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Groupe de Recherche sur le Système Nerveux Central, Department of Physiology, Faculty of Medicine, Université de Montréal, Montreal, Canada
Submitted 1 May 2007; accepted in final form 25 July 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). For instance, cats with muscle nerve sections show initial deficits during locomotion but recover relatively rapidly, highlighting the remarkable ability of the nervous system, and even the isolated spinal cord to quickly compensate for these losses (Bouyer et al. 2001; Pearson et al. 1999). As a result, denervations have been used to study compensatory mechanisms within the nervous system involved in offsetting the loss of motor and/or sensory nerves during locomotion (Abelew et al. 2000; Bouyer and Rossignol 2003a,b; Bouyer et al. 2001; Carrier et al. 1997; Cope et al. 1994; Pearson et al. 1999; Wetzel et al. 1973; Whelan et al. 1995).
To study this adaptive plasticity, Pearson and colleagues, in a series of seminal experiments, performed lesions of mixed nerves that innervate some ankle extensors [lateral gastrocnemius (LG), soleus, plantaris] (Misiaszek and Pearson 2002; Pearson and Misiaszek 2000; Pearson et al. 1999, 2003). In the days after the ankle extensors neurectomy, an increased ankle flexion (yield) during early stance and a decrease in maximal ankle extension at end stance were observed on the denervated side during locomotion. With time these deficits returned toward intact values, which was attributed to an increased activity of the medial gastrocnemius (MG), the primary remaining ankle extensor (Pearson et al. 1999). Functional recovery was not associated with the release of trophic factors from the lesioned nerve because injecting botulinum toxin into the LG, soleus, and plantaris, preventing transmission across the neuromuscular junction without damaging the nerve, produced similar deficits and an increased MG activity (Misiaszek and Pearson 2002). Moreover, functional recovery was use-dependent because cats whose denervated leg was immobilized for 6 days after the denervation did not have an increased MG activity and ankle yield resembled nonimmobilized cats once the splint was removed (Pearson et al. 1999). Large calibre afferents were deemed indispensable because performing a similar neurectomy in pyridoxine-treated cats, which is thought to selectively and permanently destroy large sensory afferents, prevented ankle yield from returning to normal and MG activity was either unaffected or changed slightly (Pearson et al. 2003). Thus large sensory afferents from the moving denervated leg are required for recovery.
Indeed, modified sensory feedback has been shown after partially denervating ankle extensors (Fouad and Pearson 1997; Whelan and Pearson 1997; Whelan et al. 1995). For instance, MG group I afferents had enhanced transmission to interneurons within the intermediate nucleus of lumbar segments L6/L7 (Fouad and Pearson 1997) and stimulating the MG nerve at group I strength had an accrued effectiveness in prolonging the stance phase (Whelan and Pearson 1997; Whelan et al. 1995) in decerebrate cats after partially denervating ankle extensors. It was thus hypothesized that transmission in group I reflex pathways from MG increased to reinforce the activity of this muscle after an ankle extensors neurectomy. This hypothesis was also largely based on the observations that additional stretch (e.g., increased ankle yield) and loading (e.g., increased force production) imposed on MG after the neurectomy would generate greater feedback from group Ia and Ib afferents, respectively, and because the postcontact EMG of MG thought to be mediated in part by sensory feedback (Gorassini et al. 1994; Hiebert and Pearson 1999), increased in the early days postneurectomy (Pearson et al. 1999). However, when tested more directly it was shown that MG group I pathways did not reinforce MG activity after denervating its close synergists. For example, the magnitude of homonymous and heteronymous group Ia excitatory postsynaptic potentials recorded intracellularly in MG motoneurons were unchanged within 1 wk of sectioning the LG-soleus (LGS) nerve (Fouad and Pearson 1997; Whelan et al. 1995). Furthermore, the increased MG activity recorded in spinal cats during locomotion did not parallel changes in MG muscle length after denervating the LGS, signifying that enhanced group Ia feedback did not mediate the increase (Bouyer et al. 2001). In another study, increased amplitude of stretch reflexes scaled with changes in the activity of MG after a LGS neurectomy, indicating that the increase in stretch reflex amplitude was caused by an increased motoneuronal activity and not mediated by increased fusimotor drive or Ia afferent transmission (Gritsenko et al. 2001). Therefore changes in MG group I reflex pathways do not seem to reinforce MG activity and are probably involved in another aspect of functional recovery after an ankle extensors neurectomy.
