Changes in intracortical excitability induced by stimulation of wrist afferents in man

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1 12359 Journal of Physiology (2001), 534.3, pp Changes in intracortical excitability induced by stimulation of wrist afferents in man Jean-Marc Aimonetti and Jens Bo Nielsen * Laboratoire Développement et Pathologies du Mouvement, Centre National de la Recherche Scientifique, Marseille, France and * Department of Medical Physiology, Panum Institute, University of Copenhagen, Denmark (Resubmitted 22 February 2001; accepted after revision 26 March 2001) 1. Inhibitory and facilitatory neuronal circuits may be explored in the human motor cortex by double pulse transcranial magnetic stimulation (TMS). At short interstimulus intervals (2 5 ms), conditioned motor-evoked potentials (MEPs) are reduced (intracortical inhibition, ICI), whereas they are facilitated at longer interstimulus intervals (8 25 ms; intracortical facilitation, ICF). The aim of this study was to investigate the effects of homonymous and antagonist nerve stimulation on the intracortical inhibition and facilitation in the cortical areas that control the wrist extensor and flexor radialis muscles. 2. Sixteen subjects were asked to contract either their wrist extensor or flexor muscles. The MEP evoked by a test TMS (at 1.2 w MEP threshold) and recorded in the target muscle was then conditioned by subthreshold TMS (at 0.8 w MEP threshold) 2 and 14 ms before the test TMS. The median and radial nerves were stimulated at 0.8 w motor threshold (MT). 3. In both flexor and extensor muscles, antagonist nerve stimulation 40 ms before the test TMS decreased ICI and increased ICF. In contrast, homonymous nerve stimulation had no effect on ICI and ICF. 4. The intensity of the antagonist nerve stimulation required to alter ICI and ICF was as low as 0.6 w MT, which suggests that thick diameter afferents may be involved. The nerve stimulation had to be applied ms prior to the test TMS to alter significantly the intracortical excitability. 5. Cutaneous afferents were probably not responsible for the alterations of intracortical excitability, since cutaneous stimulation had no effect on either ICI or ICF at the investigated intervals. 6. The present data suggest that antagonist muscular afferent inputs may evoke reciprocal facilitation or disinhibition at the cortical level. This pattern of antagonist sensory afferent effects may be of significance for control of the wrist extensor and flexor muscles when used as synergists during manipulatory finger movements and gripping tasks. Sensory feedback is integrated with central motor commands at different levels of the central nervous system. The convergence of sensory afferents and descending pathways on common interneurones has been investigated in detail in the cat spinal cord during the past years (see Baldissera et al and Jankowska, 1992 for reviews). Non-invasive electrophysiological experiments in humans during the past 20 years have increased our understanding of how this convergence is used by the brain to integrate spinal reflex activity in the control of voluntary movements of both the lower (Nielsen, 1998; Pierrot-Deseilligny & Mazevet, 2000) and upper limbs (Meunier & Pierrot-Deseilligny, 1998; Aimonetti et al. 1999). It is well known that sensory feedback also reaches the motor cortex and influences the discharge of corticospinal cells (Lemon, 1981; Cheney & Fetz, 1984), which is the basis of so-called transcortical reflexes (see Marsden et al and Christensen et al for reviews). However, little is known about the details of how sensory activity influences cortical circuits and how the transcortical reflexes are integrated into the central motor commands at a cortical level. This question is also of importance in relation to the cortical reorganization and plasticity that has been documented after nerve injury (Merzenich et al. 1983; Donoghue et al. 1990), amputation (Ridding & Rothwell, 1995) or transient ischaemic limb deafferentation (Brasil- Neto et al. 1992, 1993). Among the various mechanisms responsible for these plastic changes in the cortical

