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Journal of Neurophysiology logoLink to Journal of Neurophysiology
. 2022 Jan 5;127(2):493–503. doi: 10.1152/jn.00385.2021

Crossed reflex responses to flexor nerve stimulation in mice

Olivier D Laflamme 1, Marwan Ibrahim 1, Turgay Akay 1,✉
PMCID: PMC8836714  PMID: 34986055

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Keywords: crossed reflex, locomotion, mice, sensory feedback

Abstract

Motor responses in one leg to sensory stimulation of the contralateral leg have been named “crossed reflexes” and are extensively investigated in cats and humans. Despite this effort, a circuit-level understanding of the crossed reflexes has remained missing. In mice, advances in molecular genetics enabled insights into the “commissural spinal circuitry” that ensures coordinated leg movements during locomotion. Despite some common features between the commissural spinal circuitry and the circuit for the crossed reflexes, the degree to which they overlap has remained obscure. Here, we describe excitatory crossed reflex responses elicited by electrically stimulating the common peroneal nerve that mainly innervates ankle flexor muscles and the skin on anterolateral aspect of the hind leg. Stimulation of the peroneal nerve with low current intensity evoked low-amplitude motor responses in the contralateral flexor and extensor muscles. At higher current strengths, stimulation of the same nerve evoked stronger and more synchronous responses in the same contralateral muscles. In addition to the excitatory crossed reflex pathway indicated by muscle activation, we demonstrate the presence of an inhibitory crossed reflex pathway, which was modulated when the motor pools were active during walking. The results are compared with the crossed reflex responses initiated by stimulating proprioceptors from extensor muscles and cutaneous afferents from the posterior part of the leg. We anticipate that these findings will be essential for future research combining the in vivo experiments presented here with mouse genetics to understand crossed reflex pathways at the network level in vivo.

NEW & NOTEWORTHY Insights into the mechanisms of crossed reflexes are essential for understanding coordinated leg movements that maintain stable locomotion. Advances in mouse genetics allow for the selective manipulation of spinal interneurons and provide opportunities to understand crossed reflexes. Crossed reflexes in mice, however, are poorly described. Here, we describe crossed reflex responses in mice initiated by stimulation of the common peroneal nerve, which serves as a starting point for investigating crossed reflexes at the cellular level.

INTRODUCTION

Stimulation of the somatosensory afferents from one leg has been shown to elicit motor responses in the contralateral leg that was termed “crossed reflex” (1). These crossed reflex responses were shown to be elicited by the stimulation of proprioceptive sensory afferents (groups I and II) and cutaneous afferents, as well as flexor reflex afferents (FRAs), a term defining high-threshold afferents from joints, muscles, and skin involved in ipsilateral limb flexion and contralateral limb extension (2). Activation of all these afferents generates excitatory and inhibitory responses in contralateral flexor and extensor muscles in cats (3–8).

Neurons whose axons cross the spinal cord from one side to the other (commissural interneurons, CINs) involved in crossed reflexes have been described in cats (4, 9, 10). In mice, genetically and physiologically distinct classes of CINs that are important in left-right coordination during locomotion have been identified (11–13). However, further research is required to provide insights into whether or not the CINs identified in cats and those identified in mice share common pathways. A promising approach is to conduct crossed reflex experiments with mice whose CIN pathways have been genetically manipulated. A thorough description of the crossed reflex responses in normal wild-type mice is essential for this to happen.

Crossed reflex experiments have been performed, to a limited degree, in rodents using in vitro spinal cord preparations (14, 15) or in vivo decerebrate adult mouse preparations (16). We have recently demonstrated a new in vivo technique to record excitatory and inhibitory crossed reflex pathways elicited by stimulating two peripheral nerves (17). First, the tibial nerve was stimulated electrically at different intensities to activate sensory afferents from ankle extensor muscles (e.g., gastrocnemius, plantaris, and soleus) or the posterior and distal aspects of the hind leg (18). Second, we stimulated the main trunk of the sural nerve that predominantly carries afferents from the hind leg’s cutaneous regions that are more lateral (19). However, it is not known whether the motor responses of one leg are different depending on the target of the afferents stimulated.

