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Effects of extensor muscle afferents on the timing of locomotor activity during walking in adult rats
Brain Research 749 Ž1997. 320–328
Research report
Effects of extensor muscle afferents on the timing of locomotor activity during walking in adult rats Karim Fouad ) , Keir G. Pearson Department of Physiology, UniÕersity of Alberta, Edmonton, Alberta T6G 2H7, Canada Accepted 29 October 1996
Abstract The influence of hind leg extensor muscle afferents on the timing of locomotor phase transitions was examined in adult, decerebrate rats, walking on a treadwheel. Walking occurred either spontaneously or was induced by stimulation of the mesencephalic locomotor region. Large diameter muscle afferents innervating the lateral or medial gastrocnemius were electrically stimulated during walking. A stimulus was delivered either at the onset of extensor muscle activity, or randomly during the step cycle. Stimulation with a train duration of 300 ms at the onset of extension increased the duration of the extensor bursts. The subsequent flexion phase was delayed. Stimulation with a shorter stimulus train Ž150 ms. early in extension had little effect on the extension phase duration. However when delivered at the end of extension the same stimulus significantly increased the duration of the extension phase and decreased the duration of the following flexion phase. Stimulating near the end of the flexion phase delayed onset and decreased duration of the subsequent extension phase. The effects of stimulating extensor afferents during the extension phase were weaker but qualitatively similar, to those in cats, suggesting similar mechanisms. The results of this study also show major differences in the integration of extensor muscle afferents between adult and neonatal rats. Keywords: Rat; Locomotion; Walking; Muscle afferent; Decerebrate
1. Introduction Recently there has been increasing interest in the investigation of locomotion in rats. Motor patterns during various types of rhythmic behavior have been described in walking and paralyzed animals w3,10,19,33x, supraspinal structures involved in the initiation and regulation of locomotion have been identified w3,6,18x and preparations from neonatal animals have proven useful for the analysis of the organization and function of the central pattern generator for locomotion w5,8,26,29,32x. In addition, the rat is currently used extensively for examining recovery of locomotion following spinal cord injury w4,24,43x. Despite this attention on rat locomotion, very little is known about the influence of afferent input on the walking pattern. The only information comes from studies in neonates w23,27x. In these studies it has been shown that stimulation of extensor afferents during the flexion phase with high intensity prolongs the flexion phase. Stimulation during the extension phase with low or high intensity resulted either )
Corresponding author. Fax: q1 Ž403. 492-8915.
in no effect or in a truncation of the extension phase respectively w23x. One important observation has been that the integration of afferent input changes early in postnatal development w23x. Stimulation of extensor afferents with a low intensity stimulus delivered during the flexion phase revealed this developmental change. This stimulation prolonged the flexion phase in post-natal days 1–3 ŽP1–3. animals but truncated it in the P4–6 animals. Knowledge about sensory control of mammalian locomotion has come largely from studies in cats. Afferent control of walking has been examined in intact w11,13x, decerebrate w12,15,22,40x and spinal cats w35,36x, as well as during fictive locomotion in paralyzed animals w21,31,37x. Two groups of afferents are critical for the initiation of flexion in a hindlimb; one group is signaling hip extension w1,20,22,28x and the other is signaling that the extensor muscles are unloaded w14,40x. Preventing the hip extension andror loading the extensor muscles in just one hindlimb can stop stepping in this limb, while the remaining legs keep on stepping. Whelan et al. w40x demonstrated that the effect of loading a hindlimb is probably produced by feedback from group I afferents of
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 3 2 8 - 5
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extensor muscles. They found that electrical stimulation of group I afferents in extensor nerves prolonged the stance phase during walking in decerebrate cats. It is likely that activity in group Ib afferents contributes significantly to that effect, since their activation has been shown to prolong extensor burst duration in reduced spinal preparations w7,36x. However, recently it has been demonstrated that activity in group Ia afferents is also capable of prolonging the extension phase duration during fictive locomotion w21x. The aim of this study was to examine whether the mechanisms of regulating the transition from the extension to the flexion phase during walking in adult rats are similar to those found in cats. In addition, we wanted to compare these mechanisms to those described in neonatal rats. Preliminary results of this study have been published in abstract form w17x.
2. Materials and methods Experiments were carried out on adult male SpragueDawley rats Ž300–500 g.. Animals were initially anesthetized with halothane in 95% O 2r5% CO 2 . A tracheotomy was performed and the anesthetic maintained via the tracheal cannula. The carotid arteries were ligated, and one jugular vein was cannulated for i.v. injections of drugs and fluids. Atropine Ž0.5–1 mg. was administered i.v. routinely. Following this procedure the sciatic nerve of the left leg was exposed and placed in a bipolar recording cuff electrode. The cuff was approximately 4 mm long, with an inner diameter of about 1 mm. The bared ends of two Teflon-coated stainless steel wires ŽCooner Wire Co., AS631. were looped within the cuff and acted as recording electrodes to monitor the strength of electrical stimuli applied to nerves supplying the ankle extensor muscles. The nerve innervating either the lateral gastrocnemiussoleus ŽLGS. muscle or medial gastrocnemius ŽMG. muscle was exposed and transected close to the muscle. About 4 mm of this nerve was freed from the tibial nerve and threaded into a small stimulation cuff. The end of the nerve was anchored to a pin fixed on the cuff ŽFig. 1A.. The stimulation cuff was about 1 mm long with an inside diameter of roughly 0.4 mm. The stimulating electrodes consisted of bared sections of two Teflon-coated stainless steel wires ŽCooner Wire Co., AS631. divided into two strands inside the cuff. To avoid movement of the cuff during walking, the stimulation cuff was anchored to a larger cuff placed on the adjacent tibial nerve ŽFig. 1A.. This was essential because it minimized movement of the nerve in the cuff and helped to maintain constant stimulus strength during walking. It also prevented damage of the nerve by the cuff moving within the leg. Electromyographic ŽEMG. activity was recorded with bipolar Teflon-coated stainless steel recording electrodes ŽCooner Wire Co., AS631. implanted into the following
Fig. 1. A: construction of the stimulation electrode. The major parts of the electrode were two silicone cuffs, the larger served as an anchor around the distal tibial nerve, the smaller as the stimulation cuff. Bared sections of Teflon-coated wire were looped in the inside of the stimulation cuff. Either the LGS or MG nerve was thread through the stimulation cuff and fixed by tying the distal end to a minuten-pin attached to the cuffs. B: experimental setup. A decerebrated rat was placed in a stereotaxic holder and mounted above a treadwheel. Walking was initiated by stimulation of the mesencephalic locomotor region ŽMLR. and electromyograms were recorded from muscles of the hindlimbs.
