Plasticity in Reflex Pathways Controlling Stepping in the Cat

P. J. Whelan and K. G. Pearson

Department of Physiology, University of Alberta, Edmonton T6G 2H7, Canada

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Whelan, P. J. and K. G. Pearson. Plasticity in reflex pathways controlling stepping in the cat. J. Neurophysiol. 78: 1643-1650, 1997. Previous studies have shown that stimulation of group `I' afferents from ankle extensor muscles can prolong the cycle period in decerebrate walking cats and that the magnitude of these effects can be altered after chronic axotomy of the lateral-gastrocnemius/soleus (LGS) nerve. The effectiveness of LGS group I afferents in prolonging the cycle period decreases after axotomy, whereas the effectiveness of the uncut medial-gastrocnemius (MG) group I afferents is increased. The objectives of this investigation were to establish the time course of these changes in effectiveness and to determine whether these changes persist after transection of the spinal cord. The effects of stimulating the LGS and/or MG group I afferents on the cycle period were examined in 22 walking decerebrate animals in which one LGS nerve had been cut for 2 to 31 days. The effectiveness of LGS group I afferents declined progressively in the postaxotomy period, beginning with significant decreases at 3 days and ending close to zero effectiveness at 31 days. Large increases in the effectiveness of MG group I afferents occurred 5 days after axotomy, but there was no progressive change from 5 to 31 days. To test whether these changes in effectiveness were localized to sites within the spinal cord, the cord was transected in some decerebrate animals and stepping induced by theadministration of L-DOPA L-3-4 dihydroxyphenylalanine (LDOPA) and Nialamide. The effects of stimulating the MG and/or the LGS group I afferents on the cycle period were reexamined. In all four animals tested, stimulating the axotomized LGS group I afferents had a reduced effectiveness during locomotor activity in both the decerebrate and spinal states, whereas the increased effectiveness of the MG group I afferents was retained after transection of the spinal cord in two of five animals. Different mechanisms may be responsible for the changes in strength of the LGS and MG group I afferent pathways that project onto the rhythm generating sites in the spinal cord. This possibility follows from our observations of a linear relationship between the time after axotomy and decreased effectiveness of LGS group I afferents but no significant relationship between time postaxotomy and increased effectiveness of MG group I afferents; no significant relationship between the decreased effectiveness of LGS group I afferents and the increased effectiveness of MG group I afferents; and, after spinalization, consistent (4/4 cases) preservation of decreased LGS effectiveness but frequent (3/5 cases) loss of increased MG effectiveness.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Motor performance is optimized by the interplay of commands from the brain and spinal cord, and sensory feedback from peripheral receptors. Optimization of motor output depends on a continual adjustment of motor systems to changing environmental conditions, to neuronal and biomechanical changes during development, and to injury. In this regard, many studies of motor adaptation and learning have explored modification of reflex transmission. These include studies on oculomotor reflexes in mammals and fish (Du Lac et al. 1995), head-orientating responses to sound in owls (Knudsen 1994), adaptive postural responses in humans (Horak and Diener 1994), classically conditioned cutaneous reflexes in cats (Rispal-Padel and Meftah 1992), and instrumental conditioning of the H reflex in monkeys and rats (Wolpaw and Carp 1993). Finally, a variety of procedures have revealed plasticity in monosynaptic reflex pathways in the spinal cord: transection of nerves (Eccles and McIntyre 1953; Gallego et al. 1979), tenotomy (Goldfarb and Muller 1971; Kozak and Westerman 1961), limb immobilization (Maier et al. 1972; Mayer et al. 1981), blocking afferent activity by tetrodotoxin (Gallego et al. 1979; Webb and Cope 1992), and instrumental conditioning (Carp and Wolpaw 1994). The counterintuitive conclusion from these studies on the monosynaptic reflex is that the absence of activity increases the strength of the monosynaptic reflex, whereas increased activity decreases the strength of the reflex (Mendell 1984).

Recently we reported the occurrence of plasticity in group I excitatory pathways regulating the stance-to-swing transition in walking cats (Whelan et al. 1995b). Normally, stimulation of group I afferents in the lateral gastrocnemius and soleus (LGS) nerve is more effective in increasing the cycle period than stimulation of group I afferents in the medial gastrocnemius (MG) nerve. A few days after transection of the LGS nerve, however, the influence of group I afferents from the LGS and MG muscles is altered; the chronically sectioned LGS afferents become less effective, whereas the MG afferents become more effective (Whelan et al. 1995b).