Here, we aimed to extend our investigation of normal crossed reflexes by measuring crossed reflex responses elicited by electrical stimulation of muscle afferents from flexor muscles and afferents from a cutaneous region on the anterior aspect of the hind leg. To do this, we recorded electromyogram (EMG) activity from up to five hindlimb muscles, whereas we stimulated the common peroneal nerve in the contralateral hind leg to activate either proprioceptive afferents from ankle flexor muscles (tibialis anterior, peroneus longus, extensor digitorum longus, etc.) or cutaneous afferents from the anterior aspect of the hind leg (20) (Fig. 1A). Our data suggest that during resting, the overall pattern of crossed reflexes initiated by the common peroneal nerve stimulation is similar to the pattern previously shown when the tibial nerve was stimulated. Furthermore, when crossed reflexes are initiated during walking, we observed downregulation of the inhibitory crossed reflexes in knee extensor and ankle flexor muscles, similar to when the tibial nerve is stimulated. However, we also observed the downregulation of the inhibitory crossed reflex in the ankle extensor muscle when we stimulated the common peroneal nerve. These experiments add to the groundwork in the mouse model to identify the neuronal pathways involved in crossed reflexes and the role of these crossed reflex pathways during motor behavior.

Figure 1.

Figure 1.

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Schematic of experimental design used to investigate crossed reflex in vivo. A: the experimental design used to investigate crossed reflex in vivo. Example of EMG recording at low- (1.2 × T; i) and high- (5 × T; ii) current stimulation from one mouse after stimulation (stim.) of the common peroneal nerve (cp. n.) using a single pulse (B) or five-pulse train (C). Shaded areas indicate stimulation. Gsr, right gastrocnemius; Ipr, right iliopsoas; sens., sensory neurons; Str, right semitendinosus; TAl, left tibialis anterior; TAr, right tibialis anterior; VLr, right vastus lateralis.

MATERIAL AND METHODS

Experiments were conducted on 20 adult wild-type (WT) mice (2–4 mo old) of either sex from C57Bl6 background mouse. None of the mice were trained on the treadmill before the experiments. All studies were performed according to the Canadian Council on Animal Care (CCAC) guidelines and approved by the local council on animal care of Dalhousie University.

Construction of the Electrodes

The electrodes were made using multistranded, Teflon-coated annealed stainless steel wire (A-M systems, Cat. No. 793200). The construction of the EMG electrode and nerve cuff was previously described in detail (21–23). One or two nerve cuff electrodes and six EMG recording electrodes were attached to the headpiece pin connector (female, SAM1153-12; Digi-Key Electronics, Thief River Falls, MN) and covered with epoxy (Devcon 5 min Epoxy Gel).

Electrode Implantation Surgeries

All surgeries were performed in aseptic conditions and on a warm water-circulated heating pad maintained at 42°C. Each mouse received an electrode implantation surgery, as previously described (17). Briefly, the animals were anesthetized with isoflurane (5% for inductions, 2% for maintenance of anesthesia), ophthalmic eye ointment was applied to the eyes, and the skin of the mice was sterilized with three-part skin scrub using hibitane, alcohol, and povidone-iodine. Before each surgery, buprenorphine (0.03 mg/kg) and ketoprofen (5 mg/kg)/meloxicam (5 mg/kg) were injected subcutaneously as analgesics, whereas the animals were still under anesthesia. Additional buprenorphine injections were performed in 12-h intervals for 48 h.