2 892 J.-M. Aimonetti and J. B. Nielsen J. Physiol representation, Jacobs & Donoghue (1991) have suggested that the changes in the motor maps observed after nerve injury may result from changes in the excitability of intracortical inhibitory interneurones. It is possible in human subjects to study such cortical interneurones by a paired-pulse transcranial magnetic stimulation (TMS) technique introduced by Kujirai et al. (1993). With this technique conditioning TMS below the threshold for elicitation of a motor-evoked potential (MEP) may be shown to inhibit a MEP evoked by suprathreshold TMS at short intervals (1 5 ms) and to evoke a facilitation at longer intervals (8 25 ms). Such intracortical inhibition (ICI) and facilitation (ICF) are produced by separate populations of cortical interneurones (Ziemann et al. 1996). The paired-pulse technique thus opens the possibility of studying the influence of sensory feedback on cortical interneurones, in much the same way as the influence of sensory feedback on spinal interneurones has been studied in the past. A few studies have already used the TMS technique for this purpose. The relationship between changes in ICI and motor cortex plasticity has been investigated after lowerlimb amputation (Chen et al. 1998) and transient ischaemic limb deafferentation (Ziemann et al. 1998). In these studies, lack of sensory feedback was supposed to alter the intracortical excitability, which in turn may have contributed to the reorganization of the motor cortex. The relationship between sensory afferent activity and intracortical excitability was also addressed by Ridding & Rothwell (1999), who suggested that activation of digital skin afferents efficiently alters ICI and ICF in the cortical areas controlling finger muscles. In the present study we were interested in investigating the effects of homonymous and antagonist nerve stimulation on ICI and ICF in the areas controlling the wrist extensor and flexor radialis muscles. Our idea was that sensory feedback from the antagonistic muscles may differentially influence cortical interneurones projecting to the corticospinal cells controlling each of the muscles. Such a specific organization would facilitate the cortical control of hand motricity. Part of this study has been previously published in abstract form (Aimonetti & Nielsen, 2000). METHODS The experiments were performed on 16 healthy right-handed subjects including one of the authors (12 males and 4 females), aged years, with the approval of the local ethics committee (KF /98). All the subjects gave their informed written consent to the experimental procedure as required by the Declaration of Helsinki (1964). The subjects were seated in an adjustable armchair. Their right forearm was placed in a cushioned groove so as to ensure that the same position was adopted from one experiment to another, and the distal end of the forearm was immobilized in a U-shaped device, leaving the wrist joint free and maintaining the hand in a semi-prone position, flexed at an angle of 10 deg. The subjects were asked either to selectively activate their wrist extensor muscles by pushing with the back of their hand while keeping their fingers relaxed or to flex their wrist (Fig. 1A). In each motor task, the subjects had to maintain a tonic isometric contraction of about 5 % of the voluntary maximal contraction with the help of visual feedback in terms of rectified and integrated electromyographic activity from extensor and flexor muscles displayed on an oscilloscope. Data recording The EMG activity of the extensor carpi radialis (ECR) and flexor carpi radialis (FCR) muscles was recorded by means of bipolar surface electrodes (interelectrode distance, 2 cm). The amplified EMG signals were filtered (bandpass, 25 Hz to 10 khz), sampled at 2.5 khz, and stored on a PC for off-line analysis. Transcranial magnetic stimulation TMS was performed with a circular coil (diameter, 13 cm) using two magnetic stimulators (Magstim 200, Magstim Company, Dyfed, UK), which were connected through a Bistim device (Magstim Company). The coil was positioned over the hand area and the direction of the current in the coil was anticlockwise when viewed from above (side A). The threshold of the MEP was determined during voluntary activation of the investigated muscles as the lowest intensity of magnetic stimulation required to evoke a clearly identifiable EMG potential with similar shape and latency in 5 out of 10 consecutive trials. The intensity of the magnetic stimulus was expressed as a percentage of the maximal stimulator output. In order to induce intracortical inhibition and facilitation, a subthreshold conditioning stimulation set at 80 % of the active MEP threshold was applied at intervals from 2 to 14 ms before the test stimulation set at 120 % of the active MEP threshold (Kujirai et al. 1993). A time course of the effect of subthreshold TMS on the test MEP was obtained at the beginning of each experiment (as illustrated from one subject in Fig. 1B). In all subjects ICI was evaluated at an interval of 2 ms between the conditioning subthreshold TMS and the test TMS. ICF was evaluated at an interval of 14 ms. Each series of recordings consisted of a pseudo-random presentation of the following seven alternatives (Fig. 1C). Test and conditioning TMS were applied alone in alternatives 1 and 2, respectively. Conditioning TMS was applied 2 and 14 ms before the test TMS in alternatives 3 and 4, respectively. Nerve stimulation was applied from 27 to 60 ms before the