In this investigation, we have extended the analysis of the plasticity in the extensor group I pathways regulating extensor duration. First, the number of experimental animals was increased to establish the time course of the changes in effectiveness of group I afferents in the MG and LGS nerves after sectioning of the LGS nerve. Second, we investigated whether a site of plasticity is located in the lumbro-sacral spinal cord by determining whether changes in effectiveness are conserved after transection of the spinal cord.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

All animals used in this study were cared for in accordance with the guidelines published by the American Physiological Society. The University of Alberta animal welfare committee approved the experimental procedures. Experiments were carried out in 22 adult cats of both sexes.

Chronic procedures

All 22 animals underwent the following minor surgical procedure. Under halothane anesthesia and aseptic conditions, the nerve supplying the LGS muscle of one hind leg was exposed and transected close to the muscle. The proximal nerve was tied with 6-0 silk to mark it for future identification. An antibiotic (Ayercillin, 1 ml) and, if necessary, an analgesic (Buprenorphine, 0.005-0.01 mg/kg) were administered for <= 1 wk after surgery. Animals were allowed out of their cages on a daily basis after surgery. The cages were large enough (dimensions: 73 wide × 69 deep × 84 cm high) to permit the animal to move freely and to jump onto a ledge. After a period of 2-31 days, the acute surgical and experimental procedures were performed.

Acute procedures

Two acute preparations were used: premammillary decerebrate cats and spinal cats treated with L-3-4 dihydroxyphenylalanine (L-DOPA). Thirteen animals were studied only after decerebration, whereas 9 animals were studied after decerebration and later that day after transection of the spinal cord at the T12 level.

Under halothane anesthesia, the nerves supplying the LGS (20 cats) and/or MG (19 cats) muscles of both hind legs were cut and tied into stimulating cuffs (see Whelan et al. 1995a for details of cuffs). The threshold of the electrical stimulus to the extensor nerves (1 × T) was taken as the minimum voltage necessary to produce a visually detectable sciatic potential. The strength of the stimulus was expressed in multiples of this threshold level. Bipolar stainless steel recording electrodes (Cooner Wire, AS632) were sewn into the following muscles of both hind legs to record electromyographic (EMG) activity: MG (in the 3 cats that did not have the MG nerve cut), vastus lateralis (VL), semitendinosus (ST), and iliopsoas (IP). The wires from both the stimulating and the EMG electrodes were led subcutaneously to a multipolar connector on the back of the cat. After finishing this procedure, the animal was placed above a motorized treadmill. A sling under the abdomen aided in weight support and maintenance of lateral stability. The animal then was decerebrated by transecting the brain stem at a 50° angle from the anterior edge of the superior colliculus. The halothane anesthesia was discontinued at this time.

In 20 of the 22 decerebrate cats, spontaneous bouts of walking occurred in response to a moving treadmill. Occasionally, manual stimulation of the perineum was used to evoke these bouts of locomotion. Although the triceps surae were denervated, most cats produced a stepping pattern that was similar to that produced by normal decerebrate cats except for yielding of the ankle during the E2 phase of stance. The speed of the treadmill was set between 0.25 and 0.35 m/s, depending on the animal's step pattern. During stance, a stimulus train to the appropriate extensor nerve (1.8-2 × T) was triggered 200 ms after the onset of the extensor EMG (MG in the first 3 animals and VL in the remainder). The duration of the stimulus train was 1,000 ms. This duration allowed for reductions in LGS efficacy and increases in MG efficacy to be observed in the experimental limb. After completion of the decerebrate protocol (1-3 h), 9 of the decerebrate cats were spinalized. Nialamide (50 mg/kg; Sigma Chemicals) dissolved in 20 ml of saline was administered during a 45-min period. Then methyl ester L-DOPA (50 mg/kg; Sigma Chemicals) dissolved in 5 ml of water was infused. Approximately 20-30 min after the infusion of L-DOPA, rhythmic contraction of hind leg muscles occurred either spontaneously or could be evoked by pinching the skin of the perineum. During periods of rhythmicity, the LGS and MG nerves of each hindlimb were stimulated using stimulus parameters similar to those used during decerebrate walking.

