Hindlimb Locomotor and Postural Training Modulates Glycinergic Inhibition in the Spinal Cord of the Adult Spinal Cat

R. D. de Leon,1 H. Tamaki,3 J. A. Hodgson,2 R. R. Roy,2 and V. R. Edgerton1,2

 1Department of Physiological Science and  2Brain Research Institute, University of California, Los Angeles, California 90095; and  3Department of Physiological Sciences, National Institute of Fitness and Sports, Kagoshima 891-23, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

de Leon, R. D., H. Tamaki, J. A. Hodgson, R. R. Roy, and V. R. Edgerton. Hindlimb Locomotor and Postural Training Modulates Glycinergic Inhibition in the Spinal Cord of the Adult Spinal Cat. J. Neurophysiol. 82: 359-369, 1999. Adult spinal cats were trained initially to perform either bipedal hindlimb locomotion on a treadmill or full-weight-bearing hindlimb standing. After 12 wk of training, stepping ability was tested before and after the administration (intraperitoneal) of the glycinergic receptor antagonist, strychnine. Spinal cats that were trained to stand after spinalization had poor locomotor ability as reported previously, but strychnine administration induced full-weight-bearing stepping in their hindlimbs within 30-45 min. In the cats that were trained to step after spinalization, full-weight-bearing stepping occurred and was unaffected by strychnine. Each cat then was retrained to perform the other task for 12 wk and locomotor ability was retested. The spinal cats that were trained initially to stand recovered the ability to step after they received 12 wk of treadmill training and strychnine was no longer effective in facilitating their locomotion. Locomotor ability declined in the spinal cats that were retrained to stand and strychnine restored the ability to step to the levels that were acquired after the step-training period. Based on analyses of hindlimb muscle electromyographic activity patterns and kinematic characteristics, strychnine improved the consistency of the stepping and enhanced the execution of hindlimb flexion during full-weight-bearing step cycles in the spinal cats when they were trained to stand but not when they were trained to step. The present findings provide evidence that 1) the neural circuits that generate full-weight-bearing hindlimb stepping are present in the spinal cord of chronic spinal cats that can and cannot step; however, the ability of these circuits to interpret sensory input to drive stepping is mediated at least in part by glycinergic inhibition; and 2) these spinal circuits adapt to the specific motor task imposed, and that these adaptations may include modifications in the glycinergic pathways that provide inhibition.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The hindlimb stepping ability of cats after a complete spinal cord transection is enhanced by daily training of the hindlimbs on a motorized treadmill (Barbeau and Rossignol 1987; Belanger et al. 1996; Chau et al. 1998b; de Leon et al. 1998a; Lovely et al. 1986, 1990). Spinal cats that do not receive treadmill training recover poor locomotor ability relative to step-trained spinal cats (de Leon et al. 1998a; Lovely et al. 1986). When training is not maintained for several weeks, stepping ability declines (de Leon et al. 1999). In addition to stepping, the ability of the spinal cats to maintain a weight-bearing posture in their hindlimbs is enhanced by training the hindlimbs to perform standing (de Leon et al. 1998b; Edgerton et al. 1997a; Pratt et al. 1994). On the basis of physiological and biochemical analyses of muscle in control and spinal trained and nontrained cats, adaptations in the hindlimb musculature cannot account for the improvements in stepping and standing that occur with daily training (Roy and Acosta 1986; Roy et al. 1991, 1998, 1999). Together, these findings suggest that spinal networks that mediate hindlimb stepping and standing after spinalization are modified by repetitive practice of those motor tasks (de Leon et al. 1998a,b, 1999; Edgerton et al. 1997a,b; Hodgson et al. 1994).

There is evidence that plasticity in inhibitory neurotransmitter systems in the lumbar spinal cord has a significant impact on the ability of spinal animals to step. Pharmacologically reducing GABAergic inhibition with bicuculline, a GABA antagonist, improved locomotor performance in nontrained adult spinal cats, i.e., before bicuculline was given few full-weight-bearing steps were executed, but 30 min after its administration, continuous stepping occurred over a range of treadmill speeds, and this facilitatory effect lasted for >2 h (Robinson and Goldberger 1986). Similarly the administration of strychnine, a glycinergic antagonist, to spinal transected dogs resulted in an improved ability to walk overground <30 min after the drug was given (Hart 1971). These findings suggested that GABAergic and glycinergic inhibition in the spinal cord interfered with the generation of locomotion in spinal animals. One effect of locomotor training on stepping ability after spinalization, therefore, may be to reduce the levels of inhibition resulting in a net facilitatory effect on the spinal networks that control hindlimb stepping. There have been no studies, however, that have examined whether GABAergic or glycinergic inhibition in the spinal cord of adult cats is modified in a use-dependent manner after spinalization.