test TMS in alternatives 5 7. In alternatives 5 and 6 conditioning TMS was applied in addition at intervals of 2 and 14 ms, respectively. In alternative 7 the peripheral nerve stimulation was applied from 27 to 60 ms before the test TMS alone. In pilot experiments performed in five subjects, peripheral nerve stimulation was also applied at intervals from 16 to 30 ms before the test TMS (see Bertolasi et al. 1998). Since no significant effects on ICI or ICF were observed at these short intervals, they were not investigated in the main part of the study. In all experiments the peak-to-peak size of the MEP was measured and expressed as a percentage of the maximal M wave (M max ) evoked by supramaximal stimulation of the nerves supplying the respective muscles. It was checked in control experiments that measurement of the area of the MEPs did not change the results. Conditioned MEPs were expressed as percentages of the mean control MEP evoked by the test TMS alone (Fig. 1B). ICI and ICF were then expressed as a ratio of the conditioned MEPs over the mean test MEP. For each alternative, 10 MEPs were averaged. Stimuli were applied every 4 s. Nerve stimulation The radial nerve was electrically stimulated using a pair of spherical electrodes. The anode and cathode (cathode proximal) were placed

3 J. Physiol Human cortical excitability and sensory input cm apart 8 10 cm above the elbow on the external side of the upper arm (Fig. 1A). The median nerve was electrically stimulated using another pair of identical spherical electrodes. The anode and cathode were placed 2 cm apart on the medial aspect of the arm just above the elbow (Fig. 1A). The intensity of the radial and median nerve stimulation (1 ms duration) was set at 0.8 w motor threshold (MT). In most of the experiments, the nerve stimulation was applied 40 ms before the test TMS, since this delay was found in pilot experiments to alter ICI and ICF most efficiently. In order to investigate the effect of cutaneous stimulation on ICI and ICF, the superficial radial nerve and the median nerve at the wrist were stimulated using two adhesive-band electrodes (Blue Sensor A-10-N, Medicotest, Denmark). The superficial radial nerve was stimulated just proximal to the wrist joint on the radial part of the extensor side of the arm. The median nerve was stimulated just proximal to the wrist joint in the midline on the flexor side of the arm. Single electrical stimuli (100 µs duration) were applied at an intensity of 5 w perceptual threshold (Fig. 1A). It was checked in all subjects that there was no visible or palpable contraction of any muscles in the hand at this stimulation intensity. Activity in cutaneous afferents was also evoked by a mechanical device in four subjects (see Aimonetti et al. 2000). The skin of the palm and fingertips was lightly brushed throughout the recording session using a soft rotative brush (40 rotations per minute), while the subjects were performing wrist extension or flexion (Fig. 1A). In these recordings, no nerve stimulation was applied. Statistical analysis The effects of conditioning TMS, nerve stimulation or mechanical cutaneous stimulation on the size of the test MEPs were examined in each subject using an ANOVA procedure for repeated measures. Tukey s tests were used for post hoc analysis. The significance level was set at P<0.05. RESULTS Figure 1B illustrates the effects of sub-threshold TMS on the amplitude of a test MEP (mean ± S.E.M., 9.4 ± 0.8 % of M max ) recorded in the ECR muscles of one subject. In keeping with the results of previous studies (Kujirai et al. 1993; Di Larazzo et al. 1998; Liepert et al. 1998), the depression of the conditioned MEP at short interstimulus intervals can be taken to reflect ICI, whereas the facilitation at longer interstimulus intervals can be taken to reflect ICF. In all 15 tested subjects significant ICI was demonstrated for the ECR muscle, whereas significant ICF was observed Figure 1. Demonstration of intracortical inhibition (ICI) and intracortical facilitation (ICF) by paired-pulse TMS technique A, experimental set-up. B, time course of the effect of subthreshold TMS (0.8 w MEP threshold) on MEPs evoked in the ECR muscle by suprathreshold TMS (1.2 w MEP threshold). The abscissa is the conditioning test interval (in ms) and the ordinate is the size of the conditioned MEP as a percentage of the control MEP size. Asterisks designate that the conditioned MEP was significantly different from the control MEP with a P value below Each error bar is one standard error of the mean. The data are from a single subject. C, description of the different alternatives used. The different alternatives combined test and conditioning (Cond.) TMS (conditioning test interval of 2 and 14 ms) with and without different nerve stimulations applied from 27 to 60 ms prior the test TMS.

