Differences in unconditioned and conditioned responses of the human withdrawal reflex during stance: Muscle responses and biomechanical data

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Differences in unconditioned and conditioned responses of the human withdrawal reflex during stance: Muscle responses and biomechanical data Thomas Kaulicha , Wolfram Föhrea , Dieter Friedhelm Kutza , Markus Gerwigb , Dagmar Timmannb , Florian Peter Kolba,⁎ a

Institute of Physiology, University of Munich, Pettenkoferstr. 12, 80336 Munich, Germany Department of Neurology, University of Duisburg-Essen, Hufelandstr. 55, 45122 Essen, Germany

b

A R T I C LE I N FO

AB S T R A C T

Article history:

The aim of this study was to characterize differences between unconditioned and

Accepted 17 February 2010

classically conditioned lower limb withdrawal reflexes in young subjects during standing.

Available online 24 February 2010

Electromyographic activity in the main muscle groups and biomechanical signals from a strain-gauge-equipped platform on which subjects stood were recorded from 17 healthy

Keywords:

subjects during unconditioned stimulus (US)-alone trials and during auditory

Classically conditioned lower leg

conditioning stimuli (CS) and US trials. In US-alone trials the leg muscle activation

withdrawal reflex

sequence was characteristic: ipsilateral, distal muscles were activated prior to proximal

Standing subjects

muscles; contralaterally the sequence was reversed. In CSUS trials latencies were shorter.

Sequence of muscles activated

Subjects unloaded the stimulated leg and shifted body weight to the supporting leg. In

Ground forces exerted

US-alone and in CSUS trials leg forces on each side were inversely related and

Center of vertical pressure

asymmetric, due to preparation for unloading, whilst conditioned responses (CR),

Spatial parameters of

representing the unloading preparation, were symmetric. The trajectory of the center

body movements

of vertical pressure during US-alone trials moved initially forward (a preparatory balance reaction) and to the stimulation side, followed by a large lateral shift to the side of the supporting limb. During CSUS trials the forwards shift was absent but the CR (early lateral shift) represented a preponed preparatory unloading. Electrophysiological and biomechanical responses of the classically conditioned lower limb withdrawal reflex in standing subjects changed significantly in CSUS trials compared to US-alone trials with higher sensitivity in the biomechanics. These findings will serve as a basis for a subsequent study on a group of patients with cerebellar diseases in whom the success of establishing procedural processes is known to be impaired. © 2010 Elsevier B.V. All rights reserved.

⁎ Corresponding author. Fax: +49 89 2180 75216. E-mail address: [email protected] (F.P. Kolb). Abbreviations: CS, conditioning stimulus; US, unconditioned stimulus; CR, conditioned response; UR, unconditioned response; BW, body weight; CVP, center of vertical pressure; EMG, electromyograph; TA, tibial anterior muscle; GA, gastrocnemius muscle; RF, rectus femoris muscle; BF, biceps femoris muscle, for all muscles indexes may be added: i: ipsilateral, c: contralateral to the side of stimulation, e.g. TAc refers to the contra lateral TA 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.02.051

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1.

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Introduction

Extensive studies on limb reflexes by Sherrington (1910) led to a generally accepted flexor reflex pattern caused by the electrical activation of spinal flexor reflex pathways (Eccles and Lundberg, 1959). This pattern however, is not stereotyped (Schomburg, 1990) and may depend on descending commands and on segmental conditions. It may be tonically and/or phasically facilitated by the corticospinal and rubrospinal tracts, or inhibited by the reticulospinal pathways (Lundberg and Voorhoeve, 1962; Schwindt, 1981). Detailed information about the lower limb reflexes in humans has been obtained from subjects at rest (Shahani and Young, 1971; MacKay and Murphy, 1979; Meinck et al., 1981, 1983, 1985; Torring et al., 1981; Bloedel and Bracha, 1995; Sandrini et al., 2005). In most of these reflex studies subjects were tested in the supine positions. There are however, crucial differences in the organization and in the control of the lower limb reflex when evoked in sitting or standing subjects (Rossi and Decchi, 1994; McIlroy et al., 1999; Bent et al., 2001; Andersen et al., 1999, 2003). A detailed modular organization, in which each lower leg muscle has its own receptive field, has been shown for sitting subjects by Andersen et al. (1999). During standing and locomotion, perseveration of balance is important and may modulate spinal reflexes. During standing a shift of the center of vertical pressure precedes the actual withdrawal of the limb to maintain balance (McIlroy et al., 1999). The pattern of the reflex may be modulated by the stimulus intensity (Rossi and Decchi, 1994), by the load onto the leg (Decchi et al., 1997), and by phases of walking ( Yang and Stein, 1990) or running (Duysens et al., 1993). A reversal of the reflex was observed during the transition from the swing to stance phase compared to the transition from stance to swing phase (Duysens et al., 1993; Nakajima et al., 2008). The withdrawal reflex pattern may also be modified by plastic processes tested by the method of classical conditioning (Kolb and Timmann, 1996). The involvement of the cerebellum as a putative structure for procedural learning has been studied extensively by our group, using both electrophysiological (Timmann et al., 2000; Kolb et al., 2000) and imaging (PET: Timmann et al., 1996; fMRI: Dimitrova et al., 2004) techniques. In humans, we have shown that the cerebellum, particularly the anterior and superior regions, is required for both the acquisition and the retention of this type of reflex. From this aspect an expansion of the current concept of clinically based, functional compartmentalization was suggested, namely that anterior and superior cerebellar regions must be intact to allow plastic processes to occur (Timmann et al., 2000). The aim of this study was to show characteristic differences in the unconditioned (UR) and conditioned responses (CR) of the classically conditioned lower limb reflex in standing subjects. The responses were obtained by electrophysiological methods and biomechanical techniques. Evidence will be provided that responses are different to those obtained from subjects in supine conditions in our previous studies. The increasing development of CR incidence with time throughout the recording session is accepted as a measure for a functioning underlying plastic process. The final CR incidence level is similar in muscle-derived para-

meters and in biomechanical parameters. On the other hand, since biomechanical parameters represent the final output of the system, the total sensitivity is possibly higher than that of the specific group of leg muscles from which we recorded and which may contribute only partially to the global biomechanic responses.

2.

Results

General observations: Muscles involved in the classically conditioned lower limb withdrawal reflex evoked by electrical stimulation (US) in standing subjects were activated in a stereotyped sequence, with distal muscles first on the side ipsilateral to the stimulation and proximal muscles first on the contralateral side. The sequence was similar in US-alone trials and in trials with a preceding conditioning stimulus (CS), the so-called CSUS trials. The painful electrical stimulation evoked unloading of the stimulated leg with an concomitant shift of the body weight to the side of the supporting limb. Forces exerted and the shift of the center of vertical pressure (CVP) were characteristic reactions of this reflex. Spatial parameters such as the spatial angle given by the thigh and the lower leg and particularly markers fixed at the hips, knees, and the feet provide additional information about reactive body movements in space.