hind leg muscles: ipsilateral leg – vastus lateralis ŽVL., semitendinosus ŽST., LG or MG; contralateral leg – MG and ST. The wires from the EMG electrodes and the cuff electrodes were led subcutaneously to a multipolar connector near the middle of the animal’s back. After placement of the electrodes, the animal was mounted into a stereotaxic head holder and positioned on a treadwheel Ždiameter about 60 cm, width 8.7 cm; Fig. 1B.. The treadwheel, with a negligible resistance was driven only by the animal w2x which caused a momentum of the spinning wheel, thereby stabilizing the walking speed. Both the right and the left parietal bones were removed. The sagittal and transverse sinuses were usually left intact. Cortical tissue was aspirated until the superior colliculi were visible. A vertical decerebration was performed about 2 mm rostral to the superior colliculi with a sharpened
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spatula and the remaining forebrain removed. The anesthetic was discontinued immediately after decerebration. In three animals, short bouts of walking Žup to 15 s. occurred spontaneously about 40 min. after decerebration. Longer periods of walking Žup to 53 s. were induced in all animals by stimulating the mesencephalic locomotor region w2,6,33x. The stimulus was a continuous train of 1-ms pulses at 50 Hz of 5–30 m A. Usually the intensity was slowly increased until the animal started running, then slowly decreased to attain slow walking. During periods of walking, the cuffed extensor nerve was stimulated at different intensities, relative to the lowest threshold volley recorded by the sciatic cuff. The stimuli were either triggered 20 ms after the onset of extensor activity in the left leg Žtrain: 300 ms duration, 200 Hz. or delivered at various times during the step cycle Ž0.8 Hz; train: 150 ms duration, 200 Hz.. The onset of extensor activity was detected by the on-line monitoring of a rectified and filtered EMG from the left VL muscle, controlled by an interactive computer program. All data were recorded on magnetic tape with a Vetter 4000A PCM recorder. Selected sequences were later digitized Žsampling rate 1000rs. and stored on a computer disk using the Axotape ŽAxon Instruments. data acquisition program. Analysis was carried out using custom programs. When triggering at the onset of extensor activity, the effect of the stimulation was quantified by comparing the duration of the perturbed step cycle Žonset of flexor burst to onset following flexor burst. to that of the preceding step cycle. When stimulating at various times during the step cycle, the duration of the extension and flexion phase of the perturbed step cycle were compared with the duration of this phase of the preceding cycle. The extension phase duration was defined as the duration from the onset of the VL burst to the onset of the subsequent ST burst, and the flexion phase duration as the duration between the onset of the ST burst and the onset of the subsequent VL burst. The results were divided into two groups, based upon whether the stimulus occurred during the extension or the flexion phase. These groups were further divided into five bins, depending on the timing of stimulus onset Ž1r5 of the duration of the phase of the preceding step.. Step cycle, extension phase, and flexion phase duration of the perturbed step were compared with those of the preceding step with the use of a two-tailed, paired t-test Ž P 0.05..
3. Results 3.1. General obserÕations In three out of 10 animals, walking occurred either spontaneously or in response to a non-noxious cutaneous stimulus to the tail. The walking sequences lasted up to 16 steps with a stepping frequency from 2–3 Hz. In these and
the other seven animals, locomotion was also initiated by electrical stimulation of the mesencephalic locomotor region ŽMLR.. MLR stimulation induced long walking sequences Žup to 34 bouts per animal, sometimes with ) 100 steps. with a stepping frequency of 2–5 Hz. MLR induced locomotion normally started with fast running or galloping with full weight support. Reduction of the stimulus intensity after the beginning of locomotion decreased walking speed. Full weight support was maintained for periods up to 50 s. Most bouts of walking ended with slow stepping Ž1–2 Hz. and dragging of the abdomen. These observations correspond to those from studies by Bedford et al. w2x. In spinal and decerebrate cats, it is well known that the rate of stepping adapts to the speed of the motor driven treadmill upon which the animal is walking w16x. It was not possible to establish whether this phenomenon occurs in decerebrate rats because the treadwheel was propelled by the animal and not by a motor. However, we observed that the rate of stepping slowed when the turning of the treadwheel was resisted ŽFig. 2A and B. which is similar to observations in cats walking on a friction free treadmill w38x. During these periods the magnitude of EMG activity in extensor muscles increased, and the burst duration was prolonged ŽFig. 2A.. When the treadwheel was prevented from turning, walking ceased. Another similarity to the findings in cats was that stepping in an individual limb could be interrupted by preventing completion of extension Žw14,20x; Fig. 2C.. During these periods extensor activity remained high and the other three legs continued to step, usually at a lower rate. 3.2. Stimulating extensor afferents during extension The similarities in the effects of resisting or preventing leg extension in the rat and the cat suggest that the afferent mechanisms controlling extensor activity are similar. In cats, feedback from group Ib afferents is one mechanism regulating the relation between load and activity of extensor muscles. Activity in these afferents increases the activity of extensor muscles and prolongs the stance phase w14,40x. To test whether this occurs in rats, we stimulated afferents in either the LGS Ž n s 6. or the MG Ž n s 4. nerve. The stimulus train was triggered near the onset of extensor activity with a delay of 20 ms. The train duration Ž300 ms. was set to outlast an undisturbed extension phase. This stimulation resulted in a prolongation of the duration of extensor EMG activity as well as in a delay of the following flexion phase ŽFig. 3A.. Although consistently prolonging extension phase duration there was considerable variability between the trials. Generally, the effect of the stimulus was weak and flexion was initiated during the period of stimulation ŽFig. 3B.. Sometimes, however, the generation of the flexor burst was delayed far beyond the stimulus duration ŽFig. 3C.. Fig. 4 summarizes the results of stimulating extensor
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afferents with long stimulus trains triggered at the onset of extensor activity. The histograms in Fig. 4A show data from a single animal when LGS nerve was stimulated Žnormalized values for 100% in ms: 2 = T spont., 497; 2 = T, 482; 3 = T, 462; 5 = T, 475.. These histograms are similar to those of the pooled data from six animals ŽFig. 4B; normalized values for 100% in ms: 2 = T spont., 556; 2 = T, 416; 3 = T, 462; 5 = T, 375.. The strongest effect on cycle period was found when the animals were spontaneously walking. Stimulating at 2 = T increased the step cycle duration 14.4% Ž"14.8; S.D.. on average. The effect was weaker ŽANOVA variance analysis; P - 0.05. during MLR induced walking. Here the cycle period increased 11.2% Ž"6.3. on average. Intensities of LGS nerve stimulation greater than 2 = T did not alter the effect on cycle duration significantly ŽANOVA variance analysis; P ) 0.05.. The step cycle duration during MLR induced walk-
Fig. 3. Stimulation of muscle afferents in LGS nerve during stance prolongs the duration of the extension phase. The three sets of records show the range of variation in the effect. Each set of traces shows the stimulus duration Žhorizontal bar, top. and the rectified and filtered EMG recordings from an extensor ŽVL, middle. and a flexor ŽST, bottom. muscle. The stimulation strength was 2=T. The arrows indicate where the onset of an unperturbed ST burst would have occurred. A: the stimulus prolonged the extensor burst duration, and delayed the onset of the following flexor burst until the termination of the stimulus. This effect is similar to that in decerebrate cats. B: the flexor burst is only slightly delayed, and is initiated during the stimulus. Usually the onset of flexor burst activity was more obviously delayed and occurred before the termination of the stimulus. C: in this example the extensor activity prolongation persisted well beyond the end of the stimulus.
Fig. 2. A,B: resisting leg extension prolongs extensor activity. A: EMG recordings from an animal walking on a freely spinning wheel Žleft. and when the wheel was resisted Žright.. VL, vastus lateralis; ST, semitendinosus. B: graph illustrating the significant increase in extension phase duration and the relatively small increase of the flexion phase when the treadwheel was resisted. The error bars indicate the standard deviation. C: When extension of a single leg was prevented by supporting the foot on a stationary platform, the extensor activity persisted but stepping in the other three legs continued usually at a lower rate. The traces show the EMG recordings of a VL Žtop. and ST Žmiddle. muscle in the resisted leg and a ST Žbottom. muscle of the contralateral leg.
ing was increased 6% Ž"7.3. at 3 = T, and 11.2% Ž"10.6. at 5 = T. Using these pooled data for statistical analysis, the increase of step cycle duration in the perturbed steps was significant for each of the stimulus intensities Ž P 0.05.. The minimum strength for producing an increase in step cycle duration was 1.4 = T. MG nerve stimulation had a weaker but qualitatively similar effect on the step cycle duration as LGS nerve stimulation ŽFig. 4C; normalized values for 100% in ms: 2 = T, 355; 3 = T, 323; 5 = T, 342.. Stimulation of the MG nerve significantly increased cycle period duration by prolonging the extension phase. Stimulating with 2 = T, 3 = T and 5 = T prolonged the step cycle on average by 5% Ž"31., 7% Ž"6. and 5% Ž"2., respectively. No spontaneous walking occurred in the animals in which MG nerve was stimulated.
