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1Department of Neurobiology and Anatomy, Drexel University College of Medicine; and 2Department of Rehabilitation Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania
Submitted 22 March 2006; accepted in final form 31 January 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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), as do kittens (Howland et al. 1995a; Robinson and Goldberger 1986). Understanding the mechanisms of weight support and the control of locomotion and posture after spinal injury in such rats illuminates aspects of motor control and may provide a roadmap for the design of therapies for spinal cord injury.
Many spinal reflexes and compensatory mechanisms persist below the lesion after spinalization (Bouyer et al. 2001; Hiebert et al. 1996). Analyses of spinal circuits suggest that separate interacting interneuronal populations may play specific roles in stance and locomotion in spinal cord (see Jankowska and Edgely 1993). Thus these tasks may need to be trained differently after spinal cord transection. Indeed, adult spinalized cats trained to walk often do not stand well and those that are trained to stand do not walk well (see de Leon et al. 1999; Hodgson et al. 1994; Tillakaratne et al. 2002), although with special training efforts some spinalized cats may accomplish both (for review, see Edgerton et al. 1997). However, cats spinalized as neonates and trained (Forssberg et al. 1974, 1975; Grillner 1975; Hiebert et al. 1996; Howland et al. 1995a; Smith et al. 1982) can more often perform both locomotion and stance tasks autonomously and fairly competently as adults (e.g., Robinson and Goldberger 1986). How such neonatal spinalized mammals integrate voluntary and brain-controlled postural adjustments with spinal mechanisms in both stance and locomotor tasks is unknown.
We focus here on control of stance in rats spinalized P1/P2, which also had the ability to walk independently using hindlimb weight support. Control of quiet stance requires integrated responses of both trunk and legs. Mapping of motor cortex in such rats suggested that voluntary postural control of the axial muscles may be critical for their weight-supported locomotion (Giszter et al. 1998). The task of quiet stance has not been examined in neonatal spinalized rats, nor have stance responses to external perturbations been tested in rats generally.
We trained all rats to walk on a treadmill and to stand quietly for rewards. They showed both weight-supported stance and weight-supported locomotion on all four limbs. Postural adjustments of normal and operate (spinal and transplant) rats were examined during a stance task using robot-applied perturbations similar to jostling by cage mates. We examined coordination of limb forces during stance after perturbations applied using a small robot. Robots were previously used in rats primarily for training locomotion (e.g., Timoszyk et al. 2002; see de Leon et al. 2002a) and to assess its quality and plasticity (e.g., de Leon et al. 2002b; Timoszyk et al. 2005).
Our data will show that operate and normal rats adapt their center of pressure to perturbations. However, their voluntary postural mechanisms are quite different. During quiet posture, spinalized rats miminize transmission of perturbation forces to the hindlimbs, whereas normal rats show forelimb–hindlimb cooperation. It is unknown whether this strategy can be taught to adult injured rats. Learning to use functional motor strategies that differ from normal behaviors may aid clinical rehabilitation in some injuries and diseases.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Neonatal transection/transplantation
In total, 25 rats were used in this study; 17 Sprague–Dawley rats received midthoracic (T8–T9) transections on postnatal day 2 (P2). One or two segments of spinal cord tissue were removed with sharp dissection and gentle aspiration. In eight of these rats, a 1- to 2-mm section of E14 fetal thoracic spinal tissue was transplanted into the lesion cavity (transplant rats). The remaining nine rats underwent transection only with no transplantation (spinal rats). Methods of transplantation were described previously in Miya et al. (1997) and Giszter et al. (1998). Eight additional rats served as normal controls (Normal rats). All operate rats were confirmed to have surgically complete transections using the standard histological techniques of Nissl-myelin and serotonergic immunohistochemistry (see Giszter et al. 1998; Miya et al. 1997). All rats received treadmill training and were tested in stance training.
Training
All rats entered the training program at postnatal day 21. Rats were trained biweekly to walk on a treadmill (speed 8 cm/s) for 5-min sessions and three times/wk to stand quietly on a platform for about 5 min for a water reward. Starting at postnatal day 28, water availability was limited to the test period and for an additional 30 min/day afterward. Rats were given water ad libitum from Friday afternoon until Sunday afternoon.