2.1.

Characteristic activity patterns of the tibial muscles

The muscle primarily activated by the electrical stimulation of the tibial nerve is the ipsilateral TA. During US-alone trials the activity pattern is characterized by stable responses of short latency with a small temporal jitter on the stimulated side. On the contralateral side TA amplitudes were larger at increased latencies. During CSUS trials latencies of the unconditioned responses (UR) were reduced and CR were established. Such a characteristic TA pattern, obtained from Subject #2 (Table 1), is illustrated in Fig. 1 with stack plots in A and averaged responses in B. The process of establishing conditioned responses (CR) can be seen on both ipsilateral and contralateral sides. For both sides the change in latency can be seen clearly at the time at which the trials changed from CSUS to US-alone trials. From the corresponding averaged responses in Fig. 1B the stable pattern and a small jitter is implied by the small standard deviation indicated by the gray shaded area. The responses of this subject were evoked by a 100-ms train of electrical current pulses of an intensity of 7 mA (approximately eight fold the sensory threshold tested in this subject, Subject #2 in Table 1). Although the threshold values and the intensities applied varied in different subjects, the values of this subject were characteristic for the whole group (Table 1).

2.2. General activity patterns of the leg muscles at the group level The withdrawal reflex involves numerous muscles. For this study four main groups of muscles were selected and recorded from: TA, GA, RF, BF. An intra- and interindividual comparison of the activities of these muscles requires normalization (see data analysis) with a reference value (100%) defined as the

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Table 1 – Characteristics of the individual subjects tested in this study. For all subjects sex, age, handedness (hand) and footedness (foot) are provided. The sensory threshold (threshold sens.), i.e. the stimulus intensity, at which subjects reported sensory sensation, and the stimulus intensity applied during electrical stimulation are given. Abbreviations: f: female; m: male, r: right, l: left. Subject

Sex

Age

Hand

Foot

Threshold sens. [ma]

Intensity applied [mA]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 mean std

f m m f f f m f m m f m m f m f m 8f, 9m

24.3 24.3 24.5 24.7 24.7 24.8 25.1 25.3 25.4 25.4 25.6 25.6 25.8 27.0 27.0 27.1 27.3 25.5 1.01

r r r r r r r r r r r r r r r r l 16r.1l

r l l r l r l r l l r l r r r l l 8r.9l

0.8 0.8 0.9 0.8 0.7 0.7 0.8 0.4 1.3 1.1 0.7 2.4 0.7 0.9 1.0 1.1 0.7 0.9 0.43

8.5 7.3 7.0 9.5 3.7 5.0 8.5 7.2 7.2 4.5 9.8 8.5 7.7 7.6 9.2 4.7 8.0 7.3 1.82

amplitude of the averaged response of the TA on the stimulated side during US-alone trials. The resulting scaling factor was applied to the remaining seven other muscles. As a general result, a powerful activation of flexor muscles was observed in ipsilateral muscles with a concomitant activation of the contralateral extensor muscles. The latencies were shorter in the ipsilateral (i) distal muscles and in the contralateral (c) proximal muscles (Table 2). In US-alone trials shortest latencies were found for the TAi (121.2 ± 42.0 ms) and for the RFc (148.5 ± 37.6 ms, Table 2). Normalized averaged responses are illustrated in Fig. 2. The variability of the responses was clearly larger at the group level compared with that of the individual subject in Fig. 1, and consequently, the standard deviations (shaded areas) were considerably larger. The largest amplitude was found for the ipsilateral GAi. Amplitudes of the contralateral flexors were clearly smaller than the contralateral extensors but exhibited a reasonable amplitude for establishing a stable supporting limb. The amplitude of TAi, (used as the reference value= 100%), was large because of the proximity of the site of stimulation. The very early increase of the TA activities (Fig. 2) results from stimulus artefacts which could not be suppressed completely in some subjects (see Experimental procedures, 4.3. Stimulation). All muscles expressed CR. This can be derived from the averaged responses as well as from changes in the standard deviation in Fig. 2. The activation pattern of the UR remained as described for the US-alone trials, but with onset latencies shortened significantly by approximately 22 ms on the

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ipsilateral side and 26 ms on the contralateral side (Table 2). The onset latency of CR was 15 ms shorter on the contralateral side than on the ipsilateral side (Table 2). Peak amplitudes of muscle responses (Table 2) were reached approximately 40 ms after the corresponding onsets during both US-alone and CSUS trials. All onset latencies per individual muscle and per leg in US-alone and CSUS trials were highly significantly different (p < 0.01, exception: GAc: p < 0.05). This is also true for all differences of the times to peak. The values of the CR peak times (Table 2) are presumably too short, because peaks of CR amplitudes were taken within the CSUS window only, even if the peak occurred shortly after the US. In summary, when testing the lower leg withdrawal reflex by the method of classical conditioning all main muscle groups in a given leg establish CR. The underlying plastic process reduces onset latencies by approximately 24 ms.

2.3.

Model for the sequence of UR muscle activation

Based on the measured onset latencies, the times to peaks, and the normalized amplitudes a model for UR is constructed, which summarizes the data obtained from all subjects and shows the sequence of activation of all muscles analyzed during US-alone and CSUS trials (Fig. 3). This model demonstrates clearly, as a function of time (vertical direction), the different latencies (origin of a given triangle) at which muscle activation begins, the times to peak (base point of the normalized peak amplitude) and the corresponding normalized activation strengths (maximal amplitude). The corresponding times (latencies, occurrence of the peaks) and amplitudes of the peaks may differ slightly from those in the averaged responses as shown in Fig. 2, but for the model (Fig. 3) the calculated mean values instead of those obtained by response averaging were used. Besides the ipsilateral TA with activation due to the electrical tibial nerve stimulation, the predominant pattern is activation of the ipsilateral flexor muscles and activation of the contralateral extensor muscles, both in US-alone trials and in CSUS trials. It should be noted that the RF was activated latest on the ipsilateral side, but earliest on the contralateral side, during both US-alone and CSUS trials.