Data analysis

All data were recorded using a Vetter 4000A PCM recorder. Later, selected sequences were rectified, filtered (band-pass:10-100 Hz) and stored on computer disk using the Axotape data acquisition system (Axon Instruments). Data analyses were carried out using custom programs that could retrieve data from the Axotape files. The cycle periods before, during, and after the stimulus were calculated only during regular sequences of rhythmic locomotor activity. Each cycle period was calculated as the time between the occurrence of successive ST or IP bursts. All detection of the flexor bursts was made by manually tagging the onsets of the bursts using custom written software. A spreadsheet program (Microsoft Excel 5.0) was used to calculate the mean and standard deviation for these cycle periods and Student's t-tests detected significant differences between the conditions. The data were normalized according to the following equation to allow for comparisons between cats and between control and experimental nerves:
Percentage effectiveness = [(<IT>b</IT> − <IT>a</IT>)/(<IT>c</IT> − <IT>a</IT>)] × 100
where b equals the stimulated cycle period, a represents the control cycle period, and c represents the time from the first flexor burst before the stimulated extensor burst to the offset of the stimulus train (see Fig. 1A for an illustration of the measured variables). The percentage effectiveness is a measure of how powerfully the stimulus could affect the step cycle. For example, if the stimulus was 100% effective, the next flexor burst would have been held off until the end of the stimulus train. By contrast, if the percentage effectiveness was 0%, the stimulus would have had no effect on the cycle period.


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FIG. 1. Chronic axotomy of the lateral gastrocnemius and soleus (LGS) nerve increases the ability of group I afferents in the medial gastrocnemius (MG) to prolong the cycle period. A and B: rectified and filtered electromyographs (EMGs) from the hind leg muscles showing individual trials in the control (A) and experimental (B) legs (stimulus trains: 1,000-ms duration, 200 Hz, 2 × T). Note that in the control leg, stimulation of the MG nerve had only a small effect on the cycle period compared with the relatively large increase in the cycle period in the experimental leg (LGS cut 21 days). The parameters a, b and c used for quantifying the effectiveness of the stimulus trains are shown in A. C: bar graph showing the mean percent effectiveness of MG stimulation of the control () and experimental (square ) legs for all animals tested. Error bars represent the standard deviation. * Significant difference between the 2 conditions (P < 0.05). Number on the abscissa below each bar indicates the duration of the axotomy. VL l and r, left and right vastus lateralis; IP l and r, left and right iliopsoas.

In three animals, we measured changes in the magnitude of MG EMG bursts for <= 1 wk after axotomy of the ipsilateral LGS nerve. The procedure for recording EMG activity from intact walking animals has been described elsewhere (Hiebert et al. 1994). The magnitude of the EMG was calculated by integrating the rectified and filtered EMG during a 500-ms period. Usually, 20 steps were used to calculate the mean and standard deviation of the EMG.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Time course of plastic changes after axotomy of the LGS nerve

Stimulation of the group I afferents in a chronically transected LGS nerve produces a smaller than normal increase in the cycle period, whereas the effects of stimulating group I afferents in the ipsilateral MG nerve on the cycle period are increased (Whelan et al. 1995b). The first objective of the present study was to establish the time course of these changes by pooling data (10 animals) from this initial study with an additional group of animals. In total the effects of stimulating the MG nerve were measured in 17 of 20 cats and the LGS nerve in 18 of 20 cats (both measurements were made in 15 of 20 cats).

The effectiveness of stimulating group I afferents in the experimental and control MG nerves for all the animals tested is shown in Fig. 1C (trains: 1,000 ms duration; 2 × T; 200 Hz). The formula for calculating effectiveness is shown in Fig. 1A. Stimulation of the MG nerve in the control leg (gray bars) was usually only moderately effective; the average effectiveness was <50% in 15 animals and >100% in only 1 of 17 animals (Fig. 1, A and C). By contrast, the effectiveness of group I afferents in the MG nerve of the experimental leg (white bars) was usually much larger (Fig. 1, B and C). In 14 of 17 cats, the effectiveness was significantly greater in the experimental leg compared with the control leg, and in 7 of these animals, the average effectiveness was >100%. These large increases were not observed in four of the five animals in which the LGS nerve had been cut for <= 5 days, and no significant increases were observed in two of the three animals in which the LGS nerve had been cut for <= 3 days.