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3.3. Stimulating extensor afferents at Õarious times during the step cycle Previous studies in neonatal rats w23x have used short stimulation trains to investigate the afferent input in the stepcycle. Therefore, to compare the adult with neonates we stimulated the LGS or MG nerve at different times during the stepcycle with a short stimulus train Ž150 ms, 200 Hz.. The trains were delivered repetitively Ž0.2 Hz. during long walking sequences. The effect during the extension phase was strongest when the stimulus occurred during the late extension phase. It increased the extension phase duration and decreased the duration of the subsequent flexion phase ŽFig. 5A.. When stimulated during the flexion phase, the strongest effect occurred when the stimulus train was delivered at the end of the flexion phase. Fig. 5. EMG recordings showing the effect of stimulating LGS afferents with brief trains Ž150 ms, 200 Hz. late in the extension phase ŽA. or in the flexion phase ŽB.. VL, vastus lateralis; ST, semitendinosis. Ext., extension phase; Flex., flexion phase. In both cases the stimulus train increased the duration of the phase in which it was delivered and decreased the duration of the following phase.
Fig. 4. Histograms summarizing the effects on cycle period of stimulating extensor nerve afferents during extension. The data are grouped according to stimulus strength. Data from spontaneously walking animals are shown in the left pair of histograms in A and B. A: effects Žin %. of stimulating LGS nerve in a single animal. The duration of the preceding step Žwhite bars. was normalized to 100%. Error bars indicate standard deviation. B: pooled data from six animals when LGS nerve was stimulated. Here the increase of the step cycle duration was always significant. C: pooled data of four animals when MG nerve was stimulated. In these animals no spontaneous walking occurred. The effect on the step cycle duration was weaker than that produced by LGS stimulation, but was still significant.
This did not change ST activity, but resulted in an increase in flexion phase duration and a decrease of the subsequent extension phase duration ŽFig. 5B.. The effect of stimulating LGS nerve at various times during either the extension phase or the flexion phase is summarized in Fig. 6. Stimulation during the extension phase resulted in a modest increase of the step cycle duration Žtop graph, Fig. 6A., but this was only significant when the stimulus occurred near the beginning of the extension phase Ž5.7% " 6.5; "S.D... By comparison to the modest effect on cycle period, the duration of the extension and flexion phases was modified more noticeably. Fig. 6A Žmiddle. shows that the extension phase duration increased progressively as the stimulus occurred at later times during the extension phase. In bins 3 to 5 this increase was significant Ž13.1% " 9.5, 12.2% " 7 and 16.7% " 7.5.. Correspondingly, the following flexion phase duration progressively decreased as the stimulus was delayed during extension ŽFig. 6A, bottom., although only the data from stimulation during late extension phase were found to be significant Žy9.4% " 9.5.. The opposite effects on the duration of the extension and flexion phase explain the modest effect on the step cycle duration. Stimulation during the late flexion phase also resulted in a modest increase of the step cycle duration Žtop graph, Fig. 6B.. This increase was significant when the stimulus occurred near the end of the flexion phase Žbins 4 and 5: 6.1% " 6.7 and 5.7% " 2.7.. This effect on the step cycle was mainly caused by a progressive increase of the flexion phase duration as the stimulus occurred at later times during the flexion phase. A significant increase was ob-
K. Fouad, K.G. Pearsonr Brain Research 749 (1997) 320–328
served in bins 3, 4 and 5 Ž9.6 " 7.9, 12.2% " 9.7 and 18.7% " 6.6.. The effect on the following extension phase was a modest decrease in duration in bin 1 and a modest increase in duration in bins 2, 3 and 4. The only significant effect occurred in bin 5, a decrease in extension phase duration Žy7% " 5.4.. Stimulation of the MG nerve produced similar but weaker effects. Stimulation during the extension phase never had a significant effect on the step cycle duration, but increased the extension phase duration significantly in bins 1, 3, 4 and 5 Ž6.8% " 7.3, 4.4% " 8.5, 9.2% " 9.3 and 10.8% " 7.5.. In all cases, the subsequent flexor burst duration was decreased. If the stimulation of the MG nerve occurred during the flexion phase, the cycle period and the flexion phase duration were only increased significantly in bin 5 Ž10.7% " 4.1 and 21.7% " 7.3, respectively.. The following extension phase was never changed significantly. As in the previous experiment, no difference could be detected for stimulating at different intensities.
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4. Discussion In this set of experiments, the influence of extensor afferent input on the timing of locomotor activity in adult rats was examined. One objective was to determine whether the mechanisms controlling extension phase duration are comparable to those previously described for cats, another was to establish whether there are any differences in afferent regulation of locomotor activity in adult rats and neonates. In general, our findings in the rat are qualitatively similar to those already reported in the cat w40,41x. One important result was that electrical stimulation of extensor afferents during the extension phase delayed the onset of flexor burst activity thus increasing the cycle period ŽFig. 3 and Fig. 4.. The average increase in cycle period was modest Ž5–14%, Fig. 4., but comparable to the increases produced by stimulation of large afferents from vastus lateralisrintermedius, plantaris or medial gastrocnemius
Fig. 6. Summary of the effects of stimulating LGS nerve afferents with brief trains at different times of the step cycle. Data for stimuli applied during the extension and flexion phases are shown in the left ŽA. and right ŽB., respectively. Stimulating during extension prolonged the stance phase Žmiddle. and shortened the following swing phase Žbottom.. The later during extension the stimulus occurred, the stronger the effect. The effect on the whole step cycle Žtop. was therefore modest. Stimulation during flexion increased the duration of the flexion phase Žmiddle. and shortened the subsequent extension phase when delivered near the end of extension Žbottom.. Data were collected from 6 animals, 12 to 31 steps were analyzed per data point. The points marked with an asterisk indicate a significant effect, and error bars show standard deviation.