2.4. Biomechanical parameters of the withdrawal reflex: forces exerted As a result of the painful electrical stimulation procedure, subjects involuntarily unloaded the stimulated leg slightly. For this reaction the body weight (BW) was shifted to the supporting leg (see below). The resulting individual forces of all subjects tested were normalized to their individual BW (=100%). The averaged normalized forces responsible for this shift, and obtained from all subjects, represent the final output of all muscles involved in this reflex (Fig. 4, forces). To unload the leg (dashed line in Fig. 4) an initial push-off force must be established. In US-alone trials (group level, n = 884) the peak value of the push-off force occurred 102.3 ± 50.6 ms after the US and showed a mean amplitude of 5.7 ± 5.2% BW. This initial increase was followed by the actual decrease of the unloading limb (425.1 ± 107.0 ms after US) at a mean amplitude of the minimum of − 15.3 ± 11.5% BW. (the preceding calculated values may differ slightly from the averaged responses in

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Fig. 1 – Electrically evoked lower limb withdrawal responses of the tibial anterior muscles (TA) in a standing subject: (A): Stack plot of TA responses in a single subject (#2, Table 1) (amplitude in μV) ipsilateral (upper stack plot) and contralateral (lower stack plot) to the side of the electrical stimulation. The analysis time was 1400 ms. The beginning of the conditioning stimulus (CS) and that of the unconditioned stimulus (US) is marked by cursors. The duration is also given by differently shaded bars above the time scale. Responses were recorded during approximately 80 CSUS trials, followed by approximately 50 US-alone (US-al) trials. The sequence is from top to bottom. (B): Corresponding averaged responses constructed during CSUS and US-alone trials. The averaged responses are shown by a thick solid line, the corresponding standard deviations by shaded areas.

Fig. 4). In the supporting limb the changes were inverse and occurred approximately at the same time and had the same amplitude (417.1 ± 130.3 ms; mean amplitude of the maximum peak of 14.8 ± 11.2% BW). During CSUS trials (n = 1218) the pushoff forces did not occur after the US, as observed during USalone trials, but were recorded as CR within the CSUS window (113.9 ± 143.2 ms before US at a mean peak amplitude of 1.9 ± 3.0% BW). The corresponding CR in the supporting limb (dotted line in Fig. 4) occurred at approximately the same time and with the same absolute value of the amplitude. The UR of the supporting limb in these CSUS trials was bimodal with the first peak (10.6 ± 10.3% BW) at 163.7 ± 69.7 ms and the second (14.2 ± 11.9% BW) at 380.9 ± 107.3 ms. The unloading limb showed a single minimum of − 17.0 ± 11.5% BW at 333.4 ± 117.9 ms. The stimulus-induced forces, as the final output of all muscles involved, showed clear CR in both limbs. The temporal jitter of the occurrence of the peaks in the response curves yielded different results depending on whether they were obtained by calculating the arithmetic mean value of the times of occurrences (values given above) or by computer aided averaging the response curves (Fig. 4).

2.5.

Center of vertical pressure (CVP)

Averaged trajectories of the CVP, obtained by analyzing forces exerted by the legs and calculated during CSUS (n = 1278) and US-alone (n = 938) trials from all subjects are illustrated in Fig. 4. During US-alone trials the CVP started with an initial shift forwards and to the side of the unloading leg, followed by a fast shift to the side of the supporting leg and back. A direct comparison (with equal numbers of trials), and to remain approximately in the same period within the recording session, only data from 20 final CSUS trials and 20 initial USalone trials per person were compared. For the period following the US (700–1400 ms) in US-alone trials the response component of the total excursion of the CVP of the whole group was 19.3 ± 35.8 mm in the frontal plane (UR(Ax)) and 15.1 ± 23.3 mm in the sagittal plane (UR(Ay), Table 3). During the corresponding period in CSUS trials a smaller excursion was measured in the frontal plane and a significantly smaller excursion in the sagittal plane (Table 3). The shortening was related in part to the fact that the initial shift to the front, as observed in US-alone trials, was not present in CSUS trials

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Table 2 – Latencies and times to peak during US-alone trials and CSUS trials. The values are obtained from the main muscles (abbreviations as in Fig. 2, i ipsilateral; c for contralateral (to the side of stimulation)). The temporal parameters are presented as mean values and standard deviations (std), including n as the number of trials taken into account. The mean values of the ipsilateral and that of the contralateral side are given separately. All UR onset latencies and all UR times to peak of each muscle and those of a given leg obtained during US-alone trials are significantly different from those during CSUS trials. UR Onset latencies [ms] US-alone trials Mean TAi GAi RFi BFi mean TAc GAc RFc BFc mean

121.3 136.8 169.3 139.8 141.8 152.1 165.5 148.5 151.9 152.5

± ± ± ± ± ± ± ± ± ±

CSUS trials

std

n

Mean

42.0 47.3 52.1 39.8 49.0 47.4 52.1 37.6 47.4 45.0

850 826 844 745 3265 791 302 884 402 2379

99.4 117.4 145.3 119.1 119.8 122.8 155.9 114.4 135.5 126.0

315.2 308.8 346.6 340.0 320.5 310.1 297.7 316.3 264.7 305.2

± ± ± ± ± ± ± ± ± ±

US-alone trials

std

n

Mean

39.4 39.9 41.6 33.6 42.3 50.2 62.6 38.4 59.0 51.5

1121 1141 1027 895 4184 1079 349 981 465 2874

159.6 180.7 204.2 181.1 181.4 203.3 205.6 194.1 190.7 198.1

± ± ± ± ± ± ± ± ± ±

CSUS trials

std

n

Mean

48.1 49.5 49.6 43.8 50.5 48.7 64.1 43.4 50.5 49.7

850 826 844 745 3265 791 302 884 402 2379

139.3 160.0 180.9 153.7 158.2 166.9 184.6 152.2 165.8 163.9

± ± ± ± ± ± ± ± ± ±

CR Onset latencies [ms]

CR times to peak [ms]

CSUS trials

CSUS trials

Mean TAi GAi RFi BFi mean TAc GAc RFc BFc mean

UR times to peak [ms]

± ± ± ± ± ± ± ± ± ±

std

n

Mean

69.9 74.0 72.4 73.0 74.0 81.7 111.8 78.2 83.0 84.4

298 497 195 116 1106 377 50 272 110 809

350.1 348.8 370.1 368.4 355.0 353.8 333.0 354.7 300.1 345.5

(Fig. 4, CVP). Despite these smaller excursions the total length of the CVP trajectory calculated for the total time range (0– 1400 ms) was significantly longer during CSUS trials (Table 3). This was due to additional excursions during the CSUS window (250–699 ms) in which the preponed preparatory phase of the unloading leg occurred, representing the CR (Fig. 4, CVP, inlet). The resulting length of the trajectory during this period was significantly shorter during US-alone trials (time range 250– 699 ms, Table 3). The changes in the Ax and Ay components and changes in the lengths of the trajectories obtained in CSUS trials compared with those in US-alone trials were highly significant (p < 0.001) for the CSUS window — reflecting the CR. This was also true for the whole analysis time for the Ay component (Table 3).