In the present study, we hypothesized that the recovery of hindlimb stepping in spinal transected cats depended on the extent that glycinergic inhibition in the spinal cord was enhanced or reduced by repetitively training the hindlimbs to perform a motor task. To test this hypothesis, the ability of trained spinal cats to perform treadmill stepping was examined before and after glycinergic inhibition was reduced pharmacologically with strychnine. To determine if the levels of glycinergic inhibition in the spinal cord were related to the type of motor training that each cat experienced, strychnine was administered to the spinal cats after they initially were trained to perform one task, i.e., stepping or standing, and after they were retrained to perform the other task. The present findings provide evidence that the spinal circuits that mediate hindlimb locomotion after spinalization adapt to the specific motor task imposed on it, i.e., locomotion recovered when the spinal cats were trained to step, whereas locomotion was poor when the cats were trained to stand. Moreover strychnine was effective in inducing stepping only during the stand-training periods when locomotor performance was poor, suggesting that at least part of the neural changes underlying locomotor recovery involve plasticity in glycinergic synapses. Preliminary data have been published in abstract form (Hodgson et al. 1998).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental procedures

Electromyographic (EMG) electrodes were implanted in selected hindlimb muscles of eight adult female cats. EMG and kinematic data were collected from the hindlimbs while each cat performed bipedal treadmill locomotion, i.e., only the hindlimbs stepped while the forelimbs rested on a platform. After data from a minimum of 20 consecutive step cycles were collected (0.2-0.8 m/s) the spinal cords of the cats were completely transected (T12-T13).

Beginning 1 wk after spinalization, four spinal cats received daily hindlimb locomotor training on a treadmill while the remaining four spinal cats were trained daily to perform hindlimb standing. After 12 wk of training, the ability to perform treadmill stepping was tested before and after the administration of strychnine. To determine if there were any residual effects of strychnine on locomotor performance, treadmill stepping in six cats was retested 1-3 days later.

After the initial period of training and testing, each of the spinal cats was retrained to perform a new hindlimb task. The spinal cats that originally were trained to stand were retrained to step, whereas the cats that originally were trained to step were retrained to stand. The effect of strychnine on locomotor ability was tested after 12 wk of retraining. In addition to the postspinal testing, the locomotor response to strychnine administration also was examined in two cats before they were spinalized. During all tests, EMG activity from selected hindlimb muscles and hindlimb kinematic data were recorded.

Surgical procedures

During all surgical procedures, pentobarbital sodium (35 mg/kg intraperitoneal) was administered following pretreatment with atropine (intraperitoneal) and acepromazine (intramuscular). Supplemental doses of anesthesia were administered as needed during surgery to maintain surgical levels of anesthesia (Roy et al. 1992).

Intramuscular recording electrodes were implanted chronically in selected hindlimb muscles in the right hindlimb [deep region of the distal compartment of the semitendinosus (St), lateral deep portion of the vastus lateralis (VL), distal portion of the ilio-psoas (IP), midbelly of the soleus (Sol), medial deep portion of the medial gastrocnemius (MG), and midbelly deep portion of the tibialis anterior (TA)] as previously described (de Leon et al. 1994; Pierotti et al. 1989).

The spinal cords were transected completely at the T12-T13 junction as described in detail previously (Roy et al. 1992). Briefly, a skin incision was made on the back to expose the vertebral processes between ~T10 and L1. A partial laminectomy was performed to expose the spinal cord at the T12-T13 junction. Fine scissors and forceps were used to cut the dura and to perform the transection beginning on the dorsal surface of the cord between T12 and T13. After the transection, no spinal cord matter was visible between the two cut ends of the cord: the ends of the cord retracted leaving a clear space between the two cut ends. This procedure allowed for the preservation of the large ventral artery of the spinal cord. Gel foam was inserted in the space and the muscle and skin above the lesion site were closed with sutures.

Animal care procedures

Postspinalization management of the spinal cats has been detailed elsewhere (Roy et al. 1992). Cats were housed in spacious cages, two to four cats per cage, with the cage floors covered with shredded newspaper. The bladders and colons of the cats were expressed manually twice daily for the duration of the experiment. Dry kibble and water were given ad libitum and wet food was given once daily. All procedures were performed in accordance with the American Physiological Society Animal Care Guidelines and were approved by the Animal Use Committee at the University of California, Los Angeles.

Preparation and administration of strychnine solutions

Strychnine hydrochloride (Sigma) was dissolved in sterile water (0.5 mg/ml) the same day in which the strychnine solutions were to be administered. Subconvulsive doses (0.03-0.1 mg/kg) were administered intraperitoneally, and the cats were returned to their cages for observation. The doses used in the present study effectively reduce glycinergic inhibition of spinal neurons in cats that receive systemic injections of strychnine (Curtis et al. 1971; Pratt and Jordan 1987; Rudomin et al. 1990). Tests of stepping were performed 30-45 min after strychnine was administered.

Hindlimb training and testing procedures

Before spinalization, the cats were trained to perform long episodes of stable hindlimb stepping on a motorized treadmill (range of 0.2-0.8 m/s) while their forelimbs rested on a platform that was raised above the treadmill belt (for details, see de Leon et al. 1998a). After spinalization, training of hindlimb standing and bipedal hindlimb treadmill stepping was performed for 30 min/day, 5 days/wk as described previously (de Leon et al. 1998a,b; Hodgson et al. 1994).