A set of six bipolar EMG electrodes and one or two nerve stimulation cuffs were implanted in a total of 20 wild-type mice, as previously described (22, 23). Small incisions were made on the shaved areas (neck and both hind legs), and each bipolar EMG electrode and the nerve cuff electrodes were led under the skin from the neck incisions to the leg incisions, and the headpiece connector was stitched to the skin around the neck incision. The EMG recording electrodes were implanted into the right (ipsilateral) hip flexor (iliopsoas, Ipr), knee flexor (semitendinosus, Str) and extensor (vastus lateralis, VLr), and ankle flexor (tibialis anterior, TAr) and extensor (gastrocnemius, Gsr) as well as the left ankle flexor (tibialis anterior, TAl) or the left ankle extensor (gastrocnemius, Gsl) depending on whether the common peroneal (13 mice; 8 males and 5 females) or the main trunk of the tibial nerve (7 mice; all males) were stimulated, respectively. Nerve stimulation electrodes were implanted to the left common peroneal or tibial nerves to activate proprioceptive and cutaneous feedback that produce crossed reflexes and the right sural nerve (the main trunk) to evoke ipsilateral cutaneous reflexes. The anesthetic was discontinued, and mice were placed in a heated cage for 3 days before returning to a regular mouse rack. Food mash and hydrogel were provided for the first 3 days after the surgery. Any mouse handling was avoided until mice were fully recovered, and the first recording session started at least 10 days after the electrode implantation surgeries.

Crossed Reflex Recording Sessions

After the animals fully recovered (∼10 days) from the electrode implantation surgeries, crossed reflexes were recorded as follows: under brief anesthesia with isoflurane, a wire to connect the headpiece connector with the amplifier, and the stimulation insulation units (ISO-FLEX; AMPI, Jerusalem, Israel, or the DS4; Digitimer, Hertfordshire, UK) were attached to the mouse. The anesthesia was discontinued, and the mouse was placed on a mouse treadmill (model 802; custom built in the workshop of the Zoological Institute, University of Cologne, Germany). The electrodes were connected to an amplifier (model 102; custom built in the workshop of the Zoological Institute, University of Cologne, Germany) and a stimulus isolation unit. After the animal fully recovered from anesthesia (at least 5 min), the minimal (threshold) current necessary to elicit local monosynaptic reflex responses was determined. This was done by injecting single impulses of 0.2-ms duration into the peroneal nerve or the tibial nerve while recording the EMG response in the tibialis anterior or the gastrocnemius muscle, respectively (average ±SD threshold current: 111.8 ±82.3 µA; range: 22.5–250 µA). Following the determination of threshold currents, the current injected into the common peroneal nerve was set as either 1.2 times the monosynaptic reflex threshold current (1.2 × threshold) or five times the monosynaptic reflex threshold current (5 × threshold).

EMG signals from five muscles of the right leg and the tibialis anterior or gastrocnemius muscle of the left leg were simultaneously recorded (sampling rate: 10,000 kHz), whereas the common peroneal or tibial nerve of the left leg was electrically stimulated with five brief impulses (impulse duration: 0.2 ms, frequency: 500 Hz) using the ISO-FLEX (AMPI, Jerusalem, Israel) and DS4 (Digitimer, Hertfordshire, UK) stimulation insulation units. In some experiments, the right sural nerve was also stimulated in combination with contralateral tibial or sural nerve stimulation. The crossed reflexes were recorded while the mice were resting on the treadmill or moving at 0.2 m/s constant speed. The EMG signals were amplified (gain 100), band-pass filtered from 400 Hz to 5 kHz, and stored on the computer using Power1410 interface and Spike2 software (Cambridge Electronic Design, Cambridge, UK).

Statistical Analysis

All graphical representations of data were made using GraphPad Prism 5 and processed using Illustrator CS5 (Adobe). All data are presented as means ± standard deviation. One-to-one statistical comparisons of the data were made with the t test or Mann–Whitney test using GraphPad Prism 5 depending on whether the normality was present (t test) or not (Mann–Whitney test) using omnibus K2 or Kolmogorov–Smirnov tests. All statistical tests were two-tailed, and differences were considered statistically significant when the P value was <0.05.