2.6. Spatial positions of body segments and the corresponding angles The spatial position of the body was given by the corresponding markers at the hip, the knee and the foot (Fig. 7). During electrical stimulation these joints, and consequently the markers, of the unloading limb performed trajectories. The mean of the maximal lengths of the trajectory of the knee marker during US-alone trials was 93.9± 70.8 mm. For compar-

± ± ± ± ± ± ± ± ± ±

std

n

42.4 41.1 41.0 36.6 43.2 50.3 67.3 34.3 58.9 53.0

1121 1141 1027 895 4184 1079 349 981 465 2874

std

n

71.0 71.3 72.8 70.9 72.0 80.8 116.4 78.1 82.1 84.6

298 497 195 161 1106 377 50 272 110 809

ison the overall length, given by the sum of the individual lengths of the hip, the knee, and the foot trajectories was set to 100%. The corresponding relative lengths of the trajectories were maximal for the knee, followed by the foot and smallest for the hip (Table 3). This was true for all the different time ranges from which they were calculated (Table 3). The relative lengths of the trajectories were significantly different during CSUS trials compared with those obtained during US-alone trials (Table 3). It is worth noting that in CSUS trials the relative length of trajectory of the knee marker was reduced compared with that in US-alone trials (Table 3). In relation to the change of the position of the joints of an extremity due to the electrical stimulation a change in the spatial angle α, between thigh and lower leg, as well as its projection β to the horizontal plane occurred (Fig. 7). Spatial angles were derived from the positions of the markers. A characteristic sample of the changes of angles α and β during CSUS- and US-alone trials is illustrated in stack plots (Fig. 5A). The mean decrease of the angle α during US-alone trials was −25.4 ± 4.5° and the mean increase of angle β was +109.3 ± 10.7° in this subject (#3, Table 1). During CSUS trials the change in α was − 28.7 ± 6.0° and in β was + 103.4 ± 15.9°. In this subject all angles were found to be rather stable and large throughout the experiment whereas at the group level the angles scattered

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Fig. 2 – Comparison of normalized averaged responses of all muscles [tibialis anterior, (TA); medial gastrocnemius (GA); rectus femoris (RF) and biceps femoris (BF) muscles] of subjects tested. For comparison, muscle responses were normalized such that the maximal amplitude of the TA of the stimulated side obtained during US-alone trials was set as 100% (dashed lines). The scale factor derived from this TA normalization was applied to all muscle, ipsilateral and contralateral to the side of electrical stimulation. The thick solid line in each panel represents the averaged response, and the shaded area the standard deviation. The occurrences of the CS and the US are marked by vertical cursors and the duration by differently shaded bars above the time scale. The responses of the upper panels were obtained during paired CSUS trials and those of the lower panel during US-alone trials.

clearly more, particularly β. The preponderant pattern observed however, was similar to that shown in the single subject in Fig. 5A with rotating the leg in the hip joint inwards, reducing α and increasing β (sample in Fig. 7B). However, other subjects responded by rotating the leg outwards, reducing both α and β. Some of them changed their movement pattern

during the experiment from inward to outward rotations. Consequently, the standard deviations of these angles were considerable. The mean decrease of α during US-alone trials was 9.6 ± 10.3° and the mean increase of β was 42.6 ± 52.2° at the group level. During CSUS trials the change in α was − 9.5 ± 10.7° and in β was + 44.3 ± 50.2°. The corresponding averaged

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46.7% (Block 6). The mean CR incidence of the biomechanical parameter was approximately 12% higher than the corresponding electrophysiological value, particularly after block 2, indicating that a change in β – disregarding the type of rotation (inwards or outwards) – was more sensitive than the muscle response.

3.

Fig. 3 – Model of the occurrences (latencies) and the times to peaks of the normalized amplitudes of unconditioned responses of all muscles ipsilateral and contralateral to the side of stimulation (for abbreviations of the muscles see Fig. 2). Each muscle activation is represented by a triangle along a vertical time scale with the starting point representing the latency, the base point representing the time to peak and the peak as the normalized peak amplitude. Left panels: Responses obtained during CSUS trials. Right panels: Responses obtained during US-alone trials. Dashed lines represent normalization to 100%, with the ipsilateral TA obtained during the US-alone trials (thick solid line) as the muscle, from which the scaling factor was derived (see Fig. 2).

changes in angles α and β are illustrated in Fig. 5B. The large scatter of these angles notwithstanding, CR can be seen clearly within the CSUS window of the group data (Fig. 5B). Moreover, the process of conditioning is seen best in the individual responses of the single subject (Fig. 5A). These biomechanical responses, represented by the forces subjects exerted during electrical stimulation, the trajectories of the CVP, the changes of the position of the markers, and the changes of the angles derived from the markers' positions illustrate the final output of the withdrawal reflex during stance.

2.7.

Conditioned responses

As has been shown above, CR were detectable in both the electrophysiological and biomechanical data. The mean TArelated CR incidence of the stimulated side of the single subject (#2, Table 1) shown in Fig. 1 was 83.3 ± 21.4% (range: 40– 100%). These were the highest values observed in this study. The mean value at the group level was 28.5 ± 26.4%. The development of the CR incidences at the group level during eight blocks of 10 trials each is illustrated in Fig. 6. The values ranged between 11.1% (Block 1) and 45.2% (final block). The angle β-related CR incidence of the single Subject #3 (Table 1) in Fig. 5 was 61.1 ± 19.7% (range: 22.1 ± 100.0%) whereas the mean CR incidence at the group level was 23.9 ± 15.7%. The temporal development of the biomechanical CR incidences is shown in Fig. 6, ranging from 14.5% (Block 1) to

Discussion

The aim of this study was to characterize the differences between the electrically evoked unconditioned and the classically conditioned painful lower limb withdrawal reflex in a group of healthy, young subjects, standing on a forcerecording platform. The sequence of leg muscle activation was found to be similar in US-alone trials and in CSUS trials. Ipsilateral to the stimulation side, distal muscles were activated prior to proximal muscles whereas the sequence was reversed on the contralateral side. Shorter latencies were observed in CSUS trials. When unloading the stimulated leg subjects shifted body weight to the supporting leg. Unconditioned leg forces on each side were – due to preparation for unloading – inversely related in US-alone and in CSUS trials but were asymmetric, whilst conditioned responses (CR), representing a learned unloading preparation, were symmetric. Consequently, the excursions of the resulting trajectory of the center of vertical pressure (CVP) were different during USalone and CSUS trials. The characteristic forward shift in USalone trials was absent in CSUS trials. The early lateral shift in those trials occurred as an established CR and represented the preponed preparatory unloading. This discussion will address firstly the electrophysiological responses of the main groups of leg muscles, secondly the simultaneously recorded biomechanical signals and finally the plastic processes expressed by CR in the electrophysiological and biomechanical responses.

3.1.