A standard procedure was used to test bipedal hindlimb stepping after spinalization (de Leon et al. 1998a). Briefly, the ability of the hindlimbs to generate stepping without assistance was monitored for 45 s at a given speed. Testing began at the slower treadmill speeds (0.2-0.4 m/s) and proceeded at progressively faster speeds, usually in increments of 0.2 m/s, up to 1.0 m/s. If contact with the moving treadmill belt failed to trigger any hindlimb movements, paw placing, perianal or tail stimulation was provided by the trainers to attempt to elicit locomotor activity.

Assessment of locomotor ability

Stepping ability during a locomotor test was measured by the maximum speed of stepping (for details, see de Leon et al. 1998a). The maximum speed was defined as the fastest speed at which >= 20 consecutive full-weight-bearing step cycles were executed on the plantar surface of one paw. To ensure that the maximum speed was an accurate measurement of stepping ability over a range of speeds, it also was required that >= 20 consecutive steps were executed at all speeds below the fastest speed. In cases of inconsistent stepping performance, i.e., the inability to execute 20 consecutive steps at any speed, a maximum speed of 0 m/s was recorded. Bouts of stepping that were facilitated by paw placing or perianal or tail stimulation were not included in the assessment of the maximum speed. In addition, the number of full-weight-bearing steps and plantar-surface steps during the tests of stepping at speeds of 0.2 or 0.4 m/s were counted. The percentage of full-weight-bearing step cycles [(full-weight-bearing steps/full-weight-bearing steps + nonfull-weightbearing steps)*100] and the percentage of plantar steps [(plantar/plantar + dorsal)*100] were calculated.

Data recording and analysis

EMG and kinematic data during stepping were recorded as described previously (de Leon et al. 1994). Briefly, raw EMG signals were amplified and recorded on an FM tape recorder (TEAC Model XR-510, TEAC Corporation, Montebello, CA) while a camera and video cassette recorder (Panasonic System Camera, WV D5100; Panasonic AG1280P Panasonic, Cypress, CA) were used to record the video signals. An SMPTE time code generator (Model F30, Fast Forward Video, Irvine, CA) was used to synchronize video frames with the EMG signals recorded on FM tape.

EMG activity during 10- to 45-s bouts of stepping were sampled into an Amiga computer at 2 kHz and calibrated. For waveform analyses, >= 10 EMG bursts from each muscle were rectified and smoothed using a nine-point moving average, i.e., 110-Hz low-pass filter, and analyzed by computer (de Leon et al. 1994). Briefly, computer software designed in-house was used to detect and display the start and end of each burst based on a given threshold level above the baseline noise for a channel. The starting and ending points of bursts were used to determine the relative timing of EMG activity recorded from different muscles. Burst durations were calculated as the time between the start and the end of each burst. Mean EMG amplitude was calculated by dividing the integrated area of each unsmoothed rectified burst by the burst duration. To analyze the relationship between TA and Sol EMG amplitudes, EMG signals from each muscle during 10-s bouts of stepping were sampled (2 kHz), rectified and smoothed (25-point moving average, i.e., 40-Hz low-pass filter). TA and Sol average waveforms were generated from the smoothed bursts triggered from the start of Sol activity. The average waveforms were plotted against each other to produce a scatterplot (de Guzman et al. 1991; Hutchison et al. 1989a,b). The EMG data presented in the paper correspond to activity recorded during cyclical hindlimb movement including weight-bearing and nonweight-bearing step cycles unless specified otherwise. If no stepping movements were observed without assistance, EMG activity that corresponded to trainer-assisted steps (e.g., tail stimulation of stand-trained cats) were included in the waveform and scatterplot analyses.

The videotaped stepping sequences were viewed on video monitors, and the number of successful steps at a given speed was counted. Kinematic analyses were performed on >= 10 full-weight-bearing cycles from which EMG activity was analyzed (for details, see de Leon et al. 1994). Briefly, bony landmarks on the hindlimb were digitized and calibrated, and stick figure representations were plotted. Stance was defined as the period beginning with paw contact and ending before forward paw movement. Swing was defined as the period starting with forward movement of the paw (toe-off) and ending with, but not including, paw contact (touchdown). After spinalization in cats, the paw drags before it is lifted during swing (Belanger et al. 1996; de Leon et al. 1998a). The lengths and durations of the drag and lift phases of swing were measured by digitizing the x coordinate of the paw marker. The lengths and durations of E2 and E3 (Philippson 1905) were measured by the horizontal displacement of the x coordinate of the paw marker (head of the 5th metatarsal) during each phase.

Statistics

Comparisons of mean values before and after strychnine administration were statistically analyzed using paired t-tests. A repeated measures ANOVA was used to analyze changes in the stepping characteristics between the first and second training periods (significance at the P <=  0.05 and P <=  0.01 levels).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Reducing glycinergic inhibition in the spinal cord improves hindlimb stepping in spinal cats when they are trained to stand but not after they are retrained to step

Poor locomotor recovery was observed in the four spinal cats that received hindlimb stand training beginning 1 wk after spinalization (see Pre-STR, Fig. 1A). During treadmill tests performed after 12 wk of training, three of the four cats failed to produce any stepping movements, and their hindlimbs dragged with no weight-bearing stepping performed on the moving treadmill belt. We were able to elicit weight-bearing stepping in these cats using tail and perianal stimulation. When stepping movements were executed, only 51% of the step cycles were full-weight bearing, and the dorsal surface of the paw rather than the plantar surface contacted the treadmill belt when stance was initiated in 73% of these step cycles. None of the four cats was able to execute five consecutive, full-weight-bearing step cycles at any treadmill speed because of frequent step failures, i.e., stumbling (Fig. 2A). Each cat, however, was able to maintain full-weight-bearing extension in their hindlimbs during tests of standing posture (see de Leon et al. 1998b; Edgerton et al. 1997a).