Testing paradigm
The testing platform consisted of three pieces of 0.5-in.-thick, clear Plexiglas each attached to a FT3/10 force transducer (range of force = 3 lb, range of torque = 10 lb-in., resolution of force = 0.04 oz, resolution of torque = 0.08 oz-in.; ATI Industrial Automation, Garner, NC). There was one sensor each for the right and left hindlimb (RHL and LHL sensors, respectively) and a third for both forelimbs (FL sensor). Each force transducer, or sensor, recorded the three-dimensional forces and torques exerted on the support plate and thus allowed a calculation of the ground reaction forces resulting from the pressure of the limb, or limbs, on that sensor. Initial trials involved collecting ground reaction forces while the rat was standing freely and quietly. In later trials, a haptic interface device, the Phantom (SensAble Devices, Cambridge, MA), attached to the rat by a saddle and jacket (made in house), was used to deliver perturbations to the rat. See Fig. 1A.
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A Phantom robot (Model T/A, Sensable Device, and originally designed for human haptics applications) running in a control loop at 1.5 kHz delivered perturbations to the rat. The robot was programmed to simulate isotropic Cartesian elastic fields in the horizontal plane with an extremely weak field component vertically. Field stiffness in the horizontal plane was 76.91 N/m. Perturbations consisted of a constant-velocity motion of the elastic fields equilibrium, to the new target position specified by the perturbation direction. The velocity of the motion was proportional to the distance to be traversed (0.75, 1.5, or 2.25 in.; 1.9, 3.8, or 5.7 cm) so that all perturbations were completed in the same time (500 ms). Thus in larger perturbations the equilibrium motion and rise in force were proportionally faster. If a rat yielded perfectly and moved to track the equilibrium, the interaction forces with the robot would be zero. If a rat completely resisted the perturbation and remained immobile, the interaction force with the robot would reflect the equilibrium motion and the rat's distance from equilibrium. In this case (for an apparently infinitely stiff object) the peak horizontal plane force in the largest perturbation would have been 4.38 N (about 429 g). Typical net horizontal forces generated at the force plates in large perturbations were about 150 to 200 g. Our perturbation paradigm was in some regards (constant-velocity motions set by the peak excursion) similar to that of Shadmehr and Mussa-Ivaldi (1993), but in their case a proportional derivative (PD) controller was used rather than a simple elastic field.
Data collection
Using the robot, a cycle of eight perturbations was delivered. These were arranged uniformly, counterclockwise around a circle centered on the rest position of the rat. A perturbation caudal (toward the tail) was designated as 0°, whereas a 90° perturbation was toward the right side of the rat (see Fig. 1B). For all measures, results were separated into groups based on the direction of perturbation. These groups were rostral group, caudal group, or lateral group. Responses to perturbations at 135, 180, and 225° were classified as rostral group, those at 0, 45, and 315° were classified as caudal group, and those at 90 and 270° were classified as lateral. The cycle was repeated three times, using a different size perturbation for each cycle, with excursions of the elastic field of 1.9, 3.8, or 5.7 cm. The Phantom recorded the resulting interaction force between the rat and the robot—i.e., the force that was delivered as a result of the location of the robot tip in the programmed elastic field. The force thus represented the difference between the field equilibrium and the robot tip position and this force was experienced as a perturbation by the rat. Interaction force at the saddle was saved throughout the trial at a sampling rate of 63 Hz (i.e., synchronized to the three force-plates data collection). Simultaneously, the reaction of the rat to the perturbation was measured from the ground reaction forces on the force plates and the EMG activity recorded. Ground reaction forces of the limbs were collected from the ATI force transducers at a sampling rate of 63 Hz. This was sufficiently rapid for the stance task and was also limited by the serial communication mode with three sensors used in this set of experiments and version of our apparatus. The response of the rat to perturbation ramp onset and plateau was smooth and continuous. We believe this was essential to the tests here because other perturbation tests could cause stepping. For example, we observed that withdrawal or stepping motions could be induced with greater likelihood in operate rats by the more rapid release from applied force as the perturbation terminated. Ground reaction force data and the Phantom force data were all synchronized and collected simultaneously using in-house C programs. The data collection and control program illuminated a light-emitting diode (LED) within the video camera field, allowing video data to be synchronized with the rest of the data. Video data were collected at 60-Hz single field rate with a shutter speed of 500 Hz.