Examples of the effects produced by stimulation of group I afferents in the LGS nerve of control and experimental legs (trains 1,000 ms; 2 × T; 200 Hz) are shown in Fig. 2, A and B, whereas the average effectiveness of LGS nerve stimulation in all animals (n = 18) is shown in the bar graph in Fig. 2C. Stimulation of the group I afferents in the control LGS nerve usually had a strong effect on the cycle period (Fig. 2A); in 12 of 18 animals, the average effectiveness was >100%. Beyond 5 days postaxotomy, the effectiveness of the experimental LGS nerve was reduced significantly compared with control in all 12 animals tested. No significant reduction in effectiveness was found for LGS in three of the six animals examined within 5 days postaxotomy.


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FIG. 2. Chronic axotomy of the LGS nerve reduces the ability of group I afferents in the LGS nerve to prolong the cycle period. A and B: rectified and filtered EMG from hindlimb muscles showing individual trials in the control (A) and experimental (B) legs (stimulus trains: 1,000-ms duration; 200 Hz; 2 × T). Note that in the control leg, stimulation of the LGS nerve produced a large increase in the cycle period. In the experimental leg, in contrast, stimulation of the nerve using similar parameters only modestly increased the cycle period (LGS cut 21 days). C: bar graph showing the mean percent effectiveness of LGS stimulation of the control () and experimental (square ) legs for each individual experiment. Error bars represent the standard deviation. * Significant difference between the 2 conditions(P < 0.05). Numbers on the abscissa under each bar indicate the duration of the axotomy.

The time course of the changes in effectiveness that occurred postaxotomy were examined by plotting the effectiveness of group I afferents in the LGS and MG nerves of the experimental and control legs versus postaxotomy time (Fig. 3). As expected, for the control leg, there was no correlation between the effectiveness of MG [Fig. 3A; R2 = 0.022; slope not significantly different from 0 (P = 0.57)] and LGS [Fig. 3C; R2 = 0.0062; slope not significantly different from 0 (P = 0.76)] nerve stimulation and the time after axotomy of the contralateral LGS nerve. For the LGS nerve in the experimental leg (Fig. 3D), a decrease in effectiveness with time occurred [R2 = 0.26; slope significantly (P < 0.05) <0]. By ~30 days after axotomy, the effectivenesshad decreased to almost zero. By contrast there was no significant correlation [R2 = 0.10; slope not significantly >0(P = 0.22)] between the effectiveness of MG nerve stimulation in the experimental leg and postaxotomy time(Fig. 3B).


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FIG. 3. Time course of changes in the effectiveness of MG and LGS group I afferents after chronic axotomy of the LGS nerve. A-D: scatterplots of the data shown in Figs. 1C and 2C with the abscissa indicating the number of days after axotomy and the ordinate indicating the effectiveness of the stimulus. Best fitted linear regression lines (------) are shown along with the regression coefficient and equation of the line for each graph. Each data point represents the mean effectiveness from an individual experiment.

In 15 of the 20 cats, we were able to examine the effects of stimulating both the LGS and MG group I afferents during the same experiment. This allowed us to examine whether decreases in the effectiveness of LGS group I afferents were associated with increases in the effectiveness of the MG group I afferents. The absence of any significant correlation between differences in effectiveness of the experimental and control LGS nerves and the experimental and control MG nerves can be seen clearly in the scatter plot of the differences for the two pairs of nerves (Fig. 4). When a linear regression line was fitted to this data, the correlation coefficient was small (R2 = 0.05), and the slope of the line was not significantly greater than zero (P = 0.42). When the outlying point on the far right of Fig. 4B was removed, the regression coefficient increased (R2 = 0.09), but the slope was still not significant (P = 0.30). Another indication of the absence of a strong correlation between the changes in effectiveness in the experimental LGS and MG nerves was that in four animals, a large difference in effectiveness of one pair of nerves was associated with a small difference in the effectiveness of the synergistic pair [these points are indicated (black-diamond ) in Fig. 4].