Electrophysiological responses

3.1.1. Muscles involved in the lower limb reflex and its modulation The lower limb withdrawal reflex is a polysynaptic and multisegmentally organized spinal reflex that is controlled and modified by descending signals from supraspinal structures. It has been carefully studied by numerous groups (Lundberg, 1979, Schomburg, 1990) and many of the human studies have focused particularly on the spinal component with supine subjects. However, electrically evoked reflexes may be considerably different in subjects tested under natural conditions, such as standing or during active (Yang and Stein, 1990) and passive (Nakajima et al., 2008) walking. Reflexes are modulated depending on the movement being performed or on the phase within a cyclic movement (Yang and Stein, 1990; Stein et al., 1993; Stein and Thompson, 2006). Moreover, a change of the reflex pattern depends on whether the stimulus is non-painful (Burke et al., 1991; Duysens et al., 1993) or painful (Rossi and Decchi, 1994; Decchi et al., 1997; Bent et al., 2001; review by Sandrini et al., 2005). Painful electrical stimulation applied to the cat's paw evokes a large flexor

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Fig. 4 – Biomechanical data of all subjects tested: Data in the upper row of the panels represent the averaged responses obtained during CSUS trials and those of the lower panels during US-alone trials of all subjects tested. Left panels: averaged changes in the vertical ground forces (normalized to the individual body weight = 100%) exerted by the subjects on the platform during electrical stimulation. Thick dotted line results from recording of the forces exerted from the supporting leg, thick dashed line from the unloading leg. The beginning of the conditioning stimulus (CS) and that of the unconditioned stimulus (US) are marked by vertical cursors. Right panels: Averaged trajectories of the center of vertical pressure (CVP) derived from the vertical ground forces. The inset in the CVP plot during CSUS trials shows the beginning of the deviation of the CVP on a larger scale with the occurrence of the CS and US marked by arrows. Forces are presented as amplitude [% of body weight] against time [ms], whereas CVP represent spatial parameters only, front, rear, left, right [mm]).

reflex response which may interrupt the support action of the stimulated limb (Forssberg, 1979).

3.1.2.

Load-dependent modulation

The withdrawal reflex in humans however, is load dependent. During sitting the balance of modular activation of antago-

nistic distal muscles is shifted to plantar flexor muscles and the larger soleus response (compared with the TA response) was assumed to be due to the load onto the limb (Andersen et al., 1999). This is consistent with our results with larger ipsilateral UR in GA than in TA during US-alone trials (Figs. 2 and 3). This load aspect was also studied in the TA (Rossi and

Table 3 – Characteristic parameters of the spatial positions of the markers and the CVP: The movement of the center of vertical pressure (CVP) is presented in an excursion Ax (in the frontal plane), in an excursion Ay (in the sagittal plane), and in the trajectory length (CVP t-length). Values are given for the following time windows: CSUS window, i.e. window preceding the US (pre US): 250–699 ms; window after the US (post US): 700–1400 ms; complete analysis window (total range): 0–1400 ms. The individual lengths of trajectories (t-length) of the different markers, fixed at the hip, the knee, and the foot are given in %, with 100% as the overall length as the sum of the three lengths of trajectories. All values are given for 20 initial US-alone trials and for 20 final CSUS trials calculated for all 17 subjects resulting in degrees of freedom of 678. Shaded areas represent a significant difference (p < 0.01) between the values obtained during US-alone and CSUS trials.

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Decchi, 1994). The TA pain reflex response was depressed significantly when most of the body weight was carried by the leg stimulated; conversely, clearly increased ipsilateral TA responses were found when the body weight was transferred to the contralateral leg (Rossi and Decchi, 1994). This is consistent with the results presented in this study. Since the amplitude of the ipsilateral TA UR in US-alone trials was used for normalization at 100%, a depression cannot be derived; however, the averaged TA UR was larger on the contralateral side, both for the single subject (Fig. 1) and for the whole group (Figs. 2, 3).

3.1.3.

Stimulus-dependent modulation

The reported inhibition of the ipsilateral soleus muscle was found at an intensity 3.4 and 4.0 times the perception threshold intensity (Decchi et al., 1997). In the present study an intensity of 8 times the perception threshold was applied. A clear inhibition was observed in single trials as well whereas in averaged responses at the group level this inhibition was less pronounced because of the temporal jitter (Fig. 2, contralateral GA in US-alone trials). The differences between ipsi- and contralateral TA amplitudes in our study were clearly smaller than those reported by Rossi and Decchi (1994), which may be due to the dynamics of loading and unloading. In the present study subjects unloaded the leg (unload mean value 14.2% of BW, Fig. 4) due to the stimulus applied whereas in the study of Rossi and Decchi (1994) subjects unloaded before stimulus application.

3.1.4.

Phase-dependent modulation

A strong dynamic, phase-dependent modulation of cutaneous reflex amplitudes has been reported during walking (Yang and Stein, 1990), running (Duysens et al., 1993) or passive walking (Nakajima et al., 2008). The reflex was electrically, albeit nonpainfully, evoked with the stimulus applied during different phases of the step cycle. A strong increase was found during the transition from stance to swing phase and a reversal during the transition from swing to stance phase (Yang and Stein, 1990; Duysens et al., 1993; Nakajima et al., 2008). The maximal increase of the TA response was approximately of + 20% compared with that measured in the completely unloaded situation (Nakajima et al., 2008). This value is comparable with our results since subjects unloaded the stimulated leg up to a certain amount of the body weight resulting in an increased load in the contralateral leg, visible in an increased TAc amplitude (Fig. 2).

3.1.5.

Coordination and sequence of muscle activation

Corresponding reflex responses for proximal muscles have been described by Yang and Stein (1990) and Decchi et al. (1997). The lack of contralateral responses of the TA, quadriceps and biceps femoris muscles may be due to the lower stimulus applied compared with that used in our study. In upright standing subjects the TA is usually silent (Rossi and Decchi, 1994). This is consistent with our ipsilateral and contralateral TA (Figs. 2, 3, US-alone trials). The ipsilateral distal flexor muscles (GAi) were clearly activated and proximal flexor (BFi) and extensor muscles (RFi) also, but less so. On the contralateral side, distal and proximal extensor muscles were most prominent with a

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concomitant suppression of the distal and proximal flexor muscles (Figs. 2, 3 US-alone trials). This basic pattern with concomitant co-activation of different muscle groups is required for stable standing, as has been shown for cats (Schomburg, 1990) and humans (Andersen et al., 2003). The sequence of activation at the ipsilateral side was TA–GA–BF– RF (Table 2, Fig. 3), which is identical to the sequence published in our previous study with supine subjects (Kolb et al., 2007). Latencies of muscles of the contralateral side were longer in distal and shorter in proximal muscles (Table 2, Fig. 3). These values are slightly different to those given in Kolb et al. (2007) assuming a different activation of muscles in standing subjects. This is also true for the amplitudes recorded. Amplitudes on the non-stimulated side from supine subjects were clearly smaller (Fig. 2 in Kolb et al., 2007) than those on the contralateral side in the standing subjects in this study (Figs. 2, 3, US-alone trials). In the study of Decchi et al. (1997) latencies in muscles of the stimulated leg (only these values are published) were shorter in general and had a different sequence (soleus – biceps capitis brevis – TA – quadriceps femoris). This may be due to the location of the recording electrodes placed and to the method of detection the onset of a muscle response. More important, however, is that longer latencies of the withdrawal reflex are required for stabilizing the subject prior to instability evoked by the unloading process of the withdrawal (see below).