However, because sensory feedback is critical to functional recovery, it could be that several reflex pathways are modified after partially denervating ankle extensors. In this study, to evaluate whether changes occur in other reflex pathways, we stimulated the Tibialis (Tib) nerve at the ankle, which evokes responses in multiple hindlimb muscles during locomotion (Abraham et al. 1985; Duysens and Stein 1978; Loeb 1993; Pratt et al. 1991), before and after a LGS neurectomy. The Tib nerve was chosen because denervating ankle extensors increases the amplitude and duration of the yield at the ankle and reduces maximal ankle extension at end-stance (Pearson et al. 1999), in effect modifying how the plantar surface of the paw interacts with the ground during locomotion. It would thus be anticipated that transmission in reflex pathways from pressure-sensitive afferents of the paw would be altered. Moreover, it has recently been shown that Tib nerve reflexes are enhanced after denervation of other skin afferents supplying adjacent cutaneous territories of the paw (Bernard et al. 2007), indicating that a remaining cutaneous nerve can alter its activity to compensate for the sensory loss of other skin inputs. Because proprioceptive and cutaneous afferents are thought to have complementary roles during locomotion it is possible that the loss or reduction in one leads to an increase in the other (Duysens and Pearson 1976). Increased transmission in cutaneous afferents from the Tib nerve could offset the loss of proprioceptive information incurred by sectioning the LGS nerve. Consequently, we hypothesized that Tib nerve reflexes would be modified after the loss of ankle extensors to promote functional recovery. Preliminary results have been published in abstract form (Frigon et al. 2006).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Three adult cats of either sex (weight, 3.0–7.0 kg) were first selected based on their ability to walk for prolonged periods on a treadmill and trained for a few weeks at their preferred speed (0.35–0.5 m/s). Cats were subsequently implanted with chronic electrodes for EMG recordings and nerve stimulation, allowed to recover from the implantation, and baseline values of EMGs, Tib nerve reflexes, and kinematics were recorded. After stable control data were obtained, a neurectomy of the left LGS nerve was performed and recordings resumed, at the same treadmill speed, 24 h later and at different times thereafter to plot changes over time. The number of days studied after the neurectomy differed between cats (cat 1 = 43 days; cat 2 = 116 days; cat 3 = 54 days). A total of 78 recording sessions were made.
The experimental protocol was in accordance with the guidelines of the animal Ethics Committee of the Université de Montréal. All surgical procedures were performed under general anesthesia and aseptic conditions. Before surgery, cats were injected with an analgesic (Anafen, 2 mg/kg; sc) and premedicated (Atravet, 0.1 mg/kg; glycopyrrolate 0.01 mg/kg; ketamine, 10 mg/kg; im). Cats were intubated and maintained under gaseous anesthesia (isoflurane 2%) while heart rate and respiration were monitored. After surgery, an analgesic (buprenorphine, 0.01 mg/kg; sc) was administered subcutaneously. An oral antibiotic (cephatab or apo-cephalex, 100 mg/d) was given for the 10 days after surgery.