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FIG. 4. Changes in effectiveness of MG and LGS effectiveness are not correlated. Each point is the difference in percent effectiveness between experimental and control legs for MG and LGS in a single animal. Measurements were taken in animals from 2 to 31 days postaxotomy. black-diamond , animals in which there was a large difference in effectiveness of 1 pair of nerves and only a small difference in the synergistic pair. A best fitted linear regression line (------) is shown along with its regression coefficient and the equation of the line.

Effects of spinalization on expression of plasticity

To establish whether one site mediating the changes in effectiveness is located within the spinal cord, the following protocol was used. In nine decerebrate animals for which there were significant changes in effectiveness for either the experimental MG or LGS nerves, the spinal cord was transected and locomotion was induced by applyingL-DOPA and Nialamide. Generally, the rhythm produced in the spinalized animals did not result in stepping movements that were as powerful as those obtained when the animal was walking in the decerebrate state. Even though the stepping was generally poor in the spinal state, the timing of the extensor and flexor EMG bursts in the VL and IP muscles were close to normal during periods of rhythmicity (Figs. 5, A and B, and 6, A and B).


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FIG. 5. Spinalization can abolish differences in the effectiveness of the MG group I afferents. A and B: rectified and filtered EMG traces (knee extensor VL and hip flexor IP) from a stepping L-3-4 dihydroxyphenylalanine (L-DOPA) spinal cat (LGS nerve axotomized 21 days previously). A: control leg. Note that stimulation of the MG group I afferents modestly increased the cycle period (up-arrow , expected time of termination of VL activity in the absence of stimulation). B: Effects of stimulating the experimental MG nerve in the same animal during L-DOPA-induced stepping. In this animal, the effect on the cycle period was similar to A, i.e., the difference in effectiveness between control and experimental legs before spinalization (not shown) was not expressed following spinalization. C: bar graph summarizing the effects of stimulating the MG group I afferents in the experimental and control leg during decerebrate and spinal walking for each animal tested. Note that only 2 out of 5 animals showed a conservation of the changes in effectiveness after spinalization. * Significant difference between the 2 legs (P < 0.05). Error bars indicate the standard deviation. Numbers on the abscissa indicate the duration of the axotomy of the LGS nerve.


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FIG. 6. Axotomy of the LGS nerve decreases the effectiveness of stimulating the LGS group I afferents in the decerebrate walking cat; this effect is preserved after spinalization. A and B: rectified and filtered EMG traces from a stepping L-DOPA spinal cat (data from the same animal as shown in Fig. 5). A: control leg. Stimulation of the LGS group I afferents produced a large increase in the cycle period. B: experimental leg. Stimulation of the LGS nerve had only a modest effect on the cycle period compared with A (up-arrow in A and B indicate the expected onset of the flexor burst in the absence of stimulation). C: bar graph summarizing the effects of stimulating the LGS group I afferents in the experimental and control legs during decerebrate and spinal walking for each animal tested. Note that in all animals, the changes in effectiveness observed during decerebration locomotion persisted after transection of the spinal cord. * Significant difference between the 2 legs (P < 0.05). Error bars indicate the standard deviation. Numbers on the abscissa indicate the duration of the axotomy of the LGS nerve.

In all spinal animals, stimulation of the extensor nerves at group I strengths prolonged the cycle period. In those spinal animals in which both MG and LGS nerves were tested, the effectiveness of LGS group I afferents was significantly greater than that for the MG group I afferents in the control leg [mean effectiveness LGS 91 ± 23%, MG33 ± 9% (means ± SD); n = 3; P < 0.05). Thus the normal decerebrate pattern of greater LGS compared with MG effectiveness (Figs. 1 and 2) was preserved after transection of the spinal cord.