RESULTS

Crossed Reflex Motor Activity in Flexor and Extensor Muscles

First, the left common peroneal nerve was electrically stimulated with a single pulse (0.2 ms) at different current intensities to determine the threshold current that would elicit motor responses on the ipsilateral TA muscle (Fig. 1B). Following the determination of the threshold current, currents at 1.2 × T and 5 × T were used to deliver five pulses at 500 Hz to investigate the role of proprioceptive and cutaneous afferents in crossed reflexes in the mouse, respectively (Fig. 1C). In these experiments, the low-current stimulation (1.2 × threshold) would predominantly activate group Ia (from muscle spindles) and Ib (from Golgi tendon organs) proprioceptive afferents from ankle flexor muscles (24, 25). Our results showed that low-amplitude motor responses were evoked in all contralateral leg’s recorded flexor or extensor muscles (Fig. 1Ci). High-current stimulation (5 × threshold), which activated proprioceptive afferents (the Group Ia, Ib, and II), flexor reflex afferents, and cutaneous afferents (groups βand γ) (25) from the anterolateral aspect of the leg, evoked more robust motor responses simultaneously in right flexor and extensor muscles (Fig. 1Cii). Overall, the observed muscle activity pattern, as a response to the common peroneal nerve stimulation at different intensities, was qualitatively similar to the observations with tibial nerve stimulation (17).

We then investigated the crossed reflex responses by analyzing the rectified EMG signals and averaging them over multiple trials at either 1.2 × T (Fig. 2i, Supplemental Fig. S1; see https://doi.org/10.6084/m9.figshare.17096603) or 5 × T (Fig. 2ii). Stimulation of the common peroneal nerve elicited crossed reflex responses in every recorded muscle, regardless of stimulation intensity. However, we noticed that low-current stimulation elicited considerably weaker responses when the common peroneal nerve was stimulated compared with when tibial nerve was stimulated. This weaker response in the average EMG traces was due to less robust responses in individual muscles (Supplemental Fig. S1). Therefore, we next analyzed the probabilities of contralateral muscle activation with the common peroneal nerve stimulation and compared them with the probabilities of muscle response to tibial nerve stimulation (Fig. 3). Confirming the observations described in Fig. 2, the occurrence of activity in all contralateral muscles was lower when the common peroneal nerve was stimulated at 1.2 × T compared with the tibial nerve. It is noteworthy that the differences were only statistically significant for St, TA, and Gs (Fig. 3A). We observed no difference in the occurrence of local reflex response in the ipsilateral TA for the common peroneal nerve stimulation and Gs for the tibial nerve stimulation. In contrast, the occurrences of crossed reflex responses in any recorded muscles were not different when the common peroneal nerve and tibial nerves were stimulated at 5 × T (Fig. 3B). These data suggest that proprioceptive afferent activation from flexor muscles elicits weaker crossed reflex responses than proprioceptive afferents from extensor muscles.

Figure 2.

Figure 2.

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Average crossed reflex responses from different muscles after stimulation of the common peroneal nerve from 13 animals (8 males and 5 females). i: low (1.2 × T) current stimulation. ii: high (5 × T) current stimulation. The gray lines are averages of 20–40 stimulations in each animal. The black lines correspond to the average across all animals. Shaded areas indicate stimulation. cp. n. stim., stimulation of the common peroneal nerve; Gsr, right gastrocnemius; Ipr, right iliopsoas; Str, right semitendinosus; TAl, left tibialis anterior; TAr, right tibialis anterior; VLr, right vastus lateralis.

Figure 3.

Figure 3.

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Occurrence of motor responses following the common peroneal (n = 13; 8 males, 5 females) and tibial nerve (n = 7; all males) stimulation. Bar graph showing the percentage of time a motor response was triggered following stimulation of either the common peroneal nerve (gray) or tibial nerve (black) at low current (A) and high current (B). Occurrence was calculated using 40 nerve stimulations for the same current strength. Each dot represents one animal. GSl, left gastrocnemius; Gsr, right gastrocnemius; Ipr, right iliopsoas; Str, right semitendinosus; TAl, left tibialis anterior; TAr, right tibialis anterior; VLr, right vastus lateralis. Statistical comparisons were made using Student’s t test if normality was present. Otherwise, Mann–Whitney test was applied. Open circles on the bars indicate averages from individual animals.