Data analysis
For this study, only the data collected from normal rats and weight-supporting operate (including both spinal and transplant) rats were used. A weight-supporting rat is defined as a rat that underwent neonatal transection or transplantation surgery, but could stand and walk unassisted, and with more than 60% of its step cycles executed without its belly, knees, or hips touching the support surface.
The three-dimensional forces and torques collected from the force sensors were used to calculate the center of pressure (CoP), or foot placement, for each sensor. x and y coordinates of the foot placement on each sensor were obtained using the following formula. The x and y coordinates, for each foot placement, were based on the center of the respective sensor. Offsets were added to the individual coordinates, based on the location of the sensors to each other, to have them all related to the same world coordinate system. The z value is the offset needed because of the thickness of the Plexiglas
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Statistics
To compare distributions we used t-test parametric statistics and Fisher distribution circular statistics, using unit vectors from data samples in planar circular or in spherical coordinates (Fisher distribution test; Fisher 1996; Fisher et al. 1987).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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and Giszter et al. (1998). Center of pressure (CoP)
COP IN UNPERTURBED INITIAL TRIALS.
In initial trials, the rats stood freely on the platform and their ground reaction forces were recorded without the saddle. We then examined the effect of the saddle placement. Rats were attached to the Phantom robot using the saddle, but no perturbations or forces were delivered (quiet stance). For both of these sets of data for each animal the individual foot placements on each force plate and the resultant CoP were calculated for comparison with subsequent test trials. The CoP's small fluctuations through time during stance form the "stabilogram" (e.g., Collins and DeLuca 1993). The mean position of the CoP in the base of support, the statistics of the deviations of CoP, and the time series of the motions of the CoP can all give insight into control (Collins and Deluca 1995; Grzegorzewski and Kowalczyk 2001; Zatsiorsky and Duarte 1999). We focused here simply on the mean CoP location in unperturbed stance, deferring detailed analysis of CoP excursions to future work. There was no significant difference in CoP between the two sets of data for each individual rat when unencumbered and then when saddled before any use of the robot (P > 0.3). This constancy indicates that neither the robot's small unbalanced weight nor the sensation of the harness significantly perturbed the individual rat's posture or stance in the unperturbed pretrial condition.
There was a group difference in CoP between normal rats and operates during quiet stance. The position of the CoP relative to the individual foot placements (Fig. 2 and Table 1) showed a forward shift of the CoP in the injured rats. This shift in CoP is attributed to a change in the forelimb–hindlimb force distribution. This was also previously seen in chronic spinal cats (Pratt et al. 1994). In operate rats, the CoP is almost midway between the forelimb and hindlimb foot placement, whereas in normal rats the CoP is closer to the hindlimb foot position. This could arise from an altered mass distribution in the rat or represent a motor strategy—it is likely to reflect both. The hindlimbs are always significantly lighter in neonatal spinalized rats. We demonstrated this by testing the total hindlimb muscle mass in these rats, after our experiments were completed and the rats perfused for histology. Major muscles were removed and weighed and summed. The net hindlimb muscle mass as a fraction of body weight in the weight-supporting operates differed significantly compared with the fractional weight of muscles in normal animals (two-tailed t-test, P = 0.0032). The haunches were roughly 15–20% lighter in weight-supporting operates.
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). The force component differences are likely to arise, at least partly, from these configuration differences. COP IN PERTURBED TRIALS. For the test trials, when the Phantom was attached and producing perturbations to the rats' stance, the center of pressure during rest periods or during the quiet stance in the first 0.5 s (preperturbation) was again calculated. During this time, the Phantom was active but centered at the rat's chosen posture. However, if the rat leaned, the robot would then apply small holding forces, but these were always well <0.05 N.