EMG
Because Tib nerve stimulation during locomotion evokes responses in multiple hindlimb muscles, chronic EMG electrodes were implanted bilaterally in the following: semitendinosus (St: knee flexor/hip extensor), anterior part of sartorius (Srt: hip flexor/knee extensor), vastus lateralis (VL: knee extensor), lateral gastrocnemius (LG: ankle extensor/knee flexor), medial gastrocnemius (MG: ankle extensor/knee flexor), soleus (ankle extensor), and tibialis anterior (TA: ankle flexor). Recording from a large subset of muscles enabled us to determine whether a LGS neurectomy generated changes in several muscles or was limited to synergistic ankle extensors. A pair of Teflon-insulated multistrain fine wires (AS633; Cooner wire, Chatsworth, CA) was directed subcutaneously from head-mounted 15-pin connectors (Cinch Connectors, TTI, Pointe-Claire, Canada) and sown into the belly of each muscle for bipolar EMG recordings. The neurectomy was performed several days after chronic electrode implantations to ensure that EMG recordings were consistent for a prolonged period. Over the course of the study some EMG recordings were lost in each cat because of some unknown reasons (cat 1: right MG; cat 2: left Srt, left VL; cat 3: left MG, right TA) as determined by the disappearance of EMG signals. EMG was band-pass filtered (100–3,000 Hz) and amplified (gains of 0.5–50 K) using two Lynx-8 amplifiers (Neuralynx, Tucson, AZ). EMG data were digitized (5,000 Hz) using custom-made acquisition software. Locomotor EMG bursts were recorded during nonstimulated trials to compare changes in muscular activity before and after the neurectomy. Burst duration, amplitude, and timing relative to foot contact of locomotor EMG were calculated using custom software. Normalized mean amplitude was defined as the area under the rectified EMG burst divided by its duration and expressed as a percentage of the average control value. The EMG amplitude for extensors of both hindlimbs were also divided into two periods similar to a previous study (Pearson et al. 1999). The first period comprised the initial 120 ms of the burst, and the second included the entire integrated area after ground contact. Kinematic and EMG data were synchronized using an SMPTE time code generator (Evertz, time code master 5010).
Neurectomy
After establishing stable control (EMG bursts and Tib nerve reflexes) recordings during locomotion (
33 days), the left LGS nerve was cut by carefully separating the nerve from its surrounding tissue in the popliteal fossa (Fig. 1). Capping the proximal end with flexible vinyl polysiloxane (Reprosil, Dentsply International, Milford, DE) was used to prevent nerve regeneration. No sham operations were included in this study because it was shown (Pearson et al. 1999) that using similar surgical procedures but leaving the LGS nerve intact did not produce deficits in locomotion. Contrary to a previous study (Pearson et al. 1999) but like others (Bouyer et al. 2001; Gritsenko et al. 2001) the plantaris nerve was left intact to minimize the damage done and because both types of denervations produce similar deficits. The anesthesia and surgery for the neurectomy lasted under an hour, and cats were tested the next day to allow sufficient recovery. Postmortem analysis confirmed that the LGS nerve was cut and did not regenerate in each cat.
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Kinematics of the left hindlimb were captured during treadmill locomotion using a Panasonic digital 5100 camera (1/1,000 s shutter speed, 30 frames/s or 60 de-interlaced fields/s = time resolution of 16.7 ms) and a Sony RDR-GX315 DVD recorder. Small reflective markers were placed over prominent bony landmarks at each joint of the left hindlimb including the iliac crest, greater trochanter, lateral epicondyle, lateral malleolus metatarsophalangeal (MTP) joint, and the tip of the fourth toe. Joint angles (hip, knee, ankle, MTP) were reconstructed off-line using custom-made software with a resolution of 60 fields/s from 10 to 20 step cycles.
Tibial nerve stimulation
A chronic stimulating electrode composed of bipolar wires (AS633, Cooner Wire) embedded in a polymer (Denstply International) cuff (Julien and Rossignol 1982) was placed around the left Tib nerve at the ankle adjacent to the Achilles' tendon (Fig. 1). The Tib nerve was stimulated (Grass S88 stimulator) at varying intensities using constant current through an isolation unit during locomotion with a single 1-ms pulse at a constant time (100 ms after onset of St burst) to determine the threshold for obtaining a small yet consistent short-latency (
10 ms) response in TA. Stimulation current was then set at 1.2 times this threshold to evoke consistent yet nonnoxious reflexes in several hindlimb muscles. During testing sessions, a stimulus was given once every three cycles at pseudorandom times to elicit responses at different epochs of the step cycle for a total of 120 stimulations. Once reflexes were qualitatively and quantitatively reproducible from one session to another for a few weeks, the same stimulation current was used for the remainder of the study and assumed to remain constant (see DISCUSSION). Reflexes were evoked before and at different times after the LGS neurectomy. M-wave amplitude from intrinsic foot muscles innervated by the Tib nerve could have been recorded and maintained at a similar size throughout the study to confirm the consistency of stimulation. However, because we were interested in how reflexes from the foot change as a result of altered interactions between the paw and the surface of the treadmill, we did not want to add any extraneous factors related to implanting small intrinsic foot muscles.