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Fig. 1. Maximum treadmill speed in 4 spinal cats after stand training (A) and after step training (B). Cats initially were trained to perform hindlimb standing. After 12 wk of training, stepping ability was tested before (Pre-STR) and 30 min after (STR) the administration of strychnine. Each cat then was retrained to perform stepping for 12 wk, and locomotor ability was retested. Maximum speed is defined as the fastest treadmill speed at which >= 20 consecutive full-weight-bearing steps were performed (in addition, the animals must have performed 20 consecutive full-weight-bearing steps at each speed below the peak speed). Means ± SE are shown. *, significantly different from Stand, Pre-STR (P < 0.05).



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Fig. 2. Raw electromyogram (EMG) recorded from selected hindlimb muscles in a stand-trained cat during tests of treadmill stepping (0.4 m/s) before (Pre-STR; A) and 30 min after (STR; B) strychnine administration. St, semitendinosus; VL, vastus lateralis; IP, ilio-psoas; Sol, soleus; MG, medial gastrocnemius; and TA, tibialis anterior. Lines drawn under the EMG records indicate the stance phases in which full-weight-bearing occurred on the plantar surface of the paw. Horizontal calibration, 1 s; vertical calibration is 1 mV for all muscles except for the Sol, which is 2 mV.

To determine if glycinergic inhibition in the spinal cord interfered with the generation of stepping, strychnine, a glycinergic receptor antagonist, was administered to the stand-trained cats and stepping ability was retested. Pharmacologically reducing inhibition improved stepping performance, i.e., the maximum speed of treadmill stepping significantly increased 30-45 min after strychnine was administered (see STR, Fig. 1A). The majority (86%) of step cycles were full-weight-bearing, and dorsal surface stepping occurred in only 34% of these step cycles. The locomotor pattern was more consistent because fewer step failures occurred (Fig. 2B). Moreover the amount of assistance that was provided by the trainers to induce stepping decreased, i.e., three of the four cats were able to execute full-weight-bearing step cycles without tail or perianal stimulation. The facilitatory effects of strychnine were not long lasting, however, because stepping performance returned to baseline (prestrychnine) levels during treadmill tests performed 1-3 days later.

To determine if glycinergic inhibition in the spinal cord of the stand-trained cats was reduced by locomotor training, each cat was retrained to step on a treadmill, and the effects of strychnine on stepping ability were retested. The range of treadmill speeds at which stepping occurred increased from 0 m/s after stand training to 0.6 m/s after the cats were trained to step for 12 wk (compare Pre-STR in Fig. 1A vs. Pre-STR in Fig. 1B). Three of the cats consistently executed full-weight-bearing, plantar-surface stepping without requiring any tail or perianal stimulation to initiate or maintain stepping. Only one cat failed to produce stepping without trainer assistance. Pharmacologically reducing inhibition with strychnine had no effect on locomotor performance after the cats received locomotor training. The maximum speed of stepping was unchanged (Fig. 1B), and consistent, alternating stepping movements in the hindlimbs were maintained. Symmetrical gaits, e.g., galloping, did not occur at the range of speeds tested.

Reducing glycinergic inhibition in the spinal cord has no effect on stepping in spinal cats when they are trained to step but improves stepping after they are retrained to stand

A second group of four spinal cats was trained to perform treadmill stepping beginning 1 wk after spinalization. After 12 wk of training, these animals could perform consistent full-weight-bearing stepping with plantar surface paw contact during stance at a range of treadmill speeds (see Pre-STR, Fig. 3A). Stepping was initiated and maintained easily in three of the cats when their hindlimbs contacted the treadmill belt, whereas one cat required tail stimulation to step. Reducing glycinergic inhibition in the spinal cord with strychnine had no significant effect on the maximum speed of stepping (see STR, Fig. 3A). Consistent patterns of stepping were observed before and 30-45 min after strychnine was administered (Fig. 4) and during treadmill tests performed 1-3 days later.



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Fig. 3. Maximum treadmill speed in 4 spinal cats after step training (A) and after stand training (B). Cats initially were trained to perform hindlimb stepping. After 12 wk of training, stepping ability was tested before (Pre-STR) and 30 min after (STR) the administration of strychnine. Each cat then was retrained to perform standing for 12 wk, and locomotor ability was retested. Maximum speed is defined as in Fig. 1. Means ± SE are shown. *, significantly different from Step, Pre-STR or Stand, Pre-STR (P < 0.05).