3.2.

Biomechanical responses

Center of vertical pressure: Only few studies have reported biomechanical parameters during lower limb reflexes in standing subjects (McIlroy et al., 1999; Bent et al., 2001; Andersen et al., 2003). In this context the loading or unloading process seems to be crucial. Decchi et al. (1997) observed load-dependent parameters of reflexes. Unloading due to the electrical painful stimulation is associated with an excursion of the CVP to maintain the standing posture and withdraw from the stimulus (Andersen et al., 2003). In that study the maximal unloading was found at the heel (one out of 12 stimulus locations) of the stimulated leg with 10.4 ± 2.3 N and the highest loading at the corresponding site of the non-stimulated leg. This value cannot be related directly to our measurements since we measured the total unloading, normalized to the subject's body weight (Fig. 4) and not a partial unloading at different sites of the foot sole. Associated with the unloading Anderson and colleagues observed a shift of the CVP in the anterior direction of the stimulated leg and a posterior shift of the contralateral leg (Andersen et al., 2003) to maintain balance during the leg withdrawal period. Similar results were obtained by McIlroy et al. (1999) and Bent et al. (2001). The transversal shift to the side of the stimulation has been interpreted as preparatory balance reaction prior to the actual withdrawal (McIlroy et al., 1999). This is consistent with the results of the present study (Fig. 4, CVP, US-alone trials). The lateral displacement, i.e. the preparatory balance adjustment due to noxious stimuli was 6.6 cm (McIlroy et al., 1999) and thus larger than our values (<10 mm in Fig. 4). McIlroy et al. (1999) assumed an habituation process and tried to overcome it by limiting the number of trials (3× 5 trials). In the present study 70 CSUS trials and 50 US-alone trials per subject were performed during which a habituation process cannot be

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Fig. 5 – Changes in the angles α and β: A: Stack plots of changes Δ in angle α (upper panels) and β (lower panels) during n = 80 CSUS and n = 50 US-alone trials (from top to bottom) of a single subject (#3, Table 1). B: Angles as in A at the group level as averaged responses (solid lines) and standard deviations (shaded areas).

excluded and which may possibly result in a reduced CVP excursion. The preparatory balance reaction resulted in a significant delay of limb withdrawal which is required for the stabilization of the subjects balance (McIlroy et al., 1999; Bent et al., 2001). In this view the longer latencies provided in the present study (Table 2) may be related to this preparatory balance adjustments occurring prior to the limb withdrawal revealing a complex control of balance.

3.2.1. Spatial position of body segments and the corresponding angles Following the stimulus subjects performed specific body movements that were recorded using an ultrasonic system.

We focused on movements of the hip, knee and the foot on the stimulated side. In US-alone trials the largest trajectory was found in the knee, followed by the foot and was smallest in the hip (Table 3). As Andersen et al. (2003) pointed out, the CVP shift to anterior of the stimulated leg most likely facilitate the upward propulsion of the trunk and pelvis. The shift to posterior of the non-stimulated leg serves to improve the leverage for center of mass stabilization following the perturbation. Thus, the shift of the CVP in any direction is a complex result of changes in the positions of different parts of the body. Even if the CVP of a given subject resulted in similar trajectories the individual sites of the body may not. This is relevant for the angles α and particularly β, introduced in this study. Although the single subject in Fig. 5A produced very homogeneous changes in the angles, other subjects did not. This can be derived from the averaged responses in Fig. 5B which are clearly smaller in amplitude than those of the single subject in Fig. 5A. The overall characteristic at the group level is a reduction in α and a concomitant increase in β.

3.3. Underlying plastic processes indicated by the CR incidence

Fig. 6 – Development of the CR incidences of TA (black solid dots) and the angle β (beta, open diamonds) across blocks of 10 trials, obtained from all subjects. The standard error of the mean is provided in one direction only.

As far as we are aware, no study has yet addressed the modulation of the lower limb reflex in standing subjects tested during classical conditioning. Classical conditioning is an accepted method for testing for plastic processes in different nervous structures. There is still ongoing intensive debate about the contribution of the cerebellum in new motor learning (Christian and Thompson, 2003; Bracha et al., 2009), however there is no doubt that the cerebellum is

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involved in both the online processing of motor-related processes, such as the nociceptive lower limb withdrawal reflex (Maschke et al., 2002; Dimitrova et al., 2003; Kolb et al., 2007) and motor learning, such as eye blinks in animals (Perrett et al., 1993; Mauk, 1997; Bracha, 2004; Jimenez-Diaz et al., 2004) or in humans (Yeo and Hesslow, 1998; WoodruffPak and Steinmetz, 2000, Steinmetz and Woodruff-Pak, 2000; Linden, 2003; Timmann et al., 2005; Gerwig et al., 2007) or postural reflexes (Kolb et al., 2002, 2004). The involvement of the cerebellum in the lower limb reflex of supine subjects, tested electrophysiologically by the method of classical condition, has been studied extensively by our group using different techniques (Kolb and Timmann, 1996; Timmann et al., 1996; Timmann et al., 2000; Kolb et al., 2000; Dimitrova et al., 2004). Several studies have reported differences in the organization of the lower limb reflex when tested in standing subjects (Rossi and Decchi, 1994; Andersen et al., 2003). This led to our assumption for a more specific contribution of the cerebellum in the modification and performance of this reflex in standing subjects.