The methodology for quantifying reflexes is outlined in Fig. 2. The EMGs were grouped into stimulated or control (nonstimulated) trials. The step cycle was divided into 10 phases by synchronizing the cycle to the onset of a flexor burst (St). Averaged responses of EMGs with or without stimulation during reflex testing were separated into these 10 bins according to the time they were evoked in the cycle. An average of 50 nonstimulated cycles provided a template of baseline locomotor (blEMG) during the step cycle, and from the 120 stimulations, 10 reflex responses were grouped in each of the 10 phases. This provided about 10 reflexes in each of the 10 phases superimposed on the blEMG. Onset and offset of reflexes, delineated as a prominent negative or positive deflection away from the blEMG, were determined using predefined latencies as guidelines (Abraham et al. 1985; Duysens and Stein 1978; Loeb 1993; Pratt et al. 1991) such as P1 or N1 (8–10 ms) and P2 (25 ms), where P and N denote positive (excitation) and negative (inhibition) responses, respectively. For extensors, onsets and offsets were manually determined because P2 latency varied at different phases of the step cycle (see Fig. 2), whereas for ipsilateral flexors, time windows were fixed—St: P1 = 10–25 ms, P2 = 25–55 ms; TA: P1 = 10–30 ms, P2 = 30–55 ms). To measure reflex amplitude, the EMG from onset to offset was rectified and integrated and divided by the blEMG occurring in the same phase of the step cycle. This enabled us to compare changes in reflexes after the neurectomy independent of changes in the level of EMG activity (Duysens et al. 1993; Matthews 1986). To show the phase-dependent modulation of reflexes during the step cycle, reflexes for all days are expressed as a percentage of the maximal control value occurring in 1 of the 10 phases. Note that N1 responses in Figs. 8E and 9C represent inhibitory responses expressed as a percentage of control.
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To determine statistical differences after the LGS neurectomy, data from three separate control trials were pooled and compared with data recorded on each of the many postneurectomy days using a one-way ANOVA followed by Dunnett's post hoc test for many comparisons against a control group if significant changes were detected by the ANOVA (Bouyer et al. 2001). Reflexes were analyzed identically except that each response in each phase of the step cycle was treated independently. For example, N1 responses evoked in the fifth bin of the step cycle in the left MG in the intact state were compared against N1 responses evoked in the fifth bin of the step cycle in the left MG for each and every day after the neurectomy.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Locomotor EMG of several hindlimb muscles
Figure 3 shows rectified locomotor EMG bursts for selected muscles in the three cats in the intact state and at 2, 14, and 40–45 days postneurectomy. As can be seen, the EMG of several hindlimb muscles in all cats was increased after the neurectomy, whereas others were largely unaffected. In most cases, changes peaked at 2 days postneurectomy before returning to intact values thereafter. For example, large increases in EMG for MG occurred 2 days after the neurectomy before returning toward intact values (cats 1 and 2). Substantial increases in muscle activity could also be observed for other extensors including the left VL (cats 1 and 3) and the right VL (cat 1). More modest increases in EMG were also seen 2 days after the neurectomy in contralateral ankle extensors (cat 2). Changes in locomotor EMG were also apparent for some ipsilateral flexors such as the St, with the occurrence of an additional burst during stance (cats 1 and 3) and in TA at 2 days postneurectomy (cats 2 and 3).
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0.05) at 8 (–70.77 ± 22.28 ms) and 14 (–68.17 ± 22.28 ms) days postneurectomy. In cat 2, step cycle duration was significantly different (P 0.05) at 2 (–61.60 ± 19.75 ms) days postneurectomy but at no other day, including 1 day after. In cats 1 and 2, step cycle duration changed by <10 ms the day after the neurectomy. In cat 3, step cycle duration was significantly different (P 0.05) at 1 (–83.92 ± 8.20 ms), 2 (–27.26 ± 8.35 ms), 5 (–61.61 ± 8.35 ms), 8 (–28.01 ± 8.35 ms), 12 (–34.86 ± 8.35 ms), 14 (–37.91 ± 8.35 ms), and 27 (–40.12 ± 8.20 ms) days postneurectomy but not (P 0.05) at 6, 19, 22, and >30 days postneurectomy. Therefore changes in the timing and structure of the step cycle were not responsible for changes in reflexes described later on.