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Fig. 4. Raw EMG recorded from selected hindlimb muscles in a step-trained cat during tests of treadmill stepping (0.4 m/s) before (Pre-STR; A) and 30 min after (STR; B) strychnine administration. Muscle abbreviations, calibrations and lines drawn under the EMG records are the same as in Fig. 2.

To determine if blocking glycinergic receptors would affect stepping when spinal cats lost the ability to step, the cats were retrained to stand, and the effects of strychnine on stepping ability were retested. After 12 wk of stand training, a significant decline in stepping ability occurred (compare Pre-STR in Fig. 3A vs. Pre-STR in Fig. 3B). In three cats, there was little hindlimb movement during the treadmill tests in the absence of tail or perianal stimulation. Only one of the four cats maintained stepping ability after undergoing stand training. When stepping was induced by the trainers, 95% of the cycles were full-weight bearing but the bouts of stepping were disrupted by frequent step failures, i.e., dorsal surface steps. The decline in stepping ability was associated with greater inhibitory function because reducing inhibition with strychnine improved the range of treadmill speeds at which the cats could step (see STR, Fig. 3B). Furthermore tail stimulation was no longer necessary to initiate and maintain the episodes of stepping. On the basis of comparisons of the changes in maximum speed, strychnine restored the level of stepping ability that had been acquired after the spinal cats were originally trained to step (compare Pre-STR in Fig. 3A with STR in Fig. 3B).

Summary of the effects of strychnine on stepping performance

The effects of strychnine on stepping performance are summarized in Fig. 5. The range of speeds at which stepping occurred improved in seven of eight cats when they received strychnine after a stand-training period (Fig. 5A). Strychnine induced stepping in five of six cats that exhibited little or no stepping movements in the absence of tail or perianal stimulation (prestrychnine maximum speed = 0 m/s), while an increase in maximum speed occurred in one cat that had recovered moderate stepping function (prestrychnine maximum speed = 0.6 m/s, Fig. 5A). On the basis of analyses of the data pooled from the stand-training periods, the number of weight-bearing step cycles (73% of the cycles before and 92% of the cycles after strychnine) and plantar surface steps (52% of the cycles before and 80% of the cycles after strychnine) increased after the stand-trained cats received strychnine.



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Fig. 5. Maximum speed of treadmill stepping before and after strychnine administration in the stand-training period (A) and step-training period (B). Maximum speed before (Pre-STR) and after strychnine (Post-STR) are plotted for 8 spinal cats. Data from the initial training and the retraining periods were pooled. open circle , data from initial training (Period 1); triangle , data from retraining (Period 2). - - -, line of unity. Maximum speed is defined as in Fig. 1.

When the cats were trained to step, pharmacologically reducing inhibition with strychnine had no effect on the range of treadmill speeds achieved in seven of eight cats (Fig. 5B). Six cats already had performed stepping at a range of treadmill speeds during the prestrychnine tests, and there was no further improvement in these animals. Two step-trained cats were unable to execute stepping without tail stimulation, i.e., prestrychnine maximum speed = 0 m/s, and strychnine was effective in inducing unassisted stepping in only one of these cats (Fig. 5B).

EMG activity

WAVEFORM CHARACTERISTICS. Irregular EMG burst waveforms were recorded during the tests of stepping in the stand-trained spinal cats because much of the EMG activity corresponded to nonweight-bearing hindlimb cycling or stumbling (Fig. 2A). For example, the cycle period and the Sol EMG burst durations were highly variable (see squares, stand-trained in Fig. 6, A and B). When strychnine was administered, the number of full-weight-bearing steps increased, and this was associated with more regular EMG burst waveforms (Fig. 2B) and more consistent burst durations and cycle periods (triangle , stand-trained in Fig. 6, A and B). The EMG patterns observed after strychnine was given to stand-trained cats were similar to the patterns that were recorded when they acquired the ability to step through locomotor training (compare triangle , stand-trained vs. , step-trained in Fig. 6, A and B). No effect was observed on the EMG burst patterns when strychnine was administered after a step-training period (step-trained, Fig. 6, A and B). Strychnine also had no effect on burst durations and cycle periods in two cats that received strychnine before spinalization (Fig. 6C).



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Fig. 6. Cycle period and soleus EMG burst duration values during treadmill stepping before () and after (triangle ) strychnine administration. Data are from 1 spinal cat after 12 wk of stand training followed by 12 wk of step training (A), from 1 spinal cat after 12 wk of step training and followed by 12 wk of stand training (B), and from 1 cat, before spinalization (C). Values for cycle period and burst duration were calculated from EMG activity recorded during 10 s of treadmill testing at a speed of 0.4 m/s.

On the basis of comparisons of full-weight-bearing cycles before versus after strychnine administration, the amplitudes of the St (knee flexor) and IP (hip flexor) EMG bursts significantly increased after strychnine was administered to stand-trained cats (see stand-trained, Fig. 7). However, when strychnine was given to cats during a step-training period, there were no significant changes in flexor EMG amplitudes (see step-trained, Fig. 7). Of the three hindlimb extensors studied, only the Sol EMG amplitude increased during stand training and the MG amplitude increased during step training. The burst duration and cycle period of full-weight-bearing step cycles were unaffected by strychnine (data not shown).