Figures 5C and 6C summarize the results of stimulating the MG (n = 4) and LGS nerves (n = 5), respectively, in the control and experimental legs during locomotor activity in decerebrate animals before and after transection of the spinal cord. In the decerebrate state, the effectiveness was larger for the MG group I afferents of the experimental leg compared with the control leg. This difference in effectiveness was maintained in only two of five animals after transection of the spinal cord (P < 0.05). Figure 5, A and B, shows examples of comparable increases in cycle period produced by stimulation of the MG nerve in the control and experimental legs during stepping in one of these spinal animals. In a single animal in which no differences (P > 0.1) between the effectiveness of the MG nerve in the experimental and control legs were found during decerebrate walking, there were also no differences in effectiveness after transection of the spinal cord (P > 0.1, data not shown). The effectiveness of LGS nerve stimulation was less in the experimental leg compared with control in all nine animals, and this difference was preserved after transection of the spinal cord in four of these animals (P < 0.05). In the remaining five animals, the rhythm was either too irregular to evaluate the effects of LGS nerve stimulation or had ceased before we had the opportunity to stimulate. Figure 6, A and B, shows examples of the effects of stimulating the LGS nerves in a spinal animal. Stimulation of the LGS nerve at group I strengths in the control leg more effectively prolonged the cycle period (Fig. 6A) compared with the experimental leg (Fig. 6B).

Axotomy of the LGS nerve increases activity and weight of MG

In three animals, the magnitude of EMG bursts in the MG muscle were monitored before and after axotomy of the LGS nerve. In all animals, a significant increase in the amplitude of the integrated MG EMG in the experimental limb occurred on the first day after axotomy (P < 0.05; data not shown). Figure 7 shows the average EMGs before (thin lines) and 3-7 days after axotomy (thick lines) for three animals. A significant increase in the amplitude of the EMG occurred ~100 ms after the onset of the MG burst for two cats (Fig. 7, A and B) and 200 ms for the third animal (Fig. 7C). The mean differences between the post- and preaxotomy MG EMGs were 56% for the cat in Fig. 7A, 14% for the cat in Fig. 7B, and 12% for the cat in Fig. 7C.


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FIG. 7. In intact walking animals, the amplitude of the MG EMG bursts during stance increased after axotomy of the LGS nerve. A-C: averages of rectified and filtered EMGs (n = 20) recorded from the MG muscle of 3 animals as they walked bipedally on a treadmill (0.4 m/s). Thin traces show the average amplitudes of MG EMG before the LGS nerve was cut. Thick traces show the amplitude of the MG EMG on the day of the terminal experiment.

In all animals [including animals that did not produce any data], the lateral gastrocnemius (LG), soleus, MG, and the plantaris muscles from the control and experimental legs were weighed after the terminal experiment. Figure 8 shows plots of the percentage difference in weight of the MG and LG muscles of the experimental and control legs as a function of postaxotomy time. As expected, there was an atrophy of the LG muscle in the experimental leg (Fig. 8B) that increased in the days after axotomy of the LGS nerve[R2 = 0.71; slope significantly less than 0 (P < 0.0001)]. By contrast, a progressive hypertrophy occurred in the MG muscle of the experimental leg [R2 = 0.29; slope significantly greater than 0 (P < 0.05); Fig. 8A], increasing by ~20% after 34 days. Even at 5 days, the weight of the MG muscle increased by ~5-8%. There was also a smaller hypertrophy of PL [R2 = 0.11; slope not significantly greater than 0 (P = 0.18)] and atrophy of soleus [R2 = 0.098; slope not significantly greater than 0 (P = 0.22)] after axotomy of the LGS nerve.


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FIG. 8. Changes in the weights of the MG and LG muscles after axotomy of the LGS nerve. A and B: scatterplots showing the percentage change in the wet weight of each muscle after axotomy of the LGS nerve. Each data point represents an individual animal. Best fitting linear regression lines with regression coefficients are shown along with their regression coefficients and equations. Weights of the MG and LG muscles in the experimental leg increased and decreased respectively with time after axotomy of the LGS nerve.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

The data presented in this study support and extend a previous report that changes in LGS and MG group I pathways regulating stepping occur after axotomy of the LGS nerve (Whelan et al. 1995b). The results of this investigation show that decreases in the effectiveness of the LGS group I afferents controlling cycle period are not correlated with increases in the effectiveness of MG group I afferents. In addition, changes in effectiveness often were conserved after transection of the spinal cord, thus indicating one site of plasticity lies within the spinal cord.