4 894 J.-M. Aimonetti and J. B. Nielsen J. Physiol in only 10 subjects. For the FCR muscle significant ICI was demonstrated in all 14 tested subjects. Significant ICF was found in nine of the subjects. The effects of antagonist and homonymous nerve stimulation on intracortical excitability Figure 2 shows the effect of median nerve stimulation on ICI and ICF estimated from the ECR MEP in a single subject. When applied 40 ms before the test TMS (10.2 ± 0.9 % of M max ), median nerve stimulation had no effect on the test MEP (Fig. 2A and D). Nevertheless, ICI (measured at an interval of 2 ms between conditioning TMS and test TMS) was strongly decreased (from 57 to 79 % of the control MEP; P=0.007; Fig. 2B and E) and ICF (measured at an interval of 14 ms between conditioning TMS and test TMS) was strongly increased (from 121 to 161 % of the control MEP; P=0.02; Fig. 2C and F). Similar experiments were done in 14 subjects for the ECR muscle and 14 subjects for the FCR muscle. In all experiments the effect of both the radial and median nerve stimulation on ICI and ICF was tested. All the data in Fig. 3 were obtained with an interval of 40 ms between the conditioning peripheral nerve stimulation and TMS. The intensity of the peripheral nerve stimulation was 0.8 w MT. Data from the ECR muscle are shown in Fig. 3A D, whereas Fig. 3E H shows data from the FCR muscle. For the ECR muscle, the mean test MEP amplitude was 10.5 ± 0.8 % of M max. The antagonistic median nerve stimulation did not alter the mean test MEP amplitude (10.7 ± 0.9 % of M max ) but significantly decreased ICI (from 62 ± 10 to 85 ± 12 % of the control MEP; P<0.001; Fig. 3A) and increased ICF (from 117 ± 17 to 144 ± 19 % of the control MEP; P=0.006; Fig. 3C). The stimulation of the homonymous radial nerve did not alter the mean test MEP amplitude (10.8 ± 0.7 % of M max ) and had no significant effect on either ICI (from 62 ± 10 to 58 ± 6 % of the control MEP; Fig. 3B) or ICF (from 117 ± 17 to 112 ± 22 % of the control MEP; Fig. 3D). For the FCR muscle, the mean test MEP amplitude was 11.2 ± 0.9 % of M max. Stimulation of the antagonist nerve (in this case the radial nerve) did not alter the mean test MEP amplitude (11.5 ± 1.0 % of M max ), but decreased ICI (from 56 ± 9 to 80 ± 20 %; P= 0.007; Fig. 3E) and increased ICF (from 112 ± 18 to 134 ± 15 %, P= 0.02; Fig. 3G). The stimulation of the homonymous nerve (in this case the median nerve) did not alter the mean test MEP amplitude (11.6 ± 1.2 % of M max ), but tended to increase ICI (from 56 ± 9 to 46 ± 14 %; not significant; Fig. 3F) and decrease ICF (from 112 ± 18 to 98 ± 26 %; not significant; Fig. 3H). It was checked for all combinations of peripheral nerve stimulation and MEP that the peripheral nerve stimuli had no effect on the test MEPs regardless of their size. The effect of peripheral nerve stimulation on ICI and Figure 2. Effect of median nerve stimulation on ICI and ICF estimated from the ECR MEP The data are from a single subject and show averaged traces of ECR EMG (n = 10) following different combinations of TMS and median nerve stimulation. A C show the ECR MEP in the control situation without conditioning stimuli (A), with conditioning subthreshold TMS at an interval of 2 ms (B) and with conditioning subthreshold TMS at an interval of 14 ms (C). In D F median nerve stimulation was applied 40 ms before TMS, but otherwise the three traces represent the same combinations of TMS as A C. The intensity of the median nerve stimulation was 0.8 w MT. The values to the right of each trace give the size of the MEP recorded in that situation as a percentage of the control MEP (in A).