3.3.1. Motor learning expressed by CR in electrophysiological and biomechanical signals The success with which a plastic, motor-related process can be established is revealed by the occurrence of conditioned responses (CR). In the present study such a plastic process has been demonstrated in both electrophysiological and biomechanical signals. In an early electrophysiological study (Kolb and Timmann, 1996) CR incidence levels reached 90%. Such a high incidence we have never observed since. Although individual subjects may reach such high CR incidence levels, group means are considerably lower (59.1 ± 26,3% in Timmann et al., 2000). Maximal values for eye blink related CR incidences have been reported at approximately this level (56.7%: Solomon et al., 1989; 60.0%: Woodruff-Pak and Ivry, 1996; Gerwig et al., 2003). TA-related CR incidence values in classically conditioned postural reflexes are generally lower (38.7%: Kolb et al., 2002; 45.0%: Kolb et al., 2004). In the present study CR were observed in all of the measured electrophysiological and biomechanical parameters and in all derived parameters (Fig. 1: TA on both sides; Fig. 2: TA, GA, RF, BF on both sides; Fig. 4: forces; CVP; Fig. 5: angles α and β). The temporal development of the CR incidence of the TA and angle β is shown in Fig. 6. The maximal TA-related CR incidence level was 45.2 ± 8.63% (SEM) and the maximal angle β-related incidence level was 46.7 ± 8.33% (SEM) and thus, were within the range of other motor-learning-related incidences as mentioned above. Although the final CR incidence levels were similar in TA and in the angle β, the sensitivity is larger in the biomechanical parameter (compare the increases in Fig. 6, particularly after block 2), most likely because it is linked to the final output of the whole stabilization system. The conditioning process clearly changed the UR latencies of the muscles tested and, even more importantly, it reorganized the activation particularly on the contralateral side (Fig. 3). The preparatory balance reaction in CSUS trials did not show this initial shift to anterior as observed in US-alone trials; the initial lateral shift to the stimulated side was smaller and occurred as CR within the CSUS window clearly earlier (Fig. 4, CVP, Table 3). Taken together, it is clear that the conditioning

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process affects various aspects of a very complex balance task in standing humans. In summary our results provide sets of electrophysiological and biomechanical data obtained during classical conditioning of the electrically evoked painful lower limb reflex in standing subjects. The unconditioned and the conditioned responses of this reflex in standing subjects showed characteristic differences between the stimulated (ipsilateral) leg, which was withdrawn compared to the contralateral supporting leg. Latencies of the unconditioned responses were clearly longer than those of the conditioned responses, both however, were longer compared to those obtained from the withdrawal reflex evoked in supine subjects. This reveals a complex control of balance because an appropriate balance adjustment must be performed prior to the withdrawal of the leg, to maintain postural stability. In this group of subjects all the electrophysiological and biomechanical parameters showed conditioned responses indicating that a plastic, motor-related process was established. Although neuromuscular excitation processes cause, and hence precede, the biomechanical (final) output of the whole system, the sensitivity seemed to be higher in the biomechanical responses indicating that we had not recorded from all muscles potentially involved. The conditioning process changed the time of occurrence of the unconditioned responses and reorganized the activation of muscles resulting in a characteristic change in the trajectory of the center of vertical pressure as the final output of a complex control of the balance in standing subjects. These are important findings and will serve as a basis for a subsequent study on a group of patients with cerebellar diseases in whom the success of establishing procedural processes is known to be impaired.

4.

Experimental procedures

4.1.

Subjects

This study was performed with the permission of the ethics committee of the Ludwig-Maximilians University of Munich (Nr. 310/00). A total of 17 young and healthy subjects participated after giving written informed consent. The group consisted of 9 males and 8 females, mean age: 25.5 ± 1.01 range: 24.3–27.3 years (Table 1) with 16 of them right handed. The footedness (9 were left footed, Table 1) was defined by the foot performing a corrective step forwards after an unexpected push on the subject's shoulder. No subject presented a history of neurological or orthopedic disturbances. They were not receiving any medication.

4.2.

Paradigm

The standard delay paradigm was used to study classically conditioned withdrawal reflexes. Following the protocol suggested by Gormezano and Kehoe (1975) a time-locked sequence of a preceding conditioning stimulus (CS) and an unconditioned stimulus (US) was given. The CSUS window was fixed at 450 ms, the inter-trial interval varied randomly from 15 to 45 s. The withdrawal reflex was elicited electrically in standing subjects. This approach required specific stimulation and recording procedures.

92 4.3.

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Stimulation

The US consisted of a 100-ms train of bipolar rectangular current pulses (200 Hz, pulse duration 0.2 ms) applied through a specific custom-built electrode (see below) to the tibial nerve at the position of the medial malleolus. This stimulus was given to the subjects' right side only. In some subjects, despite careful grounding procedures, stimulus artefacts could not be eliminated completely during recording, resulting in an ‘immediate increase of the unconditioned response’. Since this ‘response’ started with the onset of the electrical stimulus, it could be distinguished easily from an evoked physiological response. Nevertheless, it may be visible primarily in the averaged TA responses of Figs. 1, 2. The CS consisted of a sinusoidal tone (1000 Hz, 75 dB SPL, 550 ms), given via head phones to the right ear and preceded the US by 450 ms. The CS was superimposed on a continuous pink noise of 50 dB SPL, applied bilaterally, to mask environmental noise. At the beginning of the session 5–10 US-alone and 5–10 CSalone trials were given (not shown) followed by 70 paired trials and then 50 US-alone trials (Fig. 1). To verify the expected learning effect – the primary goal of this study – a sufficiently large number of paired (CSUS) trials is required. On the other hand since the trials are painful for the subjects, the number of trials must be kept to the minimum necessary for data analysis. To characterize the less variable UR a lower number of trials is adequate, so that the number of CSUS and US-alone trials is not necessarily equal.

4.3.1.

Stimulation electrode

The protocol for eliciting the lower limb withdrawal reflex in stance required a different method of stimulation than that used in our previous study in subjects supine on a comfortable day bed (Kolb and Timmann, 1996; Timmann et al., 2000). Since intradermal needle electrodes penetrating the sole of the foot, as used in those studies, are clearly unsuitable for standing subjects, a surface stimulation electrode was used. It consisted of a block of plastic material (30 mm × 10 mm × 10 mm) with two sintered Ag–Ag–Cl pellets (diameter: 2 mm, distance 20 mm, Model EP2, WPI, Sarasota, Fl, USA).The surface of the plastic block was covered with a cap with two small holes (diameter: 2 mm) over the electrodes providing chambers that are filled with an electrolytic paste (Hellige, Freiburg, Germany). After finding the position of the lowest sensory threshold following electrical stimulation (Table 1) the electrode was taped to the foot and secured with an anklet (Malleo 717, Art. 3512; Juzo, Aichach, Germany). The mean of the sensory threshold strength measured in this study was 0.9 ± 0.43 mA (Table 1) and was approximately 7 times higher than that measured in our previous studies with needle electrodes (0.13 ± 0.05 mA, Kolb et al., 2007). The pain threshold was very variable in our subjects but was approximately 2–4 times the sensory threshold, and thus consistent with published values (Decchi et al., 1997). The stimulus applied in our study was approximately 9 times the sensory threshold (Table 1).

4.4.