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muscles Ž14–20%. in decerebrate walking cats w40,41x. One clear quantitative difference with cats was the effect of stimulating the LGS nerve. In cats, stimulation of large afferents in the LGS nerve powerfully influences extensor bursts, usually prolonging these bursts for the duration of the stimulus train w40x. This strong effect was observed only rarely in rats Žan example is shown in Fig. 3A.. Even though the effect of LGS nerve stimulation was weak in the rat compared to the cat, we did observe that LGS nerve stimulation produced more powerful effects than MG nerve stimulation Žaverage increase in cycle period 11.2% for LGS compared to 5% for MG.. On most trials stimulation of either the LGS or MG nerve during extension had a proportionally larger effect on extensor burst duration than on the cycle period ŽFig. 6.. Usually the prolongation of the extensor burst duration was compensated to some extent by a shortening of the following flexion phase. The relatively weak effect on cycle period is probably due to the overall timing of cycle period being dominated by the rate of stepping in the other three unperturbed legs. Although the recruitment order of different groups of afferents in hind leg nerves of the rat have not been defined for electrical stimuli, we attribute the effects we have observed to stimulation of afferents arising from muscle spindles and Golgi tendon organs. The effects on the timing of the locomotor activity were apparent at a stimulus strength of 1.4 = T and were usually maximal at 2 = T ŽFig. 4.. This range of stimulus strengths would have preferentially activated large muscle afferents. The largest afferents from the gastrocnemius muscles in the rat are considered to arise from muscle spindles and Golgi tendon organs w30x. There was no possibility of effects being produced indirectly by evoked muscle contractions because the nerves were cut distally. A comparison with data from the cat also supports our conclusion that the effects of stimulating the LGS and MG nerves are due to activation of spindle and tendon organ afferents. The minimal stimulus strength for influencing the duration of extensor bursts in decerebrate walking cats is about 1.5 = T and maximal effects are produced at 2 = T w40x. Over this range of stimulus strengths most of the afferents from primary spindle endings and Golgi tendon organs, and some afferents from secondary spindle endings, are recruited w25x. Further similarities with the cat were found by loading extensor muscles. Resisting the turning of the treadwheel on which the rats were walking preferentially increased the duration of extensor muscle activity ŽFig. 2A,B.. Qualitatively similar observations have been made in decerebrate cats walking on a friction free treadmill w38x. Furthermore, preventing the extension of a hind leg in a walking rat prevented stepping in just this limb ŽFig. 3C.. This is comparable to the observation in the cat, where it has been shown that supporting a leg on a stationary object inhibits the initiation of the flexion phase w20x. The functional
interpretation of these observations in the cat is that a leg must be unloaded and extended for the swing phase to be initiated w34x. The corresponding observations in the rat indicate that this may also be true for the rat. This is supported by our finding that extensor burst duration can be prolonged by electrical stimulation of extensor group I afferents ŽFig. 3, Fig. 4.. In the cat there is now considerable evidence that this effect is due primarily to activation of Ib afferents from the Golgi tendon organs w34x. If this is true in the rat, then a general rule for mammalian locomotion may be that stance Žextension. is maintained by feedback from tendon organs. Unloading of the extensor muscles near the end of stance Ždue to the animal’s weight being borne by other legs and to shortening of extensor muscles. is required for the initiation of swing Žflexion.. One further similarity to the cat was that the effects of stimulating the extensor nerves during the extension phase were greater during spontaneous locomotion, compared to those produced during MLR-induced locomotion ŽFig. 4.. Recently, Whelan observed that increasing the stimulus strength to the MLR in decerebrate walking cats reduces the effectiveness of group I afferents on extensor burst duration w42x. In both rats and cats this difference is not due to differences in walking speed, because it is observed at the same cycle periods during spontaneous and MLR induced walking. Although the numerous similarities in the data from cats and rats imply similar mechanisms for afferent regulation of stepping, we did find one major difference. In the adult rat, stimulating the extensor afferents during the flexion phase prolonged the flexion phase, whereas in cats the step cycle is reset by termination of the flexion phase w7,40x. Our observations in adult rats differ in some important aspects from those in neonates. In neonates of age less than P6, low and high intensity stimulation of extensor afferents delivered during the extension phase has either no effect or causes a resetting of the step cycle to flexion by truncating the extensor burst, respectively w23x. In adults we observed that stimulation of the extensor afferents with different intensities Ž2–5 = T. always increased the duration of the extension phase. Another difference is that in adults stimulation during the flexion phase at all intensities always prolonged the flexion phase. The same effect was observed in neonatal rats only when stimulated with high intensities Ž) 5 = T.. Low intensity stimulation Ž- 3 = T. of the extensor afferents prolonged the flexion phase just in P1–3 animals, but truncated it in P4–6 animals, suggesting a developmental change in the integration of the extensor afferents w23x. The observed differences between adult rats and neonates might be explained by the occurrence of a second developmental change in the integration of afferent information. The idea of such a change is strengthened by the fact that P6 neonates have not started to support their body weight w39x and by the finding that direct cortico-motoneuronal synaptic contacts are not established until about P7 w9x. Another possible explanation
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for differences in the effects between neonatal rats and adults could be the different preparation. In the neonate, afferents from the quadriceps muscle, a knee extensor, were stimulated, whereas in this study ankle extensor afferents were examined. Another difference was that neonatal rats were paralyzed and fictive locomotion was examined, whereas adult animals were walking on a treadwheel. This explanation, however, appears unlikely, because in cats these differences have not been found to qualitatively influence the results. Similar effects are produced when extensor group I afferents from knee or ankle extensors are stimulated w21,40x, and there are no major difference between the results obtained in walking animals w40x and in paralyzed preparations w21x. In conclusion, our results suggest that similar sensory mechanisms regulate the transition from the extension to the flexion phase in adult rats and adult cats. Further, differences between older neonates and adult rats indicate developmental changes of these mechanisms sometime after neonatal day 6.
Acknowledgements The authors thank R. Gramlich for excellent technical assistance. We would also like to thank D. Bennet, G. Hiebert and J. Misiaszek for reading the manuscript. This work was supported by grants of the Deutsche Forschungsgemeinschaft and the Medical Research Council of Canada.
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w10x de Leon, R., Hodgson, J.A., Roy, R.R. and Edgerton, R.V., Extensor- and flexor-like modulation within motor pools of the rat hindlimb during treadmill locomotion and swimming, Brain Res., 654 Ž1994. 241–250. w11x Drew, T. and Rossignol, S., A kinematic and electromyographic study of cutaneous reflexes evoked from the forelimbs of unrestrained walking cats, J. Neurophysiol., 57 Ž1987. 1160–1184. w12x Duysens, J., Reflex control of locomotion as revealed by stimulation of cutaneous afferents in spontaneously walking premammillary cats, J. Neurophysiol., 40 Ž1977. 737–751. w13x Duysens, J. and Loeb, G.E., Modulation of ipsi- and contralateral reflex responses in unrestrained walking cats, Brain Res., 44 Ž1980. 1024–1037. w14x Duysens, J. and Pearson, K.G., Inhibition of flexor burst generation by loading ankle extensor muscles in walking cats, Brain Res., 187 Ž1980. 321–332. w15x Duysens, J. and Stein, R.B., Reflex induced by nerve stimulation in walking cats with implanted cuff electrodes, Exp. Brain Res., 32 Ž1978. 213–224. w16x Forssberg, H. and Grillner, S., The locomotion of the acute spinal cat injected with clonidine i.v., Brain Res., 50 Ž1973. 184–186. w17x Fouad, K. and Pearson, K.G., Effects of stimulation of extensor muscle afferents during walking in adult rats, Soc. Neurosci. Abstr., 22 Ž1996. p. 1097. w18x Garcia-Rill, E., The basal ganglia and the locomotor regions, Brain Res. ReÕ., 11 Ž1986. 47–63. w19x Goudard, I., Orsal, D. and Cabelguen, J.-M., An electromyographic study of the hindlimb locomotor movements in the acute thalamic rat, Eur. J. Neurosci., 4 Ž1992. 