Temporal Characteristics of Muscle Activation Pattern during Crossed Reflex Initiated by the Common Peroneal Nerve Stimulation

We next compared the muscle activation pattern during crossed reflexes by measuring the delays between the stimulation onset and the on and offsets of activities in each recorded muscle when the common peroneal nerve (Fig. 4, A and B) or the tibial nerve (Fig. 4, C and D) was stimulated. When we compared the pattern at 1.2 × T in both nerves, onsets of activity did not exhibit considerable differences (Fig. 4E). In contrast, the duration of activities in all muscles was shorter when the common peroneal nerve was stimulated than when the tibial nerve was stimulated with 1.2 × T stimulation (Fig. 4Fi). The pattern at 5 × T was primarily similar regardless of which nerve was stimulated (Fig 4, Eii and Fii). Moreover, we observed a silent period with no activity in any EMG recordings after common peroneal nerve stimulation (Fig. 4, A and B) and tibial nerve stimulation (Fig. 4, C and D). This was similar to our previously reported observations (17). Overall, the muscle activation pattern during crossed reflex was qualitatively similar regardless of the nerve -stimulated or stimulation intensity, with minor differences mainly reflected in the duration of activities.

Figure 4.

Figure 4.

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Crossed reflex muscle activation pattern. A: the muscle activation pattern at low- and high-current stimulation following the common peroneal nerve stimulation (data from 13 experiments; 8 males and 5 females). B: latencies of activity onset in different muscles at low- and high-current stimulation following the common peroneal nerve stimulation. C: the pattern of muscle activation at low- and high-current stimulation following tibial nerve stimulation summarizing data from seven experiments (all male). D: latencies of onsets of activity in the recorded muscles following tibial nerve stimulation. E and F: latencies of onset and duration of activities in each recorded muscle as a response to common peroneal nerves (gray bars) or the tibial nerve (black bars) at low (i) and high (ii) current. Open circles on the bars indicate averages from individual animals. The shaded area in A–D represents the stimulation area. The striped area represents 10 ms after the stimulation. Filled and empty circles represent latency onset at low and high current, respectively. In B and D–F, statistical comparisons were made using Student’s t test if normality was present. Otherwise Mann–Whitney test was applied. cp. n. stim., common peroneal nerve stimulation; Gsl, left gastrocnemius; Gsr, right gastrocnemius; Ipr, right iliopsoas; Str, right semitendinosus; TAl, left tibialis anterior; TAr, right tibialis anterior; tib. n. stim., tibial nerve stimulation; VLr, right vastus lateralis.

Short Latency Inhibition in Crossed Reflex Initiated by the Common Peroneal Nerve Stimulation

The observation of a silent period after the common peroneal nerve stimulation at 5 × T indicated the presence of an inhibitory crossed reflex pathway. To investigate this possibility further, we used a paired stimulation protocol where peripheral nerves on both legs were stimulated with different delays, as previously shown (17). For this, nerve stimulation electrodes were implanted to the common peroneal nerves of the left leg to evoke crossed reflex motor responses. In addition to the five EMG recording electrodes in the right hind leg, we implanted a nerve stimulation electrode to the right sural nerve to evoke local cutaneous reflex (Fig. 5A). As it was shown previously (17), we reasoned that an inhibitory crossed reflex pathway should cause decreased activity with a constant delay after contralateral nerve stimulation regardless of the presence of EMG activity initiated by local reflex.

Figure 5.

Figure 5.

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Short-latency inhibition in crossed reflex initiated by tibial nerve stimulation. A: schematic presentation of the paired stimulation of the left common peroneal nerve (cp. n.) and right sural nerve (sur. n.) at different delays. The existence of a decreased electromyogram (EMG) activity immediately after cp. n. stimulation (black traces) regardless of expected activity due to sur. n. stimulation (red traces) is the evidence for the existence of an inhibitory crossed reflex pathway. Data are representative examples from 20 stimulations for each muscle for a total of six experiments (3 males and 3 females). The traces are average EMG recordings from contralateral flexor (B) and the extensor (C) muscles. Shaded areas represent stimulation of the contralateral common peroneal nerve to evoke a crossed reflex. Hatched areas represent the silent period detected previously. Red traces represent the average EMG response to local reflex activation initiated by ipsilateral sural nerve stimulation. Black traces indicate the EMG response when the ipsilateral sural nerve is stimulated with the contralateral tibial nerve with a delay indicated on the left of each set of recordings. Blue arrows represent the suppression of local cutaneous reflex by contralateral sensory stimulation. Gsr, right gastrocnemius; Ipr, right iliopsoas; Str, right semitendinosus; TAl, left tibialis anterior; TAr, right tibialis anterior; VLr, right vastus lateralis.