Rats quickly adapted their quiet stance posture, and shifted their CoP, once perturbation experiments had begun. This adaptation was made by rats in all sessions after about 1 wk. It occurred before perturbation application, and also occurred in sessions where perturbations were not applied at all. It was an adaptation by the rats. We found that the CoP for the normal rats was now closer to midway (a ratio of 0.5) between the forelimb and hindlimb foot placements than before the Phantom was attached. Normal rats had a mean CoP ratio of 0.65 compared with 0.75 before any perturbation testing with the robot. This change in their stabilogram's mean or target posture was statistically significant (P < 0.001, one-tailed t-test). Once the operates were attached to the phantom in perturbation experiments, and tested in rest periods, their CoP also moved. However, the resting CoP in operates moved caudally; it was 0.55 after testing began and 0.46 before testing (a significant difference, two-tailed t-test, P < 0.0001). The significant difference between normal rats and operates in resting CoP described earlier before testing remained during testing with perturbations (during perturbation testing a two-tailed t-test showed a P value <0.0001; also see Table 1). Both normal and operate rats adopted specific postural strategies, each adopting a posture that produced a more even load distribution between fore- and hindlimbs during perturbation testing (both moved closer to a ratio of 0.5). This weight distribution differed significantly from the initial quiet stance distributions recorded in the complete absence of perturbations and caused their CoP to move toward the center of the base of support when perturbations were expected.
Perturbation responses
The robot applied a directed elastic load to the rats during perturbation trials. The equilibrium of the elastic field generated by the robot moved gradually away from the rats' resting position (velocity of equilibrium motion was proportional to perturbation size: 3.8, 7.6, or 11.4 cm/s). The saddle then applied force to the rat if the rat did not comply with the motion. The force that the rat needed to generate to resist the perturbation ranged 
400 g but was easily within the rat's capacity. To receive the water reward throughout the trial the rat had to stabilize its head at the drinking spigot. Rats continued drinking through both the perturbed and unperturbed trials. There were concomitant small rhythmic forces in the body induced by head bobbing and lapping motions, and these were sometimes large enough to be visible in the three force sensors in several rats. However, these internally generated force variations were negligible (<5%) compared with the responses to the perturbations. Rats generally responded smoothly and continuously to the perturbations. They used a combination of resistance and motion. Rats only rarely lost balance or exhibited steps or shifts of the feet. Trials with compensating steps were rejected. Thus foot placement was maintained in the data subsequently presented. A typical perturbation response is shown in Fig. 3 in which the robot motion, interaction force at the thorax, and the ground reaction force in the hindlimb are shown for an operate rat.
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GROUND REACTION FORCE MAGNITUDE AND TUNING IN RESPONSE TO PERTURBATION. To test general compensatory responses of the rats to perturbation we examined the initial force vectors, the peak and plateau force vectors (Fig. 5), the changes in ground reaction force vectors in the vertical direction and the horizontal plane, and the center of pressure. We found that the CoP did not move out of the range of the resting (stabilogram) measurements during the perturbation; thus CoP was stabilized. This was true of both normal and operate rats. Preperturbation period and plateau stabilograms were not statistically different. Vertical forces were not altered significantly. The CoP location was dominated by these vertical antigravity forces, which were far larger than horizontal-plane forces. Accordingly, we focused subsequent analysis on the horizontal-force components that showed perturbation responses.
The data for the individual animals in Fig. 6 and subsequent figures are characteristic of the responses observed. Horizontal ground reaction force vectors of the forelimbs and each hindlimb were examined with respect to their direction and magnitude (Fig. 6). The forces are plotted every 45° around a circle centered on each sensor. The location of each force thus indicates the direction of the perturbation applied. For each rat the combined data from several days (6–8 days of complete testing) are depicted, each vector originating at a location representing a different day's response to the perturbation in that direction.
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For the normal rats' initial resting horizontal forces (e.g., J494; Fig. 6A), the forelimb-generated ground reaction forces were directed caudally whereas the hindlimb-generated ground reaction forces were directed rostrally. For the operate rats (e.g., J490; Fig. 6A), in contrast, the direction of initial forelimb forces was sometimes not as consistent, although the hindlimb forces were all rostrally directed before all perturbations. Although CoP was closer to the center of the base of support in all rats, as noted earlier, none of the animals anticipated the specific direction of the perturbation through the repeating cycle. None adjusted its preperturbation posture or horizontal limb forces trial by trial. This can be seen from the fact that there was no systematic variation in the direction and magnitude of initial forces with perturbation direction.