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Fig. 7. Mean EMG amplitudes in selected hindlimb muscles before () and after () strychnine in stand-trained and step-trained spinal cats. Data are from full-weight-bearing step cycles executed on the plantar surfaces of the paws at a treadmill speed of 0.4 m/s. Mean ± SE, n = 8 stand-trained cats, n = 7 step-trained cats. Data from the initial training and the retraining periods were pooled. Abbreviations are the same as in Fig. 2. *, significantly different from Pre-STR (P < 0.05).

RELATIONSHIP BETWEEN ANKLE EXTENSOR AND FLEXOR ACTIVITY. The relationship between Sol and TA EMG activity was analyzed to determine if reducing glycinergic inhibition improved the coordination of antagonist muscles during stepping. The EMG activity recorded in the Sol and TA during the stand-training periods was erratic (see average waveforms, Pre, Fig. 8, A and D), and there was no consistent recruitment order between the two muscles (see scatterplots, Pre, Fig. 8, A and D). After strychnine was administered, reciprocal EMG bursting occurred, i.e., TA activity decreased when Sol activity increased (see scatterplots, STR, Fig. 8, A and D) and resembled the Sol and TA recruitment patterns when the cats performed full-weight-bearing stepping during the step-training periods (compare scatterplots in STR, Fig. 8, A and D, with scatterplots in B and C). The Sol and TA recruitment patterns were unchanged in two cats that received strychnine before spinalization (Fig. 8E).



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Fig. 8. Scatterplots for Sol:TA EMG activity during treadmill stepping before and after strychnine administration. Data are from 1 spinal cat after 12 wk of stand training (A) followed by 12 wk of step training (B); 1 spinal cat after 12 wk of step training (C) followed by 12 wk of stand training (D); and from 1 cat, before spinalization (E). Scatterplots were generated by plotting the amplitudes of the averaged Sol (thin line) and TA (thick line) EMG burst waveforms (shown to the left of the scatterplots) during 10 s of treadmill stepping at 0.4 m/s. y axes increments are 0.2 mV (A), 0.35 mV (B), 0.1 mV (C), 0.2 mV (D), and 0.2 mV (E). Increments on the x axes are 0.5 s.

Hindlimb kinematic characteristics

SWING. When full-weight-bearing stepping was elicited in stand-trained cats, the swing phase of the step cycle consisted of a drag and a lift component (see Pre-STR, Fig. 9A) (see also Belanger et al. 1996; de Leon et al. 1998a). The normal, prespinal pattern of swing, i.e., lift of the paw immediately after toe off, occurred in only 13% of the cycles. Analyses of the data pooled from the stand-training periods showed that after the administration of strychnine, there was an increase in the number of cycles in which drag was absent (see STR, Fig. 9A). Overall, the length of lift was significantly greater [7 ± 1 (SE) cm before strychnine vs. 12 ± 0.4 cm after strychnine, P <=  0.05; see also Fig. 9B]. After the cats received step training, however, the relative lengths of drag and lift were unaffected by the administration of strychnine (Fig. 9, C and D).



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Fig. 9. Stick figure representations of the hindlimb during swing in a stand-trained (A) and step-trained (C) spinal cat, before (Pre-STR) and after (STR) strychnine administration. Position of the hindlimb during maximum lift and during the start and end of the drag and lift phases are shown. up-arrow  and down-arrow , toe off and touchdown, respectively. Bar graphs indicate the relative lengths and duration of drag () and lift (), before (Pre-STR) and after (STR) strychnine in stand-trained (B) and step-trained (D) spinal cats. Mean values for drag and lift lengths (n = 6 and 4 cats in B and D, respectively) are expressed as a percentage of swing length. Data from the initial training and the retraining periods were pooled.

STANCE. There were no changes in the length or duration of the E2 and E3 phases of stance when strychnine was administered during step- or stand-training periods (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Hindlimb motor training modifies glycinergic inhibition of the lumbar spinal networks that generate hindlimb stepping after spinalization

The levels of glycinergic inhibition in the lumbar spinal cord of spinal cats were modified by locomotor and postural training in the present study based on the following results: 1) in spinal cats that initially were trained to stand after spinalization, temporarily reducing glycinergic inhibition with strychnine had the same effect as retraining the hindlimbs to step, i.e., the number of full-weight-bearing steps increased; 2) when spinal cats were retrained to stand after initial step training, strychnine restored stepping ability to the level that was acquired with step training; and 3) there was a lack of behavioral and physiological motor responses to strychnine in spinal cats that initially were trained to step or were retrained to step after stand training, i.e., strychnine administration had little effect on cats that already could step. Thus it appears that the ability to perform stepping after spinalization is determined, in part, by the extent that the spinal networks that control stepping are inhibited by glycinergic pathways. It also is possible that these glycinergic pathways can be modified by specific patterns of use, i.e., repetitively training the hindlimbs to step or stand reduces or increases glycinergic inhibition in the lumbar spinal cord, respectively.