Data recording

Electromyographic activity (EMG) was recorded differentially from the main muscle groups of both legs (tibial: TA;

gastrocnemius, medial head: GA; rectus femoris: RF; biceps femoris: BF muscles). A pair of round disposable surface ECG electrodes (diameter: 55 mm, ARBO H66LG, ARBO Medizin Technologie, Braunschweig, Germany) was placed over the muscle belly. The electrodes were cut to shape such that they fitted on the buttons of the preamplifier (center to center: 30 mm). The positions of the electrodes were standardized with the length from the medial tibial condylus to the inner ankle set to 100% (reference length). The center of the patellar disc and the center of a horizontal virtual line across the knee joint cleft above the popliteal fossa were used as reference points for the ventral and the dorsal electrodes respectively. The proximal TA electrodes were placed distally from the patellar disc at a distance of 33% of the reference length, the proximal GA electrode, also distally at 30% of the length, and the RF and BF electrodes, both proximally with respect to the reference points at a distance of 38.5% of the reference length. The corresponding skin areas were shaved and cleaned with isopropyl alcohol (75%). A grounding strip was fixed between the site of stimulation and the recording site close to the ankle. The EMG signals were initially amplified by a factor of 1000, band-pass filtered (10 Hz–2 kHz, resulting from 1/T [Hz] with time constants T of 100 and 0.5 ms respectively), full-wave rectified, and finally amplified again with a gain depending on the subject and muscle activated (total gain: 5000 to 50,000). The rectification process itself generates high frequencies that have to be smoothed by integration with a bandwidth reduced to essentially 1/T (Hz) with a time constant T of 50 ms as suggested by Gottlieb and Agarwal (1970). Data were digitized at 1 kHz/channel using a 12-bit A/D recording system (MicroLink 1000, WES, Germany) and stored for offline analysis on the hard disk of a personal computer. Biomechanical Data: Subjects stood on a platform (Stopper, Burladingen, Germany, Fig. 7) consisting of two units, one for each leg. Each unit was equipped with strain gauges in each corner to record vertical forces exerted by each leg separately. These forces were processed online into a trajectory of the center of vertical pressure (CVP) with x- and y-components fed back to the subject on a oscilloscope screen situated in front of the subject at a distance of 1.2 m and at the height of the subject's face (Fig. 7A). Subjects were asked to stand quietly on the platform and to keep the CVP close to the origin of the coordinate system and to position each foot in the middle of each platform-half. The resultant distance between the heelcenters was 23 cm, with the feet forming a slight V-shape. Due to the electrical stimulation subjects usually unloaded the leg and/or performed a step. These movements were recorded as three-dimensional data by an ultra sonic system (CMSHS-8C-V10, Zebris Medical, Isny, Germany). The system is based on the evaluation of the transmission times and has a spatial resolution of 0.1 mm and has been used successfully in studies on the kinematics of extremities (Hermsdorfer et al., 1996, 1999; Laimgruber et al., 2005). Six markers (microphones, m1...m6, Fig. 7) were fixed by adhesive tapes to the spinae iliacae anteriores, to the knee joints, and some centimeters proximal to the talocalcaneon joints. The ultra sonic transmitter system was positioned at a distance of 1.50 m to the markers, such that no obstacle was between the microphones and the ultra sonic transmitter. Each of the markers was

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Fig. 7 – Experimental design: While subjects (side view in A, top view in B) stood on a platform (consisting of two units, each for one leg, see top view in B) an unconditioned stimulus (US) (train of current pulses) was applied to the medial malleolus of the right leg. The conditioning stimulus (CS) was an auditory signal (1000 Hz, 75 dB SPL) given via head phones. The activities of the main muscle groups of both legs [tibialis anterior, (TA); medial gastrocnemius (GA); rectus femoris (RF) and biceps femoris (BF) muscles] were recorded, amplified, filtered, and rectified. Vertical ground forces were recorded for each leg separately. The trajectory of the center of vertical pressure (CVP) was calculated online and fed back to the subject on a screen, positioned at the height of the eyes in a distance of approximately 1.2 m. The positions of the legs were recorded by an ultra sonic system with markers m1...m6 fixed at the hip-, knee-, and the talocalcaneon joints with the markers m1..m3 forming the space angle α of the right, i.e. stimulated leg. The projection of α in the horizontal plane resulted in angle β which is shown at a larger scale in B. Due to the US the positions of the markers m1...m3 changed from β < 180° to β > 180°.

scanned at a frequency of 50 Hz. These data were used to detect the exact spatial position of the joints and to calculate the corresponding spatial angle α which included thigh and shank, and β, the projection of α onto the horizontal plane (Fig. 7).

4.5.

Data analysis

EMG data was analyzed on a trial-by-trial basis using an interactive computer program developed for this and comparable studies (for details see Timmann et al., 2000). The onset of a response was defined as the time at which a certain level (5 mV/s) of the corresponding derivative function was exceeded (Timmann et al., 2000). For intra- and interindividual numerical comparison the EMG amplitudes were normalized. A reference value (100%) was defined as the maximal amplitude of the averaged response of the TA on the stimulated side obtained in USalone trials. This resulted in a scaling factor which was applied to the remaining seven other muscles in US-alone trials and CSUS trials. Scaled muscle responses are illustrated in Fig. 2 with the reference value (100%) marked by a horizontal dashed line. The forces subjects exert onto the

platform were also normalized by reference body weight (= 100%; Fig. 4). A movement was regarded as spontaneous when changes in the muscle activity or in the CVP were detected i) in US-alone trials before the US and ii) in CSUS trials before the CS. Trials with such movements were excluded from the electrophysiological and biomechanical analysis. Characteristic parameters of the raw data were imported into spreadsheets (Excel 2007, Microsoft) to obtain simple descriptive statistics (mean, SD, SEM). For the group analysis the corresponding parameters were treated by appropriate statistical tests (statistics package Prism 3.0, Mozilla Labs, Mountain View CA, USA). Depending on the sets of data paired or unpaired, two-tailed t-tests were employed for the comparison of the mean latencies of normalized responses (UR in US-alone trials and UR in CSUS trials), for the comparison of the normalized trajectory lengths, the normalized excursions of the trajectories during US-alone trials and during CSUS trials. Data sets were regarded as significantly different when p < 0.05 (Tables 2, 3). Data are presented as stackplots (Figs. 1, 5) and as averaged data (Figs. 1, 2, 4, 5). Trajectories of the CVP and their lengths were calculated (for more detail see Kolb et

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al., 2004) and are illustrated as a vector graphic (Fig. 4). The spatial data of the ultra sonic system were exported and processed in custom-made programs with the corresponding algorithm written in the Yorick interpreter language (v. 1.6.0.2, University of California; Oakland, CA, USA).

Acknowledgments The authors like to thank Dr. J. Davis for critical reading the manuscript and improving the English. Part of this work was performed as part of the doctoral thesis of Th. Kaulich at the Institute of Physiology, University of Munich. We are grateful also to U. Herget for the reevaluation of the temporal data.This study was supported by the Wilhelm Sander-Stiftung (AZ: 94.090.1-3), the Friedrich Baur-Stiftung (AZ: 0006/1999), and by the Deutsche Forschungsgemeinschaft (AZ: TI 239/2-3; 7-1).

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