Total forces resulting from the perturbation are shown in Fig. 6B. In normal rats (e.g., J494; Fig. 6B), there was a difference between the distribution of forces produced for rostral perturbations and those produced for caudal perturbations. During rostral perturbations, the total horizontal forces were larger in the forelimbs, and during caudal perturbations, the total horizontal forces were larger in the hindlimbs. In contrast, in the operate rats (e.g., J490; Fig. 6B), this distinction between rostral and caudal perturbations was largely absent. Forelimb total forces were adjusted in all directions, whereas hindlimb total forces were not very different from initial forces. The way in which rats compensated for perturbation forces thus differed in the normal and the spinal-injured rats.
ACTIVE RESPONSES FORCES. Active response forces in our analysis represent the change in force resulting from the perturbation. By examining active force responses we could assess in more detail how force alterations were distributed to the limbs in our animals. A range of response strategies is possible for rats in choosing total forces. The additional compensation by the rat was the vector sum of the changes in individual limb forces. Within a limb, the total horizontal ground reaction forces could be increased or decreased, or their direction could be altered. We evaluated response forces by subtracting initial from total force vectors and thus a decrease in total force vector from the initial force will be represented by an active response vector that is in the opposite direction from the initial force. It is important to keep in mind that, despite this convention, the actual total ground reaction force remained in the same direction and sense in both cases: in the one case total magnitude diminished and in the other it increased.
Response force analysis showed that in the normal rat (e.g., J494; Fig. 6C) the total force pattern observed resulted from two changes. There was 1) a rise in the forces in the limbs toward which perturbation occurred and 2) there was some decrease in the other limb pair's force, e.g., forelimb force increased and hindlimb force decreased together. In contrast, we found that in the operate rats (e.g., J490; Fig. 6C) the forelimb active response forces were always substantially larger than the hindlimb response forces, for all perturbations. Further, notice that the force changes in the right hindlimb in the operate rat J490 shown in Fig. 6C were negligible. Routinely we observed a pattern of very small hindlimb force adjustments of this type in the operates. We tested this difference in magnitude in the pattern of compensation in operates compared with the normal rats and this was statistically significant (two-tailed t-test, P < 0.05).
We plotted all total horizontal ground reaction forces to all perturbations as a population of vectors (Fig. 7). In the normal rat (J494; Fig. 7A) total forces exhibited a strong directionality in both the forelimbs and hindlimbs. The forelimb forces were always directed caudally and the hindlimb forces were always directed rostrally. In the operate (J490; Fig. 7B), the hindlimb forces clearly exhibited a directionality of response, but the ground reaction forces were generated in all directions by the forelimbs of operate rats in contrast to normal rats. To analyze this further we examined tuning of response magnitudes.
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. The magnitudes of initial preperturbation forces showed no clear tuning to the subsequently applied perturbation direction. A tuning curve of the magnitude of the total horizontal force measured by each sensor was generated for different perturbation directions (Fig. 8). Depth of modulation of force [(largest – smallest force)/largest force as a percentage] was 80–90% in forelimbs and 50–80% in hindlimbs. The tuning curves' principal directions for the three different size perturbations that we used did not differ significantly as a result of perturbation magnitude in either rat group; the three perturbation magnitudes of a normal rat are overlaid in Fig. 8A. At each perturbation direction, we then combined all trials. We normalized magnitudes of the horizontal ground reaction forces to the peak response from the three different size perturbations and then averaged these normalized responses for all three magnitude perturbation trials in each rat.
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In operate rats (J490 and J505; Fig. 8B), directional tuning of total forces was not at all strong in the forelimbs (e.g., compare Operate 505 with Normal J494). Depth of modulation of forelimb force was usually nearly 20% (rarely 50%; e.g., J490 in Fig. 8B). The forelimb forces were approximately the same magnitude whether the perturbation constituted rostral or caudal components (rostrocaudal modulation in both operate rats shown is <20%). The hindlimb total forces in the operates were also tuned to both rostral and caudal perturbations, but more weakly directed than normal rats. (However, the depth of modulation of hindlimb force was usually slightly larger than that in the forelimbs, amounting to 25–35%.) These differences in modulation pattern between normal and operate rats were significant (P < 0.05) when tested using circular statistics (Fisher distribution test; Fisher 1996; Fisher et al. 1987).