A more complete representation of changes in the mean amplitude of locomotor EMG for selected muscles is provided in Fig. 4 for each cat 50 days postneurectomy with each colored line showing a different muscle. In most extensors, increases in EMG peaked at 2 days after the neurectomy before returning toward intact values thereafter. However, whereas activity of some muscles completely returned to intact levels (left MG, left VL, right VL in cat 1) in others the EMG could remain elevated for prolonged periods without ever fully returning to prelesion values (left MG in cat 2; right MG in cat 3), and in a few cases, the EMG even started to increase a second time after the initial large increase and sharp reduction (left VL in cat 3). In all cats, the LGS neurectomy produced considerable changes in locomotor EMG in multiple hindlimb muscles but the subset of muscles affected could vary from one animal to another.
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It was previously shown that the centrally generated initial component of the MG EMG during locomotion followed a different time-course of increase than the postcontact, partially reflex-mediated component (Pearson et al. 1999). Figure 5 shows changes in the initial and postcontact components of the locomotor EMG, as denoted in Fig. 2B, in selected hindlimb extensors for the three cats. In general, both the initial and postcontact EMG components saw a large increase in the first 2 days before declining thereafter. The left MG of cat 1 was the only instance where both components did not share similar profiles. In this muscle, the initial component did not significantly (P = 0.204) increase with time after the LGS neurectomy (14 days), whereas the postcontact EMG peaked 2 days after the neurectomy before sharply declining. In cat 2, both the initial and postcontact components of the left MG significantly increased (P 0.001) immediately after the neurectomy and gradually decreased toward the intact value, although the relative increase in postcontact EMG was greater. In the left VL of cats 1 and 3, both the initial and postcontact components of the EMG significantly increased (P 0.001) and peaked at 2 days before gradually toward intact values. In contralateral extensors, the right VL of cats 1 and 3 and the right soleus of cat 2 showed large significantly increases (P 0.001) that peaked at 1 day postneurectomy followed by a sharp decrease thereafter.
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Figure 6A reconstructs the left hindlimb during swing and stance for cat 3 in the intact state and at 2 and 14 days postneurectomy. The most apparent change in limb trajectory is an increased yield (amount of flexion) at the ankle joint during the stance phase. With time, the magnitude of yield returned toward intact values. Similar to a previous study (Pearson et al. 1999) deficits resulting from the LGS neurectomy were quantified. Figure 6B shows changes in ankle yield for the three cats. Cat 3 had a large significant (P 0.001) increase in ankle yield, which returned toward the intact value but remained significantly (P 0.001) elevated for the remainder of the study. Cat 1 also had a significant (P 0.001) increase in ankle yield, but it returned to intact levels at 14 days (P = 0.962). In cat 2, although ankle yield changed significantly after the neurectomy (P 0.001) post hoc comparisons revealed that this was only significant at 2 days (P 0.05) after the denervation but not at 1 day (P = 1.00). Figure 6C shows changes in maximal ankle extension during the latter half of stance in the three cats for the same days as in Fig. 6B. The magnitude of ankle extension significantly decreased in cat 3 (P 0.001) in the week after the denervation, whereas in cat 1, it significantly increased (P 0.01). In cat 2, it was unchanged 1 day after the neurectomy (P = 1.00) before increasing on days 2 and 4 (P 0.01) and returning to intact values at 7 days (P = 0.68). In summary, in all cats, there was a significant increase in ankle yield in early stance in the first few days after the neurectomy and in maximal ankle extension attained during the latter half of stance, but the magnitude of these changes could vary from one cat to another.
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Figure 7 gives raw data for Tib nerve reflexes in the left MG of cat 1 (Fig. 7A) and the left St of cat 3 (Fig. 7B) in the intact state and for 2 selected days after the LGS neurectomy. In the left MG of cat 1, N1 responses increased in most phases of the step cycle, but this increase scaled with the level of blEMG and was not significantly different, except a significant decrease (P 0.05) in phase 0.7. The P2 responses did not significantly change (P 0.05) 2 days postneurectomy, but at 8 days they were significantly increased in phases 0.3–0.6, 300% of the maximal control value above the level of blEMG. In the left St of cat 3, P1 and P2 responses were significantly increased above the level of blEMG (P 0.05) 2 days postneurectomy, particularly from phases 0.4 to 0.7, when this muscle is normally inactive, and by 28 days, reflexes returned toward intact values.