1130–1139. w20x Grillner, S. and Rossignol, S., On the initation of the swing phase of locomotion in chronic spinal cats, Brain Res., 146 Ž1978. 269–277. w21x Guertin, P., Angel, M.J., Perreault, M.C. and McCrea, D.A., Ankle extensor group I afferents excite extensors throughout the hindlimb during MLR evoked fictive locomotion in the cat, J. Physiol., 487 Ž1995. 197–209. w22x Hiebert, G.W., Whelan, P.J., Prochazka, A. and Pearson, K.G., Contribution of hind limb flexor muscle afferents to the timing of phase transitions in the cat step cycle, J. Neurophysiol., 75 Ž1996. 1126–1137. w23x Iizuka, M., Kiehn, O. and Kudo, N., Development in neonatal rats of the sensory resetting of the locomotor rhythm induced by NMDA and 5-HT, Exp. Brain Res., Ž1996. in press. w24x Iwashita, Y., Kawaguchi, S. and Murata, M., Restoration of function by replacement of spinal cord segments in the rat, Nature, 367 Ž1994. 167–170. w25x Jack, J.J.B., Some methods for selective activation of muscle afferent fibres. In R. Porter ŽEd.., Studies in Neurophysiology, Cambridge University Press, Cambridge, UK, 1978, pp. 155–176. w26x Kiehn, O. and Kjaerulff, O., Spatiotemporal characteristics of 5-HT and Dopamine-induced rhythmic hindlimb activity in the in vitro neonatal rat, J. Neurophysiol., 75 Ž1996. 1472–1482. w27x Kiehn, O., Iizuka, M. and Kudo, N., Resetting from low threshold afferents of N-methyl-D-aspartate-induced locomotor rhythm in the isolated spinal cord–hindlimb preparation from newborn rats, Neurosci. Lett., 148 Ž1992. 43–46. w28x Kriellaars, E.I., Brownstone, R.M., Noga, B. and Jordan, L.M., Mechanical entrainment of fictive locomotion in the decerebrate cat, J. Neurophysiol., 71 Ž1994. 2074–2086. w29x Kudo, N. and Yamada, T., Development of the monosynaptic stretch reflex in the rat: in vitro study, Neurosci. Lett., 148 Ž1985. 43–46. w30x Lewin, G.R. and McMahon, S.B., Physiological properties of primary sensory neurons appropriately and inappropriately innervating skeletal muscle in adult rats, J. Neurophysiol., 66 Ž1991. 1218–1231. w31x McCrea, D.A., Shefchyk, S.J., Stephens, M.J. and Pearson, K.G., Disynaptic group I excitation of synergist ankle extensor motoneurones during fictive locomotion, J. Physiol., 487 Ž1995. 527–539. w32x MacLean, J.N., Hochman, S. and Magnuson, D.S.K., Lamina VII
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K. Fouad, K.G. Pearsonr Brain Research 749 (1997) 320–328 neurons are rhythmically active during locomotor-like-activity in the neonatal rat spinal cord, Neurosci. Lett., 197 Ž1995. 9–12. Nicolopoulos-Stournaras, S. and Iles, J.F., Hindlimb muscle activity during locomotion in the rat Ž Rattus norÕegicus. ŽRodentia: Muridae., J. Zool. Lond., 203 Ž1984. 427–440. Pearson, K.G., Proprioceptive regulation of locomotion, Curr. Opin. Neurobiol., 5 Ž1995. 786–791. Pearson, K.G. and Collins, D.F., Reversal of the influence of the group Ib afferents from plantaris on activity in medial gastrocnemius muscle during locomotor activity, J. Neurophysiol., 70 Ž1993. 1009–1017. Pearson, K.G., Ramirez, J.M. and Jiang, W., Entrainment of the locomotor rhythm by group Ib afferents from ankle extensor muscles in spinal cats, Exp. Brain Res., 90 Ž1992. 557–566. Perreault, M.-C., Drew, T. and Rossignol, S., Activity of medullary reticulospinal neurons during fictive locomotion, J. Physiol., 487 Ž1993. 211–220. Shik, M.L., Severin, F.V. and Orlovskii, G.N., Control of walking
w39x w40x w41x w42x w43x
and running by means of electrical stimulation of the mid-brain, Biofizyka, 11 Ž1966. 756–765. Westerga, J. and Gramsbergen, A., Changes in the electromyogram of two major hindlimb muscles during locomotor development in the rat, DeÕ. Brain Res., 57 Ž1990. 163–174. Whelan, P.J., Hiebert, G.W. and Pearson, K.G., Stimulation of the group I extensor afferents prolongs the stance phase in walking cats, Exp. Brain Res., 103 Ž1995. 20–30. Whelan, P.J., Hiebert, G.W. and Pearson, K.G., Plasticity of extensor group I pathway controlling the stance to swing transition in the cat, J. Neurophysiol., 74 Ž1995. 2782–2787. Whelan, P.J., Function of group I extensor feedback in the control of locomotion, Ph.D. Thesis, University of Alberta, 1996. Yakovleff, A., Cabelguen, J.-M., Orsal, D., Gimenez y Ribotta, M., Rajaofetra, N., Drian, M.-J., Bussel, B. and Privat, A., Fictive motor activities in adult chronic spinal rats transplanted with embryonic brainstem neurons, Exp. Brain Res., 106 Ž1995. 69–78.
Research report
Effects of extensor muscle afferents on the timing of locomotor activity during walking in adult rats Karim Fouad ) , Keir G. Pearson Department of Physiology, UniÕersity of Alberta, Edmonton, Alberta T6G 2H7, Canada Accepted 29 October 1996
Abstract The influence of hind leg extensor muscle afferents on the timing of locomotor phase transitions was examined in adult, decerebrate rats, walking on a treadwheel. Walking occurred either spontaneously or was induced by stimulation of the mesencephalic locomotor region. Large diameter muscle afferents innervating the lateral or medial gastrocnemius were electrically stimulated during walking. A stimulus was delivered either at the onset of extensor muscle activity, or randomly during the step cycle. Stimulation with a train duration of 300 ms at the onset of extension increased the duration of the extensor bursts. The subsequent flexion phase was delayed. Stimulation with a shorter stimulus train Ž150 ms. early in extension had little effect on the extension phase duration. However when delivered at the end of extension the same stimulus significantly increased the duration of the extension phase and decreased the duration of the following flexion phase. Stimulating near the end of the flexion phase delayed onset and decreased duration of the subsequent extension phase. The effects of stimulating extensor afferents during the extension phase were weaker but qualitatively similar, to those in cats, suggesting similar mechanisms. The results of this study also show major differences in the integration of extensor muscle afferents between adult and neonatal rats. Keywords: Rat; Locomotion; Walking; Muscle afferent; Decerebrate
1. Introduction Recently there has been increasing interest in the investigation of locomotion in rats. Motor patterns during various types of rhythmic behavior have been described in walking and paralyzed animals w3,10,19,33x, supraspinal structures involved in the initiation and regulation of locomotion have been identified w3,6,18x and preparations from neonatal animals have proven useful for the analysis of the organization and function of the central pattern generator for locomotion w5,8,26,29,32x. In addition, the rat is currently used extensively for examining recovery of locomotion following spinal cord injury w4,24,43x. Despite this attention on rat locomotion, very little is known about the influence of afferent input on the walking pattern. The only information comes from studies in neonates w23,27x. In these studies it has been shown that stimulation of extensor afferents during the flexion phase with high intensity prolongs the flexion phase. Stimulation during the extension phase with low or high intensity resulted either )
Corresponding author. Fax: q1 Ž403. 492-8915.
in no effect or in a truncation of the extension phase respectively w23x. One important observation has been that the integration of afferent input changes early in postnatal development w23x. Stimulation of extensor afferents with a low intensity stimulus delivered during the flexion phase revealed this developmental change. This stimulation prolonged the flexion phase in post-natal days 1–3 ŽP1–3. animals but truncated it in the P4–6 animals. Knowledge about sensory control of mammalian locomotion has come largely from studies in cats. Afferent control of walking has been examined in intact w11,13x, decerebrate w12,15,22,40x and spinal cats w35,36x, as well as during fictive locomotion in paralyzed animals w21,31,37x. Two groups of afferents are critical for the initiation of flexion in a hindlimb; one group is signaling hip extension w1,20,22,28x and the other is signaling that the extensor muscles are unloaded w14,40x. Preventing the hip extension andror loading the extensor muscles in just one hindlimb can stop stepping in this limb, while the remaining legs keep on stepping. Whelan et al. w40x demonstrated that the effect of loading a hindlimb is probably produced by feedback from group I afferents of
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 3 2 8 - 5
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extensor muscles. They found that electrical stimulation of group I afferents in extensor nerves prolonged the stance phase during walking in decerebrate cats. It is likely that activity in group Ib afferents contributes significantly to that effect, since their activation has been shown to prolong extensor burst duration in reduced spinal preparations w7,36x. However, recently it has been demonstrated that activity in group Ia afferents is also capable of prolonging the extension phase duration during fictive locomotion w21x. The aim of this study was to examine whether the mechanisms of regulating the transition from the extension to the flexion phase during walking in adult rats are similar to those found in cats. In addition, we wanted to compare these mechanisms to those described in neonatal rats. Preliminary results of this study have been published in abstract form w17x.