Active response forces.
Tuning curves for active response magnitudes indicate the adjustments made and are shown in Fig. 8C. In normal rats (J494; Fig. 8C) the magnitude of change of forelimb response was less dependent on whether the direction of the perturbation was rostral or caudal, or medial or lateral. Depth of force modulation was roughly 50%. (The two forelimb contributions were combined in our measurement system: any forelimb lateral tuning was averaged out.) Modulation observed was mostly rostrocaudal because lateral perturbations showed less forelimb force change compared with a similar rostrocaudal perturbation. The hindlimb response force magnitudes in the normal rats were tuned to rostral and caudal perturbations. Depth of hindlimb force modulation in normal rats was nearly 80%. There was a rotation of the principal direction or axis of tuning of each hindlimb toward the midline.
The magnitude of the operate rats' active response in the forelimb did not vary much irrespective of whether the perturbation was rostral or caudal (compare tuning of forelimbs in J490 and J505; Fig. 8C). In this they were similar to the normal rats. The depth of modulation of force was roughly 50% and also similar. However, in contrast to the normal rats, in operate rats the hindlimb response forces were both small and poorly tuned. The depth of modulation of forces in operates was usually roughly 25% of the peak value, whereas it was nearly 80% of peak in normal rats.
Taken as a whole, the response tuning in Fig. 8C suggested that operate rats differ from normal rats in having poorly tuned and small active hindlimb responses. Instead they compensate mostly with the forelimbs for all directions of perturbation.
Stiffness
Our experiments allowed us to coarsely evaluate the magnitude of the postural stiffness of the rats. Stiffness measures variations in the magnitude of restoring forces with change in position (negative stiffness is thus destabilizing). Stiffness can be linear, with force linearly related to position changes (as in a linear spring), or nonlinear—for example, muscles exhibit nonlinear stiffness during stretch. Stiffness can vary with direction, such as in a limb. This directional variation can be compatible with a linear model or incompatible. For example, if a rat behaved as a very stiff (but linear) system, and was uniform in its response to perturbation, it would not alter its posture much. As a result, it would strongly resist perturbations in all directions, and with similar magnitude. The resisting forces needed would be generated through the legs and observed as high ground reaction forces. Our data showed that there were significant nonlinearities in the rats' stiffness. These did not allow a simple linear fitting procedure with our data. First, stiffness components did not vary linearly with vector direction. Second, the magnitude of apparent stiffness at plateau was lower for the larger (and faster) perturbation excursions. It was not possible to accurately express the measured stiffness of the rat in response to our perturbations as a single linear function [i.e., a graphical stiffness ellipse, as used in Mussa-Ivaldi et al. (1985) and as used for individual perturbations in Shadmehr and Mussa-Ivaldi (1993)]. For a single extent and rate of perturbation, magnitudes of restoring forces were smallest for lateral perturbations. Rats were far less stiff mediolaterally than rostrocaudally. To evaluate stiffness coarsely, and without additional experiments, we proceeded as follows: we estimated the plateau magnitude of the stiffness of the rat in each perturbation direction by dividing the scalar magnitude of horizontal force exerted by the Phantom (during the perturbation plateau; e.g., see Fig. 2), by the scalar magnitude of the horizontal excursion of the Phantom at the plateau. Thus significant vector information in Figs. 4 and 5, for example, was lost in the approximation but our data could not readily support more complex analysis. All rats displayed a greater-magnitude stiffness when a smaller perturbation was delivered (see Fig. 9).