First, we stimulated the right sural nerve with a high-current (5 × threshold) to obtain a local reflex response (Fig. 5, B and C, red traces). Then, we stimulated the left common peroneal nerve simultaneously with the right sural nerve at varying delays (Fig. 5, B and C, black traces). We detected a period of decreased EGM activity immediately after the left common peroneal nerve stimulation (Fig. 5, B and C, blue arrows). This response could be detected consistently in all recorded flexor (Fig. 5B) and extensor (Fig. 5C) muscles. This finding suggested that similar to the previous reports, with tibial and sural nerve stimulations (17), inhibitory crossed reflex pathways were also activated with the common peroneal nerve stimulation.

Long-Latency Crossed Reflex Responses Initiated by the Common Peroneal Nerve Stimulation

One qualitative difference between the crossed reflex responses initiated by the common peroneal or the tibial nerve was the presence of a long-latency response observed when the common peroneal nerve was stimulated but not the tibial nerve (Fig. 6). The 75-ms timeframe following the contralateral tibial and the common peroneal nerves are illustrated in Fig. 6, A and B, respectively. We consistently detected a long-latency response in all recorded muscles only when the common peroneal nerve was stimulated but not when the tibial nerve was stimulated.

Figure 6.

Figure 6.

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Long-latency motor responses in the common peroneal (cp. n.) but not tibial nerve (tib. n.) stimulation (stim). Example of average crossed reflex responses from different muscles after the stimulation of tibial nerve representing similar results from seven animals (A) or the common peroneal nerve representing similar results from 13 animals (8 males and 5 females) (B). Heat diagrams of the muscle activity are shown underneath each average. Muscle response to each of 40 nerve stimulations from 1 experiment is staggered on the vertical axis as a function of time. The brighter color indicates higher muscle Gsr, right gastrocnemius; Ipr, right iliopsoas; Str, right semitendinosus; TAr, right tibialis anterior; VLr, right vastus lateralis.

Modulation of Crossed Reflex Responses during Locomotion Initiated by the Common Peroneal Nerve Stimulation

Are the crossed reflex responses to the common peroneal nerve stimulation similar when the animal is moving compared with when the animal is resting? To address this question, we stimulated the left common peroneal nerve when the animals were moving on a treadmill and recorded EMG activity from the right leg muscles (Fig. 7A). We evoked crossed reflexes by stimulating the common peroneal nerve at high current (5 × T) separately during each muscle’s active or inactive phase (Fig. 7, B and C). We observed that the crossed reflex responses during locomotion depended on the timing of the nerve stimulation relative to the activity of the muscle before the stimulation. We consistently detected the silent period after nerve stimulation when all muscles were inactive before nerve stimulation, as described during resting above. However, the silent period was absent when the contralateral nerve stimulation occurred while the Gsr, VLr, and TAr muscles were active (Fig. 7). In contrast, in the EMG activity profile of Ipr and Str, a short latency silent period was always observed after stimulation regardless of the muscle activity before nerve stimulation (Fig. 7). These data reveal that the crossed inhibitory reflex pathway is downregulated during locomotion selectively for Gsr, VLr, and TAr muscles depending on the activity before the contralateral common peroneal nerve stimulation.

Figure 7.

Figure 7.

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Crossed reflex response selectively in right vastus lateralis (VLr), right tibialis anterior (TAr), and right gastrocnemius (Gsr) depends on muscle activity status before nerve stimulation during walking. A: electromyogram (EMG) recordings from the right iliopsoas (Ipr), VLr, TAr, and Gsr at rest and the two different states during walking when the muscle was not active or active before the stimulation. These examples are representative of nine experiments (5 males and 2 females). Occurrences of muscle responses, as active or silent within the first 5 ms after stimulation of the common peroneal nerve at low current (B) or high current (C). The graphs in B and C illustrate the data from nine animals (5 males and 2 females). Str, right semitendinosus; TAl, left tibialis anterior.