5 J. Physiol Human cortical excitability and sensory input 895 ICF thus could not be explained simply by the fact that the MEPs were smaller and larger than the unconditioned MEPs. Threshold of the effect of peripheral nerve stimulation on ICI and ICF In eight subjects the intensity of the peripheral nerve stimulation was varied in order to determine the threshold of the effect of the stimulation on ICI and ICF. The interval between the peripheral nerve stimulation and TMS was kept constant at 40 ms in these experiments. Data for the ECR muscle from one subject are illustrated in Fig. 4A and B. Subthreshold TMS depressed the MEP to around 40 % of its control value (12.5 ± 1.2 % of M max ) at an interval of 2 ms (Fig. 4A; open columns) and produced a weak facilitation of the MEP at an interval of 14 ms (Fig. 4B, open columns). Stimulation of either the homonymous radial nerve (Fig. 4A and B; grey columns) or the antagonistic median nerve (Fig. 4A and B; black columns) at intensities below 0.6 w MT had no effect on ICI or ICF. However, at intensities above 0.5 w MT median nerve stimulation consistently decreased ICI and increased ICF, whereas radial nerve stimulation increased ICI and tended to decrease ICF (although this only reached a significant level at 0.8 w MT). A similar low threshold of the effect of peripheral nerve stimulation on ICI and ICF was also found in the other tested subjects (except that in the other subjects stimulation of the radial nerve had no effect on ICI and ICF in the ECR muscle). Data for the FCR muscle from the same subject are illustrated in Fig. 4C and D. Subthreshold TMS depressed the MEP to around 60 % of its control value (13.2 ± 1.6 % of M max ) at an interval of 2 ms (Fig. 4C; open columns) and produced a weak facilitation of the MEP to around 115 % of its control value at an interval of 14 ms (Fig. 4D; open columns). Stimulation of either the homonymous median nerve (Fig. 4C and D; black columns) or the antagonistic radial nerve (Fig. 4C and D; grey columns) at intensities below 0.6 w MT had no effect on ICI or ICF. However, at intensities above 0.5 w MT radial nerve stimulation consistently decreased ICI and increased ICF, whereas median nerve stimulation had no significant effect on ICI and ICF. The decrease of ICI and increase of ICF induced by radial nerve stimulation were found to have a similarly low threshold in all eight tested subjects. In three of the subjects median nerve stimulation increased ICI and decreased ICF in the FCR muscle. This effect also had a threshold around 0.7 w MT. Figure 3. Effect of median and radial nerve stimulation on ICI and ICF measured from ECR and FCR MEPs in all tested subjects The graphs give the effect of median (A,C, F and H) and radial (B, D, E and G) stimulation on ICI (A, B, E and F) and ICF (C, D, G and H) estimated from the ECR (A D) and FCR (E H) MEPs. A comparison is made of the size of ICI or ICF with (right-hand side of graph) and without peripheral nerve stimulation (left-hand side of graph). Each small circle (extensor MEPs) or square (flexor MEPs) and thin line represents one subject. In addition the mean size of ICI and ICF with and without peripheral nerve stimulation is shown as large circles or squares and thick lines in each of the graphs. In all experiments ICI was estimated at an interval of 2 ms between conditioning and test TMS, whereas ICF was measured at an interval of 14 ms. The interval between peripheral nerve stimulation and TMS was 40 ms. The intensity of peripheral nerve stimulation was 0.8 w MT. ns, not significant.