It is likely that the locomotor responses to strychnine observed before and after spinalization in the present study reflect the amounts and/or functional characteristics of the glycine receptors in the lumbar spinal cord. Greater than normal levels of the alpha 1 and gephyrin subunits of the glycine receptor were found in the lumbar spinal cords of rats 12 wk after they received a complete spinal cord transection at 7 days of age (Talmadge et al. 1996). However, training the hindlimbs of the spinal rats to step on a treadmill for 12 wk resulted in a recovery of full-weight-bearing stepping and a reduction in the levels of these glycine receptor subunits to control levels (Talmadge et al. 1996). Other evidence for spinal receptor plasticity is the supersensitivity of monoaminergic receptors that occurs in the spinal cord after the loss of descending input (Barbeau and Bedard 1987; Nygren and Olson 1976). It has been suggested that increased amounts of monoaminergic receptors in the spinal circuits that generate stepping mediate the changes in hindlimb motor reflex responsiveness and locomotor ability that occur in spinal cats after the administration of noradrenergic agonists (Chau et al. 1998a) and serotonergic agonists (Barbeau and Rossignol 1991). Likewise, in the present study, an increase in glycine receptors would be expected in the spinal cats after they received stand training relative to control cats, whereas normal levels would most likely be associated with step training. Comparisons of glycine receptor levels in the spinal cords of normal cats and trained and nontrained spinal cats are necessary to determine whether glycine receptor characteristics are modified by spinalization and motor training in these species.

Glycinergic and GABAergic neurotransmission are linked at some synapses in the CNS, i.e., GABA and glycine are colocalized in the presynaptic terminals (Ornung et al. 1994; Taal and Holstege 1994). Changes in GABAergic- and glycinergic-mediated inhibition in the spinal cord therefore may occur in parallel. The expression of the GABA synthetic enzyme (glutamate decarboxylase, GAD67) in the lumbar spinal cord increases after a complete spinal cord transection in cats (Robinson and Goldberger 1986; Tillakaratne et al. 1995). Hindlimb step training appears to normalize GAD67 levels in the lumbar spinal segments of adult spinal cats (Tillakaratne et al. 1995). In contrast, stand training results in a training-specific distribution of GAD67 between the two ventral horns, i.e., GAD67 was detected in both ventral horns or in a single ventral horn depending on whether the spinal cat was trained to stand on both hindlimbs or on a single hindlimb (Tillakaratne et al. 1995). These results suggest that the reorganization that occurs in the spinal circuits after spinalization is likely to involve adaptations in multiple neurotransmitter systems. The extent that each system can be modulated in a use-dependent manner largely will determine the level of functional recovery.

The changes in stepping ability that occurred with training were dramatic in some cases, e.g., from minimal hindlimb movements after 12 wk of stand training to full-weight-bearing stepping at a range of treadmill speeds after step training of the same animals. Improvements in locomotor performance of this magnitude do not occur spontaneously based on our studies of the long-term locomotor recovery characteristics in nontrained and trained spinal cats (de Leon et al. 1998a; Lovely et al. 1986). The extent that stepping ability deteriorated when the spinal cats were retrained to stand also cannot be explained by an absence of locomotor training. We recently have examined the changes in locomotor performance when step training was withheld in spinal cats that acquired the ability to step after 6-12 wk of treadmill training (de Leon et al. 1999). Although the maximum speed of stepping decreased significantly after the cessation of training for 3 mo, the spinal cats were capable of executing some full-weight-bearing steps without tail stimulation. Thus it appears that training the hindlimbs to stand reduces locomotor ability below the level of recovery that occurs in the absence of training (see also Edgerton et al. 1997a) and that a greater glycinergic presence in the spinal cord may contribute to the deterioration in stepping induced by stand training.

Other factors related to the systemic delivery of strychnine or to a "ceiling" effect on stepping performance may explain some of the present results. Recent studies have used intrathecal cannulas to administer noradrenergic agonists more locally to the lumbar spinal cord of spinal cats (Chau et al. 1998a,b). When the agents were delivered intrathecally, there was a reduction in overall side effects, but the effects on stepping performance were similar to the effects when the drugs were administered intraperitoneally. In the present study, strychnine was administered at doses that have been shown to reduce effectively spinal neuron inhibition in cats when strychnine was administered systemically (Curtis et al. 1971; Pratt and Jordan 1987; Rudomin et al. 1990). Although there were no effects of strychnine on treadmill stepping in the step-trained spinal cats, extensor rigidity in the hindlimbs was observed while they were in their cages after the administration of strychnine. However, once the hindlimbs made contact with the moving treadmill belt, appropriately coordinated hindlimb stepping patterns were initiated and maintained. These findings suggest that strychnine affected glycinergic synapses that were involved in the processing of proprioceptive input and the generation of treadmill-induced stepping. Pharmacological studies that can affect targeted circuits, and specific synapses in the spinal cord would provide a clearer assessment of the function of inhibitory neurotransmitters in the reorganized lumbar spinal cord of the spinal transected cat.

It is unlikely that the step or stand training resulted in adaptations in the hindlimb musculature that could account for the locomotor performances that occurred with training. The amount of and types of exercise training used in the present study are not sufficient to prevent muscle atrophy. For example, the amount of force that can be generated in the hindlimb muscles of step-trained, stand-trained, and nontrained spinal cats is similar, based on muscle mass and twitch and tetanic tension characteristics (Roy and Acosta 1986; Roy et al. 1991, 1998, 1999). We conclude that the changes in stepping performance after training and subsequent retraining were not due to an increased ability to produce sufficient levels of force in the hindlimb muscles but instead were the result of changes within the neural pathways of the spinal cord.