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; Fig. 2) in their evaluation of human arm stiffness. However, because we did not explicitly control for velocity we cannot discount velocity effects in our data. For all perturbation sizes in both rostral and caudal directions the normal rats were more stiff than the operate rats (two-tailed t-test, P < 0.05; also see Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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) or tested rats during rearing and bipedal-balance tasks (Mulligan et al. 2002). Here we described the compensations for external perturbations to quadrupedal stance in normal and spinal-injured rats, specifically those receiving spinal transection as neonates. These rats are interesting because a fraction succeed in developing autonomous weight support as adults (Miya et al. 1997; Stelzner et al. 1975). Understanding postural and locomotor mechanisms of rats that succeed in developing weight support despite spinal transection gives insights into the development and plasticity of spinal motor control mechanisms. In turn these may aid designing rehabilitative or restorative interventions. Neonatal spinalized rats and transplant rats differ in various ways (e.g., see Giszter et al. 1998). However, our data here show no difference in their biomechanical stance strategies. We selected injured rats that could both stand and locomote at >60% successfully weight-supported steps as adults and examined their postural controls compared with normal.
Our experimental paradigm is similar to the mutual jostling of rats in cages. We examine how stresses applied at the torso are resisted and how the applied stress is distributed to the limbs. This method differs from that most frequently used for quadrupedal stance, which examines reactions to linear translation of support surfaces (Fung and MacPherson 1995; Pratt et al. 1994). In our task, the animal controlled how applied force was distributed among the limbs and thus to the force plates. Rats altered trunk posture to comply with the perturbation if they desired. In contrast, support surface translation causes unavoidable changes in the limbs, partly the goal of the method. Our method allowed us to test whether the compensating adjustments of ground reaction forces in response to trunk perturbations, on a timescale in which voluntary mechanisms could act, are distributed or localized to specific limbs in normal and operate rats. For example, we determined whether compensation is localized entirely in the forelimbs or distributed equally among all limbs.
Similarities of injured and normal rats
There were common features in the postural mechanisms characterized here in all our animals, both operates and normals. 1) Both adaptated by shifting of CoP toward the center of the base of support after a few trials of the perturbation experiments, although the robot was producing only a minimal holding force (the CoP shifted rostrally in the normals and caudally in the operates). 2) Both had qualitatively similar initial stance forces, when the animal was at rest before any perturbation. 3) Both had greater magnitude whole body stiffness in the rostral–caudal direction with an hourglass tuning overall.
The similar initial preperturbation forces demonstrated by both normal and operate rats are consistent with MacPherson (1988a); the limbs working as inclined struts. They also mirror the finding of Lacquaniti and Maioli (1994a,b) of separate specification of limb configuration and postural ground reaction forces in mammals because postures of injured and normal rats were quite different whereas stationary forces were similar. All rats showed small day-to-day variations in limb configuration. Rats varied their postures while preserving CoP location and ground forces, even in the absence of perturbations. In injured rats the consistent configuration changes we observed compared with normal rats probably represented one form of compensation (e.g., see Miklyaeva et al. 1995, 1997). MacPherson (1988b) considers the force vector produced as a result of a perturbation as a "high level, task-dependent controlled variable," whereas the "selection of muscles to activate to produce the vector is controlled at a lower level."
Whole body apparent stiffness and resistance to perturbations
Whole body stiffness in the rats in response to perturbations was nonlinear. We had too few perturbation directions to accurately identify the structure, but we characterized the magnitude. Both normal and operate rats showed similarly tuned magnitudes of horizontal stiffness. Stiffness magnitude was larger for rostrocaudal perturbations and considerably smaller in lateral directions. The hourglass tuning pattern was probably attributable to the mechanical structure of the quadrupedal rat. Several simple planar mechanical models we tested captured the variation of stiffness magnitude. In part the pattern of stiffness is explained by the fact that the base of support of the rat is narrower in the mediolateral direction than that in the rostrocaudal direction. The legs, acting as four inclined struts, also change their geometric relationship to the trunk during perturbations. This effect could be further exaggerated by adding a laterally more compliant "vertebral" column. The similarity of stiffness tuning in operates and normals, despite dramatic differences in motor capability and posture, is consistent with the shape of stiffness variation being dominated by the mechanical structure.
We observed that rats showed a yielding behavior for larger perturbations, which in our design were also faster perturbations. These yielding patterns could represent local muscle properties and similar neuromechanical controls or they could represent voluntary compensations. In favor of the latter, normal rats sometimes showed additional and clearly voluntary trunk yielding. This yielding occurred during the plateau phase after perturbation, and the perturbation duration of 500 ms was clearly sufficiently long for a planned response.
