each period (Fig. 1D). 268 The EMG of P1 or N1 responses was integrated and the blEMG was subtracted to provide   316 In chronic spinal cats with their hindlimbs restrained, electrical stimulation of the SP or 317 Tib nerves generates rhythmic bursts of activity in multiple hindlimb muscles (Merlet et al., 318 2020). To determine how perineal stimulation affected these bursts, we recorded hindlimb 319 EMG before, during and after mechanically stimulating the perineal region, as shown in 320 Figure 2. Note that the EMGs for a given muscle are at the same vertical scale within a trial.

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On the right of each condition, we present the average rectified EMG trace before, during  Table 1 shows the presence of rhythmic activity unilateral and bilateral to SP and Tib 338 nerve stimulation before, during, and after vibration or pinch of the perineal region for pooled 339 data (i.e. the sum of trials over 5 weeks). Note that bilateral activity means an out-of-phase 340 alternation between the ipsilateral and contralateral sides for a given muscle. Before perineal 341 stimulation, we observed alternating bursts of activity between flexors and extensors 342 unilaterally (29-40% of the trials) and bilaterally (11-33% of the trials). Perineal stimulation 343 increased the presence of rhythmic activity unilaterally (70-83% of trials) and bilaterally (67-344 74% of trials). When vibration/pinch of the perineal region was removed, the presence of 345 rhythmic activity was slightly elevated compared to the before period (unilateral: 43-49% 346 and bilateral; 17-33%). It is important to note that perineal stimulation rarely induced 347 rhythmic activity in Cats 7 and 8. Interestingly, these cats did not recover forward hindlimb   Values are the sum of trials over 5 weeks when we observed locomotor-like activity unilateral = 5.4×10 -9 , -23%, respectively) of the perineal region, with a value of ⁓0.8 (Fig. 3D), indicating that flexor burst onset was less timed to or entrained by the nerve stimulation.

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After perineal stimulation, the phasing returned to ⁓1. 395 rhythmic activity, we compared cycle and burst durations of ipsilateral and contralateral IP 398 and LG muscles as well as the phasing of I-IP and C-IP muscles during perineal stimulation.

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The rhythms ipsilateral and contralateral to the nerve stimulation showed different types of   To facilitate comparisons in reflex responses between conditions and muscles, we  no response (-) was observed before and after (Fig. 5C-D, left). Thus, in these cases, the 495 appearance of an N1 response results in a reduction of reflex responses with our measure. respectively; Fig. 5D, right). 501 We next evaluated the modulation of ipsilateral and contralateral reflex responses by 502 perineal stimulation in the IP muscle, a hip flexor (Fig. 6). In the ipsilateral IP, stimulating induced a switch from a P1 response to an N1 response (11-27% of the trials) or an N1 515 response appeared during perineal stimulation when no response was observed before or after 516 (9-11% of the trials) (Fig. 6C, left). Vibration and pinch of the perineal region significantly 517 decreased reflex responses evoked by Tib nerve stimulation (F(2,16) = 12.30, p = 0.001, -47%; 518 F(2,20) = 7.80, p = 0.003, -73%, respectively) (Fig. 6C, right). We next evaluated the modulation of ipsilateral and contralateral reflex responses by 523 perineal stimulation the VL muscle, a knee extensor (Fig. 7). In the ipsilateral VL, we 524 observed P1 (6-47% of trials) or N1 (11-28% of trials) responses evoked by SP and Tib nerve 525 in the three periods, as well as the appearance of N1 responses (12-61% of trials) or 526 disappearance of P1 responses (11-33%) during perineal stimulation (Fig. 7A-B, left). Pinch Lastly, we evaluated the modulation of ipsilateral and contralateral reflex responses by 543 perineal stimulation in the LG muscle, an ankle extensor and a knee flexor (Fig. 8). In the 544 ipsilateral LG, we observed P1 (23-82% of trials) or N1 (7-25%) responses evoked by SP 545 and Tib nerve stimulation in all three periods or a switch from P1 to N1 responses (11-54% 546 of trials) (Fig. 8A-B, left). Vibration and pinch of the perineal region significantly decreased In summary, mechanically stimulating the perineal region with vibration or pinch 563 decreased ipsilateral and contralateral reflex responses evoked by SP or Tib nerve stimulation 564 in all studied muscles, as summarized in Table 3. In some cases, perineal stimulation also 565 caused the appearance of inhibitory responses or a switch from a positive to a negative For each muscle, ↑ or ↓ represent a significant increase or decrease, respectively, in reflex  The present study showed that vibration or pinch of the perineal region was equally 628 effective in triggering unilateral and bilateral rhythmic activity ( Table 1)  Interestingly, we also observed that perineal stimulation induced different patterns of 645 rhythmic activity between the ipsilateral and contralateral sides relative to nerve stimulation.

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Interestingly, we reported that pinching the skin of the perineal region was more effective 743 than vibration in decreasing the gain of cutaneous reflexes (     Table 3. For each muscle, ↑ or ↓ represent a significant increase or decrease, respectively, in