6 896 J.-M. Aimonetti and J. B. Nielsen J. Physiol Figure 4. Threshold of the effect of peripheral nerve stimulation on ICI and ICF estimated from the extensor and flexor MEPs The data are from a single subject. A and B show the effect of peripheral nerve stimulation on ICI and ICF, respectively, estimated from changes in the amplitude of extensor MEPs. C and D show the effect of peripheral nerve stimulation on ICI and ICF, respectively, estimated from changes in the amplitude of flexor MEPs. Open columns show the size of ICI or ICF in the control situation without peripheral nerve stimulation, whereas grey columns show the effect of radial nerve stimulation and black columns the effect of median nerve stimulation. The intensity of the peripheral nerve stimulation was varied from 0.5 to 1.0 w MT. ICI was measured at an interval of 2 ms between the conditioning subthreshold TMS and the test TMS and ICF was measured at an interval of 14 ms. The interval between the peripheral nerve stimulation and TMS was 40 ms in all cases. The ordinate is the size of the conditioned MEP as a percentage of the control MEP size. Each error bar is one standard error of the mean. The asterisks designate statistical significance at P<0.05.

7 J. Physiol Human cortical excitability and sensory input 897 Time course of the effect of peripheral nerve stimulation on ICI and ICF In nine subjects the time course of the effect of median nerve stimulation on ICI and ICF measured in the ECR muscle was investigated. Data from one of the subjects are illustrated in Fig. 5. At an interval of 2 ms, subthreshold TMS depressed the test MEP to between 40 and 60 % of its control value (Fig. 5A, open circles). Median nerve stimulation significantly decreased this inhibition when it was applied ms before TMS, but had no effect at earlier or later conditioning test intervals (Fig. 5A; filled circles). For the group of nine subjects, the onset of the effect on ICI in the ECR muscle of median nerve stimulation varied between 33 and 36 ms. The offset ranged from 44 to 48 ms. In the same subject as in Fig. 5A, subthreshold TMS increased the test MEP at an interval of 14 ms to between 115 and 130 % of its control value (Fig. 5B, open circles). Median nerve stimulation applied ms before the test TMS significantly increased this ICF, but had no effect at shorter or longer conditioning test intervals (Fig. 5B; filled circles). In the six subjects in whom this could be tested, the onset of the effect on ICF in the ECR Figure 5. Time course of the effect of median nerve stimulation on ICI and ICF estimated from the extensor MEPs The data are from a single subject. A, ICI was measured at an interval of 2 ms between the conditioning subthreshold and the test TMS. B, ICF was measured at an interval of 14 ms between the conditioning subthreshold and the test TMS. Open circles show the size of the conditioned MEP without peripheral nerve stimulation whereas filled circles show the size when preceded by median nerve stimulation at 0.8 w MT. The ordinate is the size of the conditioned MEP expressed as a percentage of the control MEP and the abscissa is the interval between the nerve stimulation and the test TMS. Each error bar is one standard error of the mean. The asterisks indicate statistical significance at P<0.05.