Ability of the existing spinal networks to interpret sensory input is hindered by glycinergic inhibition

On the basis of the present findings, the synapses among the spinal networks and the afferent pathways interacting with those networks are critical for the successful coordination of motor pools that generate stepping. In poorly stepping spinal cats, there was in many cases a lack of a hindlimb motor response to sensory stimuli associated with treadmill locomotion. In these animals, cyclical responses of the hindlimbs with occasional weight bearing occurred only when additional, nonspecific, sensory input, e.g., mechanical stimulation of the tail, was provided. The finding that strychnine induced successful weight-bearing stepping without tail stimuli in these animals suggests that strychnine enabled the spinal networks to appropriately interpret stepping-specific sensory information to drive network-generated locomotion by partially suppressing the baseline level of inhibition.

Glycinergic inhibition has been shown to play a critical role in the coordination of motor activity between the left and right sides during spinally mediated locomotion. For example, strychnine abolished alternating left and right motor discharge and induced synchronous activity during fictive swimming in the lamprey and the Xenopus embryo (Alford and Williams 1989; Dale 1985; Soffe 1987). The effects of strychnine administration were similar during in vitro neonatal rat spinal locomotor activity (Cowley and Schmidt 1995). Strychnine, however, failed to abolish right-left alternation of the scratch reflex that was elicited in spinal turtles by cutaneous stimulation (Currie and Lee 1997). In the present study, the step-trained spinal cats maintained alternating hindlimb movements after glycinergic inhibition was reduced by strychnine. The lack of an effect of strychnine on interlimb coordination in the present study may be explained by the spinal cord's ability to interpret sensory input that is derived from full-weight-bearing locomotion. Load-related proprioceptive input has been shown to initiate the transition between stance and swing during stepping in cats (Duysens and Pearson 1980), and hindlimb gait patterns in spinal kittens also are sensitive to the rate at which the treadmill belt moves, i.e., a conversion from asymmetric to symmetric gaits occurs as treadmill speed is increased (Forssberg et al. 1980). Therefore it is likely that multiple sources of sensory input associated with weight-bearing treadmill stepping are interpreted by the spinal networks and are used to maintain alternating patterns of hindlimb movements even when reciprocal inhibition is reduced pharmacologically.

Another possibility is that glycinergic pathways inhibited motor output during locomotion. The levels of flexor EMG activity and the amount of paw lift during full-weight-bearing stepping were enhanced after strychnine was administered to the stand-trained spinal cats in the present study. Renshaw cells and Ia inhibitory interneurons provide much of the inhibitory inputs to motoneurons, and pharmacologically blocking these inputs with strychnine enhanced extensor motoneuron activity during fictive locomotion in cats (Pratt and Jordan 1987). Blocking other sources of motoneuronal inhibition with strychnine, i.e., interneurons associated with muscle and cutaneous afferents, also has been shown to increase hindlimb reflex activity in cats (Rudomin et al. 1990). Based on immunocytochemical localization of glycine and glycine receptors, the motoneurons within the cat lumbar spinal cord are influenced heavily by glycinergic inhibition (Ornung et al. 1994; Triller et al. 1985; van den Pol and Gorcs 1988). Together these findings suggest that the activity in specific motoneuronal pools may be inhibited by glycinergic pathways such that an appropriate motor pattern cannot be expressed. Additional evidence, e.g., anatomic studies of glycinergic synapses on flexor motor pools in chronic spinal cats, is necessary to support these conclusions.

Clinical implications

Pharmacological studies in spinal transected animals can provide some insight into the neurochemical adaptations to spinal injury that may be useful for designing effective therapies for rehabilitation. Based on the present findings, there is a potential therapeutic use for pharmacological agents that reduce glycinergic spinal inhibition. Clonidine, a noradrenergic agonist, is being used in the rehabilitation setting, but it has not been as effective in initiating stepping in clinically complete patients as it has in spinal cats (Barbeau et al. 1998; Norman and Barbeau 1993; Stewart et al. 1991). It is important to realize, however, that the response to any drug for improving functional recovery will be affected by the physiological state of the synapses, i.e., the "milieu," of the spinal cord (Edgerton et al. 1997a). In some cases, overcoming disruptive synaptic influences that disable the spinal networks may prove to be the key to triggering stepping. Thus, in evaluating locomotor-enhancing drugs for use in treating paralysis, it will be helpful to consider factors such as training experience and the level of motor recovery which affect the neurochemistry of the pathways in the spinal cord that control stepping when supraspinal input is compromised.


    ACKNOWLEDGMENTS

The authors thank S. Lauretz for excellent care of the animals.

This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-16333.


    FOOTNOTES

Address for reprint requests: R. D. de Leon, Dept. of Physiological Science, UCLA, P.O. Box 951527, Los Angeles, CA 90095-1527.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 23 February 1999; accepted in final form 1 April 1999.


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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society