2. Materials and methods Experiments were carried out on adult male SpragueDawley rats Ž300–500 g.. Animals were initially anesthetized with halothane in 95% O 2r5% CO 2 . A tracheotomy was performed and the anesthetic maintained via the tracheal cannula. The carotid arteries were ligated, and one jugular vein was cannulated for i.v. injections of drugs and fluids. Atropine Ž0.5–1 mg. was administered i.v. routinely. Following this procedure the sciatic nerve of the left leg was exposed and placed in a bipolar recording cuff electrode. The cuff was approximately 4 mm long, with an inner diameter of about 1 mm. The bared ends of two Teflon-coated stainless steel wires ŽCooner Wire Co., AS631. were looped within the cuff and acted as recording electrodes to monitor the strength of electrical stimuli applied to nerves supplying the ankle extensor muscles. The nerve innervating either the lateral gastrocnemiussoleus ŽLGS. muscle or medial gastrocnemius ŽMG. muscle was exposed and transected close to the muscle. About 4 mm of this nerve was freed from the tibial nerve and threaded into a small stimulation cuff. The end of the nerve was anchored to a pin fixed on the cuff ŽFig. 1A.. The stimulation cuff was about 1 mm long with an inside diameter of roughly 0.4 mm. The stimulating electrodes consisted of bared sections of two Teflon-coated stainless steel wires ŽCooner Wire Co., AS631. divided into two strands inside the cuff. To avoid movement of the cuff during walking, the stimulation cuff was anchored to a larger cuff placed on the adjacent tibial nerve ŽFig. 1A.. This was essential because it minimized movement of the nerve in the cuff and helped to maintain constant stimulus strength during walking. It also prevented damage of the nerve by the cuff moving within the leg. Electromyographic ŽEMG. activity was recorded with bipolar Teflon-coated stainless steel recording electrodes ŽCooner Wire Co., AS631. implanted into the following
Fig. 1. A: construction of the stimulation electrode. The major parts of the electrode were two silicone cuffs, the larger served as an anchor around the distal tibial nerve, the smaller as the stimulation cuff. Bared sections of Teflon-coated wire were looped in the inside of the stimulation cuff. Either the LGS or MG nerve was thread through the stimulation cuff and fixed by tying the distal end to a minuten-pin attached to the cuffs. B: experimental setup. A decerebrated rat was placed in a stereotaxic holder and mounted above a treadwheel. Walking was initiated by stimulation of the mesencephalic locomotor region ŽMLR. and electromyograms were recorded from muscles of the hindlimbs.
hind leg muscles: ipsilateral leg – vastus lateralis ŽVL., semitendinosus ŽST., LG or MG; contralateral leg – MG and ST. The wires from the EMG electrodes and the cuff electrodes were led subcutaneously to a multipolar connector near the middle of the animal’s back. After placement of the electrodes, the animal was mounted into a stereotaxic head holder and positioned on a treadwheel Ždiameter about 60 cm, width 8.7 cm; Fig. 1B.. The treadwheel, with a negligible resistance was driven only by the animal w2x which caused a momentum of the spinning wheel, thereby stabilizing the walking speed. Both the right and the left parietal bones were removed. The sagittal and transverse sinuses were usually left intact. Cortical tissue was aspirated until the superior colliculi were visible. A vertical decerebration was performed about 2 mm rostral to the superior colliculi with a sharpened
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spatula and the remaining forebrain removed. The anesthetic was discontinued immediately after decerebration. In three animals, short bouts of walking Žup to 15 s. occurred spontaneously about 40 min. after decerebration. Longer periods of walking Žup to 53 s. were induced in all animals by stimulating the mesencephalic locomotor region w2,6,33x. The stimulus was a continuous train of 1-ms pulses at 50 Hz of 5–30 m A. Usually the intensity was slowly increased until the animal started running, then slowly decreased to attain slow walking. During periods of walking, the cuffed extensor nerve was stimulated at different intensities, relative to the lowest threshold volley recorded by the sciatic cuff. The stimuli were either triggered 20 ms after the onset of extensor activity in the left leg Žtrain: 300 ms duration, 200 Hz. or delivered at various times during the step cycle Ž0.8 Hz; train: 150 ms duration, 200 Hz.. The onset of extensor activity was detected by the on-line monitoring of a rectified and filtered EMG from the left VL muscle, controlled by an interactive computer program. All data were recorded on magnetic tape with a Vetter 4000A PCM recorder. Selected sequences were later digitized Žsampling rate 1000rs. and stored on a computer disk using the Axotape ŽAxon Instruments. data acquisition program. Analysis was carried out using custom programs. When triggering at the onset of extensor activity, the effect of the stimulation was quantified by comparing the duration of the perturbed step cycle Žonset of flexor burst to onset following flexor burst. to that of the preceding step cycle. When stimulating at various times during the step cycle, the duration of the extension and flexion phase of the perturbed step cycle were compared with the duration of this phase of the preceding cycle. The extension phase duration was defined as the duration from the onset of the VL burst to the onset of the subsequent ST burst, and the flexion phase duration as the duration between the onset of the ST burst and the onset of the subsequent VL burst. The results were divided into two groups, based upon whether the stimulus occurred during the extension or the flexion phase. These groups were further divided into five bins, depending on the timing of stimulus onset Ž1r5 of the duration of the phase of the preceding step.. Step cycle, extension phase, and flexion phase duration of the perturbed step were compared with those of the preceding step with the use of a two-tailed, paired t-test Ž P 0.05..
3. Results 3.1. General obserÕations In three out of 10 animals, walking occurred either spontaneously or in response to a non-noxious cutaneous stimulus to the tail. The walking sequences lasted up to 16 steps with a stepping frequency from 2–3 Hz. In these and
the other seven animals, locomotion was also initiated by electrical stimulation of the mesencephalic locomotor region ŽMLR.. MLR stimulation induced long walking sequences Žup to 34 bouts per animal, sometimes with ) 100 steps. with a stepping frequency of 2–5 Hz. MLR induced locomotion normally started with fast running or galloping with full weight support. Reduction of the stimulus intensity after the beginning of locomotion decreased walking speed. Full weight support was maintained for periods up to 50 s. Most bouts of walking ended with slow stepping Ž1–2 Hz. and dragging of the abdomen. These observations correspond to those from studies by Bedford et al. w2x. In spinal and decerebrate cats, it is well known that the rate of stepping adapts to the speed of the motor driven treadmill upon which the animal is walking w16x. It was not possible to establish whether this phenomenon occurs in decerebrate rats because the treadwheel was propelled by the animal and not by a motor. However, we observed that the rate of stepping slowed when the turning of the treadwheel was resisted ŽFig. 2A and B. which is similar to observations in cats walking on a friction free treadmill w38x. During these periods the magnitude of EMG activity in extensor muscles increased, and the burst duration was prolonged ŽFig. 2A.. When the treadwheel was prevented from turning, walking ceased. Another similarity to the findings in cats was that stepping in an individual limb could be interrupted by preventing completion of extension Žw14,20x; Fig. 2C.. During these periods extensor activity remained high and the other three legs continued to step, usually at a lower rate. 3.2. Stimulating extensor afferents during extension The similarities in the effects of resisting or preventing leg extension in the rat and the cat suggest that the afferent mechanisms controlling extensor activity are similar. In cats, feedback from group Ib afferents is one mechanism regulating the relation between load and activity of extensor muscles. Activity in these afferents increases the activity of extensor muscles and prolongs the stance phase w14,40x. To test whether this occurs in rats, we stimulated afferents in either the LGS Ž n s 6. or the MG Ž n s 4. nerve. The stimulus train was triggered near the onset of extensor activity with a delay of 20 ms. The train duration Ž300 ms. was set to outlast an undisturbed extension phase. This stimulation resulted in a prolongation of the duration of extensor EMG activity as well as in a delay of the following flexion phase ŽFig. 3A.. Although consistently prolonging extension phase duration there was considerable variability between the trials. Generally, the effect of the stimulus was weak and flexion was initiated during the period of stimulation ŽFig. 3B.. Sometimes, however, the generation of the flexor burst was delayed far beyond the stimulus duration ŽFig. 3C.. Fig. 4 summarizes the results of stimulating extensor
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afferents with long stimulus trains triggered at the onset of extensor activity. The histograms in Fig. 4A show data from a single animal when LGS nerve was stimulated Žnormalized values for 100% in ms: 2 = T spont., 497; 2 = T, 482; 3 = T, 462; 5 = T, 475.. These histograms are similar to those of the pooled data from six animals ŽFig. 4B; normalized values for 100% in ms: 2 = T spont., 556; 2 = T, 416; 3 = T, 462; 5 = T, 375.. The strongest effect on cycle period was found when the animals were spontaneously walking. Stimulating at 2 = T increased the step cycle duration 14.4% Ž"14.8; S.D.. on average. The effect was weaker ŽANOVA variance analysis; P - 0.05. during MLR induced walking. Here the cycle period increased 11.2% Ž"6.3. on average. Intensities of LGS nerve stimulation greater than 2 = T did not alter the effect on cycle duration significantly ŽANOVA variance analysis; P ) 0.05.. The step cycle duration during MLR induced walk-
Fig. 3. Stimulation of muscle afferents in LGS nerve during stance prolongs the duration of the extension phase. The three sets of records show the range of variation in the effect. Each set of traces shows the stimulus duration Žhorizontal bar, top. and the rectified and filtered EMG recordings from an extensor ŽVL, middle. and a flexor ŽST, bottom. muscle. The stimulation strength was 2=T. The arrows indicate where the onset of an unperturbed ST burst would have occurred. A: the stimulus prolonged the extensor burst duration, and delayed the onset of the following flexor burst until the termination of the stimulus. This effect is similar to that in decerebrate cats. B: the flexor burst is only slightly delayed, and is initiated during the stimulus. Usually the onset of flexor burst activity was more obviously delayed and occurred before the termination of the stimulus. C: in this example the extensor activity prolongation persisted well beyond the end of the stimulus.