DISCUSSION

Here, we presented a detailed analysis of the crossed reflex responses evoked by the common peroneal nerve stimulation in awake mice in vivo during resting and locomotion. In the following, the results are compared with crossed reflex responses evoked by tibial nerve stimulation or sural nerve stimulation (17). Our data indicate that crossed reflex responses are mostly similar regardless of which nerve is stimulated, with mainly two qualitative differences. First, the common peroneal nerve stimulation elicits a long-latency crossed reflex response that is absent when the tibial nerve [in this paper and the study by Laflamme and Akay (17)] or sural nerve (17) is stimulated. Second, when the crossed reflex responses are elicited with the common peroneal nerve stimulation while the animal is walking, we observed downregulation of inhibitory crossed reflex responses in TA, VL, and Gs muscles. This downregulation of the inhibitory crossed reflex responses was only observed in TA and VL, but not in Gs, when tibial or sural nerves were stimulated (17). These findings will be essential and serve as a starting point for future research combining the type of experiments presented here with mouse genetics to understand the spinal network underlying the crossed reflexes.

Short-Latency Crossed Reflex Responses Are Widely Similar Regardless of the Stimulated Nerve

Our results in this article indicate that short-latency activation patterns as a response to the stimulation of different nerves of the contralateral sites are widely similar when the animal is resting. The similarity of the response to the common peroneal nerve stimulation, with the tibial and sural nerve stimulation, is especially interesting when considering the muscles or cutaneous regions these nerves innervate. First, the common peroneal nerve innervates mostly flexor muscles of the ankle joint, namely the tibial anterior and the extensor digitorum longus. In contrast, the tibial nerve innervates ankle extensor muscles, such as gastrocnemius muscles, soleus, and plantaris muscles. Second, the cutaneous sensory fibers that run through the common peroneal nerve originate mainly from the anterior aspect of the leg. In contrast, the cutaneous sensory fibers that run through the tibial and the sural nerves originate from the posterior aspects of the leg (26). The similarities of the crossed reflex responses to these nerves indicate that the spinal network that conveys the afferent information is widely similar despite the difference in afferents origin.

The similarity of crossed reflex response to the stimulation of cutaneous afferents originating from different leg regions is especially interesting because clear differences are observed at the local reflex level. When cutaneous afferents from the anterior aspect of the leg are activated by electrically stimulating the superficial peroneal nerve (27) or the saphenous nerve (28), a well-defined reflex response called the stumbling corrective reaction is elicited (29–31). This stumbling corrective reaction is not observed when the tibial nerve or the sural nerve carrying cutaneous afferent fibers from the posterior aspect of the leg is stimulated. These observations indicate that even if the sensory afferent stimulated are completely different, the crossed reflex responses can be similar. This suggests that the divergence of spinal function or circuitry in one reflex (the ipsilateral reflex) can be absent in another reflex (the crossed reflex).

Apart from observing muscle activation patterns indicating excitatory crossed reflex pathways, we could find evidence of inhibitory crossed reflex pathways similar to when the tibial or the sural nerve is stimulated (17). Two observations indicated this. First, the stimulation of the common peroneal nerve at low intensity (1.2xT) initiated a staggering muscle activation on the contralateral side. When the stimulation strength was increased to recruit cutaneous afferents in addition to proprioceptive afferents, the muscle activation was more simultaneous with an ∼10-ms delay after the stimulation offset. Second, contralateral cutaneous afferent stimulation could suppress local reflex response initiated by ipsilateral cutaneous afferent stimulation. These data support the previous findings of inhibitory crossed reflex pathways activated by cutaneous afferents in cats (5) and humans (32) and in mice shown with tibial and sural nerve stimulation (17).

Collectively, our results, together with our previous data (17), suggest that proprioceptive and non-nociceptive cutaneous afferent activation initiate crossed reflex responses in mice. This is in agreement with results from research conducted on cats and humans that muscle afferents (4, 9, 32–35), as well as cutaneous afferents (5, 8, 34, 36), mediate crossed reflex responses.