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21 days in phases 0.0–0.3, where the muscle is normally active, and 0.9 (Fig. 8A). The P2 responses were significantly different in phases 0.0 and 0.2 for all selected days after the neurectomy but not in other phases except for phase 0.4 at 2 days postneurectomy (Fig. 8B). In the left St, P1 responses were increased predominantly in phases 0.4–0.7, when this muscle is inactive, for up to about 8 days after the neurectomy before returning to intact values (Fig. 8C). In general, P2 responses were decreased at 2 and 5 days in phases where the St muscle is active (0.8–0.2) before returning toward intact values later on (Fig. 8D). In the left MG, N1 responses were mostly unchanged after the neurectomy except for phases 0.6–0.7, where responses were decreased for some days (Fig. 8E). The P2 responses of the left MG were unchanged 2 days postneurectomy before increasing in phases 0.4–0.7 at 8 days, when this muscle is active, before returning toward intact values thereafter (Fig. 8F). In cat 2 (Fig. 9), P1 responses of the left TA were unchanged in the first 2 days postneurectomy before increasing at 9 days, mostly in phases 0.0 and 0.2–0.5, before increasing in all phases at 36 and 50 days (Fig. 9A). Changes in P2 responses of the left TA were more variable showing no clear trends after the neurectomy (Fig. 9B). In the left MG, N1 responses were decreased in a few phases in the first 2 days postneurectomy (Fig. 9C). Changes in P2 responses of the left MG were much more variable and only showed a decrease in the first 2 days in phase 0.5 of the step cycle. There were very large (>800% of the maximal control value) increases in P2 responses of the right MG (Fig. 9E) and soleus (Fig. 9F) after the neurectomy, which were limited to phase 0.8 of the step cycle or early stance of the right leg. Changes were gradual and only became significant at 36 days postneurectomy.
In cat 3 (Fig. 10), P1 responses of the left TA increased at 2 days postneurectomy in phases 0.2 and 0.4 before increasing in most phases at 8 days and returning toward intact values thereafter (Fig. 10A). Changes in P2 responses of the left TA were subtler and were confined to phases 0.0–0.1 and 0.4–0.6 (Fig. 10B). In the left St, P1 and P2 responses were increased in the first 2 days postneurectomy, particularly in phases where the muscle is active, before returning to intact values thereafter (Fig. 10C). In the right MG, increases in P2 were observed at 2 and 8 days after the neurectomy but only in phase 0.8 (Fig. 10E). In the right soleus, increases in P2 were seen 1 day postneurectomy in phase 0.7 and in phase 0.3 at 27 days (Fig. 10F).
Moreover, although each response is expressed as a function of the level of blEMG, in some cases, increased Tib nerve reflexes after the neurectomy were observed in muscles that showed little change in locomotor EMG [e.g., P1 responses in the left St of cats 1 (Fig. 8C) and 3 (Fig. 10C); P2 responses in the right MG (Figs. 9E and 10E) and soleus (Figs. 9F and 10F) of cats 2 and 3, respectively]. In addition, the largest changes in reflexes could be seen at days where changes in locomotor EMG were not peaking but declining (e.g., P2 responses in left MG of cat 1; Fig. 8F). Therefore modifications in reflex pathways can be independent from those seen in EMGs.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Technical considerations
When working with chronic animals, it is imperative that recordings and stimulations remain stable for the duration of the study to draw meaningful physiological conclusions. Based on several observations, we feel confident that most recordings reflected a physiological change in muscle activity. For instance, EMG activity in several extensors increased considerably in the immediate days after the LGS neurectomy before gradually returning toward control values over several weeks (Figs. 3 and 4). Moreover, reflex amplitude, at a given stimulus intensity, was modified in the days after the neurectomy but progressively returned to intact levels (Fig. 7) in some muscles, suggesting that stimulation intensity remained constant. If stimulation intensity was somehow modified, reflexes in all muscles would have changed similarly over time, but the data showed that modifications were most often limited to a particular muscle, to specific responses such as N1, P1, or P2, or to particular phases of the step cycle (Figs. 8–10).