One of the assumptions made in this paper is that changes in the effectiveness of stimulating the MG and LGS group I afferents reflect plasticity of an oligosynaptic pathway from extensor group I afferents to extensor motoneurons. This assumption is based on previous data obtained from spinal and decerebrate cats that show that during locomotion, an oligosynaptic pathway from extensor group I afferents to extensor motoneurons is opened (Gossard et al. 1994), that spatial summation between the flexor reflex afferent (FRA) system and the group I oligosynaptic pathway (Gossard et al. 1994), and that stimulation of group I extensor afferents resets and entrains the locomotor rhythm, thus demonstrating that these afferents access the rhythm generating circuitry of the spinal cord (Conway et al. 1987; Pearson and Collins 1993; Pearson et al. 1992). Although we did not directly measure changes in transmission through the oligosynaptic pathway, we did measure influences on stepping that have been attributed to transmission in this pathway (Whelan et al. 1995a). Future experiments using intracellular recording techniques (Gossard et al. 1994; McCrea et al. 1995) will test the hypothesis that transmission in group I oligosynaptic pathways is altered after axotomy of the LGS nerve.

Time course of plasticity

One of the aims of this investigation was to investigate the time course of the changes in the LGS and MG group I effectiveness after axotomy of the LGS nerve. The initial finding (Whelan et al. 1995b) that the onset of the changes in the effectiveness of both the MG and LGS group I pathways could occur within 3 days was confirmed. In addition, two differences in the expression of the plasticity in the LGS and MG group I pathways were found. First, there was no correlation between the decrease in the effectiveness of the LGS pathway and the increase in effectiveness of the MG pathway (Fig. 4). This was especially evident during the first week where, in three of five animals, there were large changes in one pathway without significant changes in the other pathway. Second, the effectiveness of the LGS group I pathway progressively declined over time, whereas the effectiveness of the MG group I pathway did not increase linearly (Fig. 3). These two results suggest that different mechanisms may underlie the plasticity in the MG and the LGS group I pathways. Different mechanisms also have been postulated to account for the opposite changes in homonymous monosynaptic reflex strength that occur after axotomy of the nerve and after interventions that leave the nerve intact but abolish afferent conduction (Gallego et al. 1979; Webb and Cope 1992). It has been suggested that the reduction in the monosynaptic reflex that occurs after axotomy may be due to pathological changes in the afferents, such as retraction of the axon terminals (Mendell 1984). Given that axotomy of the LGS group I afferents reduces the effectiveness of the presumptive oligosynaptic pathway affecting stepping and the homonymous monosynaptic pathway, it is conceivable that similar mechanisms underlie both processes. However, one difference between our findings and those on the monosynaptic group Ia pathway is the time course for the reduction in the reflex strength may be slower in the monosynaptic pathway after axotomy of LGS afferents. The earliest reported reduction in the amplitude of the homonymous monosynaptic excitatory postsynaptic potential (EPSP) is 7 days after axotomy of the extensor nerve (Gallego et al. 1979), whereas the results presented here show considerable declines in the LGS group I pathway beginning as early as 3 days.

After axotomy of the LGS nerve there is an increase in EMG activity in the MG muscle (Figs. 7). Assuming that force is related to the level of activity, it is likely that an increase in feedback from group Ib afferents from the MG muscle of the experimental leg occurs. Furthermore, the increase in yield of the ankle after axotomy (Whelan 1996) presumably increases feedback from group Ia afferents from the MG muscle during early stance. Because both Ia and Ib afferents contribute to the oligosynaptic pathway regulating cycle period (Guertin et al. 1995), an attractive possibility is that increased activity in MG group I afferents is one factor leading to strengthening of the pathway. This could be tested in future experiments by establishing whether reducing feedback from MG group I afferents during the recovery period after LGS axotomy (by tenotomy of the MG muscle, for example) has any influence on the changes in effectiveness of the MG group I afferents. If an increase in feedback from MG group I afferents is necessary for plasticity to develop, then it will be a distinctly different phenomenon than in the monosynaptic group Ia pathway where a decrease in activity leads to an increase in EPSP amplitude (Webb and Cope 1992).