Long-Latency Crossed Reflex Responses Are Only Observed When the Common Peroneal Nerve Is Stimulated

One of the most striking qualitative differences in the crossed reflex response in the resting mouse was a long-latency response only observed when the common peroneal nerve was stimulated but not when the tibial or the sural nerve was stimulated. We believe that these long-latency motor responses could be explained by the presence of a longer pathway that would involve the supraspinal centers (37). These pathways could involve main ascending pathways that carry proprioceptive and low-threshold cutaneous mechanoinformation, such as the dorsal column lemniscus pathways and the dorsal spinocerebellar pathway (38, 39). Clarification of this issue needs further experimentation in the future.

Crossed Reflex during Locomotion

Our data demonstrate that similar to the tibial and sural nerve stimulation (17), the crossed inhibitory reflex is downregulated when the animal walks selectively for the knee extensor (VL) and the ankle flexor (TA) muscles. In addition, the inhibitory crossed reflex is also downregulated in the Gs muscle during walking, only with the common peroneal nerve stimulation but not with tibial or sural nerve stimulation. This observation extends the previous observations that the inhibitory crossed reflex selectively for the VL muscle during locomotion in the cat when the superficial peroneal nerve and the tibial nerve were stimulated (40). Our present and previous (17) results add to this data that crossed inhibition is also downregulated during walking in TA muscle. We also show that during locomotion, crossed inhibition is downregulated in the ankle extensor muscle. Our present data extends previous data on inhibitory crossed reflex changes during walking to further describe crossed reflex modulation during locomotion.

Previously, it was suggested that the absence of crossed inhibitory influence in the VL was due to the more rostral location of the motor neuron pool in the spinal cord than the other muscles (40). However, our previous (17) and present results show that the inhibitory crossed reflex influence is also downregulated in TA (motor neurons located in lumbar 3–4 spinal segments) and only for the common peroneal nerve stimulation in Gs (motor neurons located mainly in lumbar 4 spinal segments) in addition to VL (motor neuron cell bodies located between lumbar spinal segments 1–3) (41). Therefore, our data do not support the view that the absence of the state-dependent modulation of the inhibitory crossed reflex response is due to motor neuron pool location.

Commissural interneurons mediating crossed reflexes from group II muscle spindle afferents have been shown to depend on the presence of serotonin. When group II muscle afferents were stimulated, short-latency inhibition of contralateral extensor motor neurons was observed when the spinal cord was intact, but excitation was observed when the animal was spinalized (4, 9). The inhibition of motor neurons in spinalized cats could be restored following activation of 5-HT receptors (3b). In addition, monoaminergic inputs are also known to interact with commissural interneuron located in lamina VIII of the spinal cord (42, 43) and that they are also involved in rhythmic motor activities such as locomotion (44). Therefore, it is likely that monoaminergic modulation might be the regulating factor of inhibitory crossed reflex in the VL, TA, and Gs during locomotion.

In this study, we provide a detailed description of the motor response to the stimulation of contralateral sensory afferents innervating the ankle flexor muscles and the skin covering the anterolateral aspect are stimulated in mice in vivo. Our results demonstrate that crossed reflexes regardless of the stimulated nerves are mostly similar with slight differences. These differences are limited to the downregulation of the inhibitory crossed reflexes to particular muscles during walking. Moreover, we found the existence of long-latency crossed reflex responses when the common peroneal nerve is stimulated (present article), but not when the tibial or sural nerve is stimulated (17). These data will be important for our future efforts to disclose the role of genetically distinct classes of commissural interneurons (11–13) in crossed reflexes.

SUPPLEMENTAL DATA

Supplemental Fig. S1: https://doi.org/10.6084/m9.figshare.17096603.

GRANTS

This work was funded by the Natural Sciences and Engineering Research Council of Canada Grant RGPIN-2015–03871 and the National Institutes of Health Grant R01 NS115900.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

T.A. conceived and designed research; O.D.L and M.I. performed experiments; O.D.L. and M.I. analyzed data; O.D.L. and T.A. interpreted results of experiments; O.D.L. prepared figures; T.A. drafted manuscript; O.D.L. and M.I. edited and revised manuscript; O.D.L., M.I., and T.A. approved final version of manuscript.

ACKNOWLEDGMENTS

We thank Brenda Ross for technical assistance. We also thank Tyler Wells for proofreading the manuscript.

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Supplemental Fig. S1: https://doi.org/10.6084/m9.figshare.17096603.

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