Reorganization of muscular activity
Previous studies have shown that functional recovery after an ankle extensors neurectomy was mediated by an increased activity of close synergists (Bouyer et al. 2001; Pearson et al. 1999). Although we found considerable increases in MG EMG, changes in the activity of several muscles of both hindlimbs, which paralleled and in some cases exceeded that of MG, were also observed (Figs. 3 and 4). Muscles frequently displaying an increased activity included the left and right VL (knee extensors) and contralateral ankle extensors. Augmented activity in extensors other than MG could reinforce weight support and propulsion during stance. In addition, the appearance of an additional burst in the left St during stance in the early days after the neurectomy (Fig. 3) could assist other extensors in supporting and propelling the denervated leg during stance, because this muscle is also a hip extensor but normally only a flexor burst is recorded during level treadmill locomotion in cats (Rossignol 1996). Presumably, the activity of other muscles, such as hip extensors/adductors that were not recorded was also altered. Therefore modified activity after a LGS neurectomy is not solely limited to close synergists but involve several muscles, as is the case with cutaneous denervations of the hindpaws (Bouyer and Rossignol 2003a; Bretzner and Drew 2005). Furthermore, although the EMG was increased in several muscles for all cats, the subset of muscles involved could differ between cats probably stemming from interanimal differences in kinematic deficits incurred by the neurectomy (Fig. 6) and in neuronal connections (Loeb 1993). Thus cats share similar but also have unique adaptive strategies after an LGS neurectomy (Bouyer et al. 2001).
Changes in the locomotor EMG of most muscles followed similar time-courses and profiles. For instance, in cats 1 and 2, changes in muscle activity in all extensors peaked 2 days after the neurectomy before gradually returning toward normal values (Fig. 4). Although muscle hypertrophy after the neurectomy was not measured, studies have shown marked and progressive hypertrophy in a muscle after denervating its synergists (Degens et al. 1995; Walsh et al. 1978; Whelan and Pearson 1997). As a result, the muscle can produce more force and less neural drive is needed. However, in the early days after the neurectomy, large increases in EMG activity could only have been mediated by modifications in neural drive to motoneurons since hypertrophy at the muscle takes 10 days to develop (Degens et al. 1995).
Like previous studies (Bouyer et al. 2001; Gritsenko et al. 2001), changes in both initial and postcontact EMG components of most extensors shared similar profiles. A previous report showed that the initial (1st 120 ms) and late (100 ms centered on peak activity after foot contact) components of the MG EMG followed different profiles after denervating synergistic ankle extensors (Pearson et al. 1999). For example, the late component increased immediately after the lesion, which could be followed by either an increase or a decrease thereafter, whereas the initial period increased more gradually after the neurectomy. We found large increases in postcontact MG activity, which peaked 2 days after the neurectomy before gradually returning toward intact values (Fig. 5, cats 1 and 2). The initial component of MG EMG was more variable showing virtually no change in cat 1 for a period of 2 wk, whereas cat 2 showed a similar profile as the postcontact EMG, albeit smaller in proportion. Thus although there might be a slight dissociation between the two components for MG, in some animals, in other extensors the two periods behave similarly. Therefore the immediate increase in postcontact EMG might in part be reflex-mediated but it can also be centrally generated, as is the early precontact EMG as previously suggested (Gritsenko et al. 2001; Pearson et al. 1999), and also be modified by transmission in reflex pathways, such as the Tib nerve pathway, to the locomotor central pattern generator (CPG). A recent model formalizes how peripheral feedback from proprioceptive and cutaneous afferents can influence the locomotor CPG and the activity of several motoneuron pools (Rybak et al. 2006). The isolated spinal cord can govern the increased muscular activity (Bouyer et al. 2001), but in the intact state, it is probable that both spinal and supraspinal structures are involved. Increased corticospinal efficacy has been shown after a cutaneous denervation of the hindpaw in intact cats during locomotion (Bretzner and Drew 2005).