Fig. 2. A,B: resisting leg extension prolongs extensor activity. A: EMG recordings from an animal walking on a freely spinning wheel Žleft. and when the wheel was resisted Žright.. VL, vastus lateralis; ST, semitendinosus. B: graph illustrating the significant increase in extension phase duration and the relatively small increase of the flexion phase when the treadwheel was resisted. The error bars indicate the standard deviation. C: When extension of a single leg was prevented by supporting the foot on a stationary platform, the extensor activity persisted but stepping in the other three legs continued usually at a lower rate. The traces show the EMG recordings of a VL Žtop. and ST Žmiddle. muscle in the resisted leg and a ST Žbottom. muscle of the contralateral leg.
ing was increased 6% Ž"7.3. at 3 = T, and 11.2% Ž"10.6. at 5 = T. Using these pooled data for statistical analysis, the increase of step cycle duration in the perturbed steps was significant for each of the stimulus intensities Ž P 0.05.. The minimum strength for producing an increase in step cycle duration was 1.4 = T. MG nerve stimulation had a weaker but qualitatively similar effect on the step cycle duration as LGS nerve stimulation ŽFig. 4C; normalized values for 100% in ms: 2 = T, 355; 3 = T, 323; 5 = T, 342.. Stimulation of the MG nerve significantly increased cycle period duration by prolonging the extension phase. Stimulating with 2 = T, 3 = T and 5 = T prolonged the step cycle on average by 5% Ž"31., 7% Ž"6. and 5% Ž"2., respectively. No spontaneous walking occurred in the animals in which MG nerve was stimulated.
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3.3. Stimulating extensor afferents at Õarious times during the step cycle Previous studies in neonatal rats w23x have used short stimulation trains to investigate the afferent input in the stepcycle. Therefore, to compare the adult with neonates we stimulated the LGS or MG nerve at different times during the stepcycle with a short stimulus train Ž150 ms, 200 Hz.. The trains were delivered repetitively Ž0.2 Hz. during long walking sequences. The effect during the extension phase was strongest when the stimulus occurred during the late extension phase. It increased the extension phase duration and decreased the duration of the subsequent flexion phase ŽFig. 5A.. When stimulated during the flexion phase, the strongest effect occurred when the stimulus train was delivered at the end of the flexion phase. Fig. 5. EMG recordings showing the effect of stimulating LGS afferents with brief trains Ž150 ms, 200 Hz. late in the extension phase ŽA. or in the flexion phase ŽB.. VL, vastus lateralis; ST, semitendinosis. Ext., extension phase; Flex., flexion phase. In both cases the stimulus train increased the duration of the phase in which it was delivered and decreased the duration of the following phase.
Fig. 4. Histograms summarizing the effects on cycle period of stimulating extensor nerve afferents during extension. The data are grouped according to stimulus strength. Data from spontaneously walking animals are shown in the left pair of histograms in A and B. A: effects Žin %. of stimulating LGS nerve in a single animal. The duration of the preceding step Žwhite bars. was normalized to 100%. Error bars indicate standard deviation. B: pooled data from six animals when LGS nerve was stimulated. Here the increase of the step cycle duration was always significant. C: pooled data of four animals when MG nerve was stimulated. In these animals no spontaneous walking occurred. The effect on the step cycle duration was weaker than that produced by LGS stimulation, but was still significant.
This did not change ST activity, but resulted in an increase in flexion phase duration and a decrease of the subsequent extension phase duration ŽFig. 5B.. The effect of stimulating LGS nerve at various times during either the extension phase or the flexion phase is summarized in Fig. 6. Stimulation during the extension phase resulted in a modest increase of the step cycle duration Žtop graph, Fig. 6A., but this was only significant when the stimulus occurred near the beginning of the extension phase Ž5.7% " 6.5; "S.D... By comparison to the modest effect on cycle period, the duration of the extension and flexion phases was modified more noticeably. Fig. 6A Žmiddle. shows that the extension phase duration increased progressively as the stimulus occurred at later times during the extension phase. In bins 3 to 5 this increase was significant Ž13.1% " 9.5, 12.2% " 7 and 16.7% " 7.5.. Correspondingly, the following flexion phase duration progressively decreased as the stimulus was delayed during extension ŽFig. 6A, bottom., although only the data from stimulation during late extension phase were found to be significant Žy9.4% " 9.5.. The opposite effects on the duration of the extension and flexion phase explain the modest effect on the step cycle duration. Stimulation during the late flexion phase also resulted in a modest increase of the step cycle duration Žtop graph, Fig. 6B.. This increase was significant when the stimulus occurred near the end of the flexion phase Žbins 4 and 5: 6.1% " 6.7 and 5.7% " 2.7.. This effect on the step cycle was mainly caused by a progressive increase of the flexion phase duration as the stimulus occurred at later times during the flexion phase. A significant increase was ob-
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served in bins 3, 4 and 5 Ž9.6 " 7.9, 12.2% " 9.7 and 18.7% " 6.6.. The effect on the following extension phase was a modest decrease in duration in bin 1 and a modest increase in duration in bins 2, 3 and 4. The only significant effect occurred in bin 5, a decrease in extension phase duration Žy7% " 5.4.. Stimulation of the MG nerve produced similar but weaker effects. Stimulation during the extension phase never had a significant effect on the step cycle duration, but increased the extension phase duration significantly in bins 1, 3, 4 and 5 Ž6.8% " 7.3, 4.4% " 8.5, 9.2% " 9.3 and 10.8% " 7.5.. In all cases, the subsequent flexor burst duration was decreased. If the stimulation of the MG nerve occurred during the flexion phase, the cycle period and the flexion phase duration were only increased significantly in bin 5 Ž10.7% " 4.1 and 21.7% " 7.3, respectively.. The following extension phase was never changed significantly. As in the previous experiment, no difference could be detected for stimulating at different intensities.