One site of plasticity is within the spinal cord

Because the oligosynaptic group I extensor pathway is open and functional in spinal animals (Conway et al. 1987; Gossard et al. 1994; Pearson et al. 1992), one locus for the plasticity may be within the circuitry of the spinal cord. Alternatively, or in addition, another locus could lie in the brain stem or cerebellum. To explore this issue, we spinalized some of the decerebrate animals in which we observed positive signs of plasticity in both the MG and the LGS group I pathway. In two of five animals, the increase in MG effectiveness persisted after spinalization (Fig. 5), and in four of four animals, a significant reduction in the LGS effectiveness was retained in the spinal state (Fig. 6). These observations demonstrate that one site for plasticity of the group I pathways regulating stepping is in the spinal cord. The conservation of plastic changes in the LGS pathway after spinalization is consistent with the idea that changes in this pathway are a direct result of axotomy. On the other hand, the abolition of differences between control and experimental MG pathways after spinalization in three of five animals indicates a role for supraspinal structures in the expression of plastic changes in the MG pathway. The simplest explanation is that transmission in modifiable spinal pathways is regulated by tonic signals from the brain stem. Considerable evidence exists for brain stem regulation of spinal reflex pathways (Baldissera et al. 1981), and Gossard et al. (1994) have shown that transmission in extensor group I oligosynaptic pathways is expressed gradually after stimulation of the mesencephalic locomotor region (MLR) in the brain stem. Also, preliminary observations from our laboratory indicate that stimulation of the MLR can alter the effectiveness of extensor group I stimulation in increasing the cycle period in decerebrate walking cats (Whelan 1996). Another, more speculative, possibility is that plasticity occurs in long-loop reflex pathways from group I afferents to supraspinal structures that contribute to regulating the cycle period. Long-loop reflex pathways are known to contribute to reflex responses evoked by perturbations of the limbs in humans (Thilmann et al. 1991), but they have not yet been shown to contribute to reflexes regulating stepping. If these pathways do exist, and are modifiable, it would support the growing evidence that adaptive modification of motor systems depends of plastic changes at multiple sites (Bloedel et al. 1991; Raymond et al. 1996; Wolpaw and Carp 1993).

Functional relevance

The nervous system is endowed with an ability to reorganize and mold synaptic connections. Within this general context, the need for adaptive reflex modification has been recognized for many years (Bloedel et al. 1991; Ito 1976). During development, after injury, and also during the learning of new tasks, adaptation of reflexes must occur if the motor output is to remain optimized. For example, the gain of the vestibulo-ocular reflex can be altered if the visual field is altered by wearing prisms for a number of days. This adaptation ensures that eye gaze remains stable even though the visual field is distorted (Raymond et al. 1996).

With the well-established examples of adaptive plasticity in motor systems, it should not be surprising that changes in the reflex systems controlling stepping occur after injury to nerves and muscles. The results from this investigation show that when the LGS nerve is cut in an adult animal, reflexes from the synergist MG muscle increase. Stimulation of the LGS nerve can affect the timing of the step cycle in the conscious (Whelan and Pearson 1996) and decerebrate cat (Guertin et al. 1995; Whelan et al. 1995a). Thus after axotomy, the increased effectiveness of the MG group I pathway may compensate for the lack of timing cues from the LGS group I afferents. Moreover, the general absence of changes in the MG pathway for a few days immediately after axotomy is appropriate because this ensures that recalibration of reflex strength occurs only after a relatively permanent change in the use of the MG muscle. Examples of situations where such changes may be induced under normal conditions include increases in weight during development and increases in muscular strength with training.

One of the unanswered questions is the role of supraspinal inputs in the plasticity of the oligosynaptic group I pathways. Although we have some evidence for supraspinal regulation of the MG group I pathway, more data are required. A logical next step is to examine whether plasticity in the MG oligosynaptic group I pathway, after axotomy of the LGS nerve, occurs in chronic spinal cats that have been trained to step with their hind legs. If supraspinal inputs are necessary for the plasticity to develop, one structure to examine would be the cerebellum because it is suspected that it is involved in the adaptive modification of reflex gains (Bloedel et al. 1991; Ito 1976)

    ACKNOWLEDGEMENTS

  We thank Dr. K. Fouad for valuable comments on the manuscript and R. Gramlich for technical assistance.

  This work was supported by grants from the Medical Research Council of Canada and the Alberta Heritage Foundation for Medical Research.

    FOOTNOTES

   Present address of P. J. Whelan, Laboratory of Neural Control, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 49, Room 3A50, Bethesda, MD 20892-4455.

  Address for reprint requests: K. G. Pearson, Dept. of Physiology, University of Alberta, Edmonton T6G 2H7, Canada.

  Received 12 February 1997; accepted in final form 22 May 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society