Plasticity in cutaneous reflex pathways
Plasticity in reflexes from plantar structures of the foot was observed in several muscles of both hindlimbs after the LGS neurectomy. At an intensity of 1.2 times the threshold for a small but consistent short-latency response in the ipsilateral TA, responses were most likely mediated by low-threshold afferents. Indeed, at this stimulation intensity, responses evoked in TA are only slightly higher in stimulation intensity than the threshold for recording an afferent volley in the sciatic nerve and are thought to be generated by the largest diameter A
afferents (Loeb 1993). However, influences from group I or II afferents cannot be eliminated because the Tib nerve also supplies intrinsic foot muscles. The stimulation did not visibly alter limb trajectory during locomotion, which is important because perturbations of the limb could introduce responses linked to the movement through proprioceptive reflexes. The pattern of responses, as opposed to amplitude, is typically invariant unless very high and clearly noxious stimulus intensities are used (Abraham et al. 1985; Loeb 1993). Therefore responses are probably mediated by low-threshold cutaneous afferents.
It has been proposed that electrically stimulating low-threshold cutaneous afferents could activate the same afferent population normally recruited during movement (Lundberg 1979; Lundberg et al. 1987; Pratt et al. 1991). Evidently, electrically stimulation differs from natural activation because multiple afferents are recruited simultaneously (Wand et al. 1980), but nevertheless, responses evoked during the time these afferents are normally active during locomotion could provide important clues as to their normal function. In other words, because cutaneous afferents from the Tib nerve are normally active during ipsilateral stance (e.g., when the paw is in contact with the ground), evaluating changes in Tib nerve reflexes during this period could provide some clues as to why they are altered after the LGS neurectomy. For example, the large increase in P2 responses in MG with time after the neurectomy, especially during the middle portion of stance, could reinforce MG activity during the propulsive portion of stance (Fig. 8F). In the left St, large increases in short-latency excitatory responses throughout stance in the early days postneurectomy (Figs. 8C and 10C) could reinforce hip extension for the purposes of weight bearing and propulsion during stance to offset the loss of ankle extensors, which are normally involved. Therefore changes in reflex pathways mediated by low-threshold afferents from the Tib nerve could serve to modify or correct the centrally generated pattern of muscle activation and promote functional recovery.
Reflexes evoked by stimulation have been shown to increase progressively with the level of preexisting voluntary activity and thus remain proportionally constant with the level of background activity, giving rise to the concept of automatic gain control (Matthews 1986). In other words, an afferent volley will evoke a larger reflex if more motoneurons are active and vice versa (this goes for excitation but also for inhibition as shown by Matthews). In this study, because changes in reflexes were in many cases independent of changes in locomotor EMG, premotoneuronal mechanisms must be involved in gating sensory feedback. The increase in force produced by knee and ankle extensors might increase the pressure on receptors of the foot and thus alter transmission in cutaneous afferents of the Tib nerve. Also, cutaneous pathways make polysynaptic connections within the spinal cord and a reorganization of pre- and/or postsynaptic inhibition at these different relays could substantially change reflex amplitude. Changes in pre- and/or postsynaptic inhibition could result from altered input originating in spinal and/or supraspinal structures to interneurons involved in these processes (Gossard et al. 1989, 1990). Last, collateral sprouting of afferents could rewire interneuronal circuitry and modify reflexes, at least in the long term (Cameron et al. 1992; Goldberger and Murray 1988; Koerber et al. 1994).
In summary, whatever the mechanism(s) may be, it is evident that reflex pathways from the Tib nerve are modified after an LGS neurectomy and that functional recovery involves a reorganized activity in several motoneuron pools. Adaptations to the loss of sensory and/or motor innervation caused by a neurectomy undoubtedly occur at multiple levels of the nervous system including reflex pathways from the periphery, locomotor generating networks within the spinal cord, and supraspinal inputs from the brain and brain stem, which in turn alters the activity of several muscles.
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Address for reprint requests and other correspondence: S. Rossignol, Dept. of Physiology, Groupe de Recherche sur le Système Nerveux Central, Faculty of Medicine, Université de Montréal, PO Box 6128, Station Centre-Ville, Montreal, Quebec, Canada H3C 3J7 (E-mail: serge.rossignol{at}umontreal.ca)
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