Operates were less stiff than normals for caudal perturbations. The injured rats consistently had much reduced hindlimb contributions to postural adjustments. Finally, in operate rats we more often observed asymmetric stiffness magnitudes. Our simple models suggested that this could arise from symmetry breaking flexing in the trunk, or scoliotic bias in the trunk, which is a problem spinal-injured rats are prone to. Injured rats might therefore show this behavior more often resulting from either skeletal distortion or weakened trunk control and diminished trunk stability.
Strategies of compensation
The most significant differences between operates and normals were in the relationship between their forelimb and hindlimb forces. Force-tuning curves of normal rats showed that the magnitude of their total forces after perturbations varied with the perturbation direction. Normal rats maintained their posture by varying the balance of horizontal forces in hindlimbs and forelimbs. The forelimbs mainly stabilized rostral group perturbations, whereas the hindlimbs mainly stabilized caudal group perturbations. In contrast, operates usually produced greater force changes with the forelimbs than with the hindlimbs in response to all perturbations. A reduced force-generating capability of the hindlimbs contributed. However, hindlimb force responses were substantially smaller than could be accounted for in this way. The forelimbs also produced forces in atypical directions compared with normal rats. We speculate that operates used a strategy that led to a different pattern of response, with compensation focused in the forelimbs. This would achieve two things: first, the forelimbs are more precise because they are under full voluntary control; second, this strategy would avoid motion in the hindlimbs. It might be expected that the local lumbar circuitry and resistance reflexes would be able to provide some predictable and useful hindlimb responses after initial loading, based on the research in cats (e.g., Ting and Macpherson 2005). However, we believe that the strategy of minimizing any transmission of perturbation force to the hindlimbs that we observed here reduces the likelihood of inappropriate stepping or reflex motions induced by the perturbation. Operates that have lost all descending voluntary control of lumbar cord may lack the ability to control or suppress such spontaneous and reflex responses. Autonomous local pattern generation and reflexes play a central role in the weight-supported locomotion of these spinally injured rats, but their spurious activations during quiet stance could cause instability and stance failures. We sometimes saw such motions induced by the rapid release from the perturbation, at trial end. Specific stance or step training in spinal cats biases spinal excitation and inhibition systems up or down (de Leon et al. 1999; Tillakaratne et al. 2002). If the spinal-injured rats are to accomplish both stance and locomotion they must find compromise strategies where both can be achieved through largely reflex hindlimb use. Our study identifies important aspects of the stance strategy they use.
In conclusion, we have quantified the responses of spinally injured rats to outside perturbations to stance. The data complement kinematic and kinetic analyses of locomotion. Results demonstrate that the voluntary motor strategies used in stance differ between normals and operates. Within operates there were no obvious differences in motor strategy of spinal and transplant rats despite potential bridging effects of transplants. In all operate rats the active compensation for perturbations occurred almost entirely in the forelimbs. Our data are consistent with a strategy of stance and compensation in the injured rats that organizes motor adjustments in forelimbs and trunk so as to isolate the hindlimbs from perturbing forces. This strategy avoids involuntary hindlimb stepping during stance. Learning this strategy may be a key feature of developing function after complete spinal transection in rats and these data may help guide future efforts to use motor training after adult spinal cord transection.
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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.
Address for reprint requests and other correspondence: S. Giszter, Department of Neurobiology and Anatomy, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, PA 19129 (E-mail simon.giszter{at}drexel.edu)
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: approximate rest location of rat before perturbations). Vectors show the peak forces generated by the rat after subtraction of resting forces. Vectors displayed were measured at the peak excursion produced by the Phantom for each perturbation. Each ring of vectors represents a perturbation size (1: 19 mm, 2: 38 mm, 3: 57 mm). Each line of 3 vectors represents a perturbation direction. Interaction forces are shown plotted as vectors at the measurement point to which the robot moved against the rat's resistance. Force rises with the size of the perturbation, although not proportionately. Note also that forces converge approximately toward the rest position of the rat, but not precisely, because these represent active response forces.