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4. Discussion In this set of experiments, the influence of extensor afferent input on the timing of locomotor activity in adult rats was examined. One objective was to determine whether the mechanisms controlling extension phase duration are comparable to those previously described for cats, another was to establish whether there are any differences in afferent regulation of locomotor activity in adult rats and neonates. In general, our findings in the rat are qualitatively similar to those already reported in the cat w40,41x. One important result was that electrical stimulation of extensor afferents during the extension phase delayed the onset of flexor burst activity thus increasing the cycle period ŽFig. 3 and Fig. 4.. The average increase in cycle period was modest Ž5–14%, Fig. 4., but comparable to the increases produced by stimulation of large afferents from vastus lateralisrintermedius, plantaris or medial gastrocnemius
Fig. 6. Summary of the effects of stimulating LGS nerve afferents with brief trains at different times of the step cycle. Data for stimuli applied during the extension and flexion phases are shown in the left ŽA. and right ŽB., respectively. Stimulating during extension prolonged the stance phase Žmiddle. and shortened the following swing phase Žbottom.. The later during extension the stimulus occurred, the stronger the effect. The effect on the whole step cycle Žtop. was therefore modest. Stimulation during flexion increased the duration of the flexion phase Žmiddle. and shortened the subsequent extension phase when delivered near the end of extension Žbottom.. Data were collected from 6 animals, 12 to 31 steps were analyzed per data point. The points marked with an asterisk indicate a significant effect, and error bars show standard deviation.
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muscles Ž14–20%. in decerebrate walking cats w40,41x. One clear quantitative difference with cats was the effect of stimulating the LGS nerve. In cats, stimulation of large afferents in the LGS nerve powerfully influences extensor bursts, usually prolonging these bursts for the duration of the stimulus train w40x. This strong effect was observed only rarely in rats Žan example is shown in Fig. 3A.. Even though the effect of LGS nerve stimulation was weak in the rat compared to the cat, we did observe that LGS nerve stimulation produced more powerful effects than MG nerve stimulation Žaverage increase in cycle period 11.2% for LGS compared to 5% for MG.. On most trials stimulation of either the LGS or MG nerve during extension had a proportionally larger effect on extensor burst duration than on the cycle period ŽFig. 6.. Usually the prolongation of the extensor burst duration was compensated to some extent by a shortening of the following flexion phase. The relatively weak effect on cycle period is probably due to the overall timing of cycle period being dominated by the rate of stepping in the other three unperturbed legs. Although the recruitment order of different groups of afferents in hind leg nerves of the rat have not been defined for electrical stimuli, we attribute the effects we have observed to stimulation of afferents arising from muscle spindles and Golgi tendon organs. The effects on the timing of the locomotor activity were apparent at a stimulus strength of 1.4 = T and were usually maximal at 2 = T ŽFig. 4.. This range of stimulus strengths would have preferentially activated large muscle afferents. The largest afferents from the gastrocnemius muscles in the rat are considered to arise from muscle spindles and Golgi tendon organs w30x. There was no possibility of effects being produced indirectly by evoked muscle contractions because the nerves were cut distally. A comparison with data from the cat also supports our conclusion that the effects of stimulating the LGS and MG nerves are due to activation of spindle and tendon organ afferents. The minimal stimulus strength for influencing the duration of extensor bursts in decerebrate walking cats is about 1.5 = T and maximal effects are produced at 2 = T w40x. Over this range of stimulus strengths most of the afferents from primary spindle endings and Golgi tendon organs, and some afferents from secondary spindle endings, are recruited w25x. Further similarities with the cat were found by loading extensor muscles. Resisting the turning of the treadwheel on which the rats were walking preferentially increased the duration of extensor muscle activity ŽFig. 2A,B.. Qualitatively similar observations have been made in decerebrate cats walking on a friction free treadmill w38x. Furthermore, preventing the extension of a hind leg in a walking rat prevented stepping in just this limb ŽFig. 3C.. This is comparable to the observation in the cat, where it has been shown that supporting a leg on a stationary object inhibits the initiation of the flexion phase w20x. The functional
interpretation of these observations in the cat is that a leg must be unloaded and extended for the swing phase to be initiated w34x. The corresponding observations in the rat indicate that this may also be true for the rat. This is supported by our finding that extensor burst duration can be prolonged by electrical stimulation of extensor group I afferents ŽFig. 3, Fig. 4.. In the cat there is now considerable evidence that this effect is due primarily to activation of Ib afferents from the Golgi tendon organs w34x. If this is true in the rat, then a general rule for mammalian locomotion may be that stance Žextension. is maintained by feedback from tendon organs. Unloading of the extensor muscles near the end of stance Ždue to the animal’s weight being borne by other legs and to shortening of extensor muscles. is required for the initiation of swing Žflexion.. One further similarity to the cat was that the effects of stimulating the extensor nerves during the extension phase were greater during spontaneous locomotion, compared to those produced during MLR-induced locomotion ŽFig. 4.. Recently, Whelan observed that increasing the stimulus strength to the MLR in decerebrate walking cats reduces the effectiveness of group I afferents on extensor burst duration w42x. In both rats and cats this difference is not due to differences in walking speed, because it is observed at the same cycle periods during spontaneous and MLR induced walking. Although the numerous similarities in the data from cats and rats imply similar mechanisms for afferent regulation of stepping, we did find one major difference. In the adult rat, stimulating the extensor afferents during the flexion phase prolonged the flexion phase, whereas in cats the step cycle is reset by termination of the flexion phase w7,40x. Our observations in adult rats differ in some important aspects from those in neonates. In neonates of age less than P6, low and high intensity stimulation of extensor afferents delivered during the extension phase has either no effect or causes a resetting of the step cycle to flexion by truncating the extensor burst, respectively w23x. In adults we observed that stimulation of the extensor afferents with different intensities Ž2–5 = T. always increased the duration of the extension phase. Another difference is that in adults stimulation during the flexion phase at all intensities always prolonged the flexion phase. The same effect was observed in neonatal rats only when stimulated with high intensities Ž) 5 = T.. Low intensity stimulation Ž- 3 = T. of the extensor afferents prolonged the flexion phase just in P1–3 animals, but truncated it in P4–6 animals, suggesting a developmental change in the integration of the extensor afferents w23x. The observed differences between adult rats and neonates might be explained by the occurrence of a second developmental change in the integration of afferent information. The idea of such a change is strengthened by the fact that P6 neonates have not started to support their body weight w39x and by the finding that direct cortico-motoneuronal synaptic contacts are not established until about P7 w9x. Another possible explanation
K. Fouad, K.G. Pearsonr Brain Research 749 (1997) 320–328
for differences in the effects between neonatal rats and adults could be the different preparation. In the neonate, afferents from the quadriceps muscle, a knee extensor, were stimulated, whereas in this study ankle extensor afferents were examined. Another difference was that neonatal rats were paralyzed and fictive locomotion was examined, whereas adult animals were walking on a treadwheel. This explanation, however, appears unlikely, because in cats these differences have not been found to qualitatively influence the results. Similar effects are produced when extensor group I afferents from knee or ankle extensors are stimulated w21,40x, and there are no major difference between the results obtained in walking animals w40x and in paralyzed preparations w21x. In conclusion, our results suggest that similar sensory mechanisms regulate the transition from the extension to the flexion phase in adult rats and adult cats. Further, differences between older neonates and adult rats indicate developmental changes of these mechanisms sometime after neonatal day 6.
Acknowledgements The authors thank R. Gramlich for excellent technical assistance. We would also like to thank D. Bennet, G. Hiebert and J. Misiaszek for reading the manuscript. This work was supported by grants of the Deutsche Forschungsgemeinschaft and the Medical Research Council of Canada.
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