8 898 J.-M. Aimonetti and J. B. Nielsen J. Physiol muscle of median nerve stimulation varied between 34 and 36 ms. The offset ranged from 44 to 48 ms. Time courses of the effect of radial nerve stimulation on ICI in the FCR muscle were obtained in seven subjects. In these subjects the onset of the effect ranged from 32 to 36 ms, whereas the offset ranged from 42 to 46 ms. In the four subjects in whom the effect of radial nerve stimulation on ICF in the FCR muscle was investigated, the onset of the increase of ICF ranged from 34 to 36 ms, whereas the offset ranged from 43 to 46 ms. In none of the subjects were significant effects of stimulation of the radial nerve on ICI or ICF found at shorter latencies. Effect of stimulation of cutaneous afferents on ICI and ICF In order to determine whether the effects of the median and radial nerve stimulation could have been caused by skin afferents, stimuli mimicking the cutaneous sensation evoked by the median and radial nerve stimuli, but without activating muscle afferents, were applied. Cutaneous afferents were activated by brushing of the palmar side of the hand (as illustrated for one subject in Fig. 6A F), stimulation of the median nerve at the wrist (as illustrated for the same subject in Fig. 6G L), and stimulation of the superficial radial nerve (data not shown). Brushing of the palm. In Fig. 6, ICI and ICF were tested in the ECR muscle. Subthreshold TMS depressed the MEP to 75 % of its control value (9.2 ± 0.8 % of M max ) at an interval of 2 ms (Fig. 6B), and facilitated it to 130 % at an interval of 14 ms (Fig. 6C). There was no change in the size of the test MEP (Fig. 6D), ICI (Fig. 6E) or ICF (Fig. 6F) when the skin of the palmar side of the hand was concurrently brushed, although this produced a cutaneous sensation that was much stronger than that induced by the median nerve stimulation. In none of the four subjects tested was a change in the size of ICI or ICF observed when brushing the palmar side of the hand. Stimulation of the median nerve at the wrist. The interval between the median nerve stimulation and TMS was 45 ms in the illustrated recording (Fig. 6G L) in order to take the extra conduction distance from the wrist into account. The intensity of the stimulation was adjusted to evoke a cutaneous sensation similar to that evoked by median nerve stimulation at the elbow. In none of the five subjects tested, including the one used for the illustration in Fig. 6G L, did this stimulation produce any significant effect on the test MEP (compare Fig. 6G, 9.6 ± 0.9 % of M max ; and J, 8.8 ± 0.7 % of M max ), ICI (compare Fig. 6H and K) or ICF (compare Fig. 6I and L). Intervals between median nerve stimulation and test TMS ranging from 30 to 55 ms were investigated. Stimulation of the superficial radial nerve. In separate experiments on seven subjects, ICI and ICF were tested in the FCR muscle and conditioning stimuli were applied to the superficial radial nerve to mimic the cutaneous sensation evoked by stimulation of the radial nerve at the elbow. Intervals between superficial radial Figure 6. Effect of cutaneous stimulation on ICI and ICF estimated from the ECR MEP The data are from a single subject. The figure is arranged in the same way as Fig. 2. Upper traces show the test MEP alone (A and G) and with brushing of the hand (D) or stimulation of the median nerve at the wrist (J). The middle traces show the test MEP when conditioned by subthreshold TMS at a 2 ms interval alone (B and H) and in combination with brushing of the hand (E) or stimulation of the median nerve at the wrist (K). The lower traces show the test MEP when conditioned by subthreshold TMS at an interval of 14 ms alone (C and I) and in combination with hand brushing (F) or median nerve stimulation at the wrist (L). The cutaneous stimulation of the palmar side of the hand was applied by a rotating brush. The intensity of the median nerve stimulation was 5 w perception threshold. The stimulation was applied 45 ms prior to TMS. All traces are the mean of 10 stimuli.

9 J. Physiol Human cortical excitability and sensory input 899 nerve stimulation and test TMS ranging from 30 to 55 ms were investigated. In five of the seven subjects the superficial radial nerve stimulation had no effect on either ICI or ICF, although stimulation of the radial nerve at the elbow significantly decreased ICI and increased ICF. However, in the remaining two subjects, the superficial radial nerve stimulation decreased ICI (0.003 < P<0.02) and increased ICF (0.01 < P<0.04). In these two subjects, the superficial radial nerve stimulation also significantly increased the size of the test MEPs (0.004 < P<0.01). This may explain the changes in ICI and ICF, since MEPs of different amplitude may have different sensitivity to inhibition and facilitation. Effect of antagonist nerve stimulation on wrist flexor and extensor EMG activity As already described, stimulation of the radial and median nerve had no effect on the ECR and FCR MEPs at the intervals investigated in the present study. This might suggest that the changes in ICI and ICF induced by peripheral nerve stimulation did not alter the output from the motor cortex. However, the MEP may not be sufficiently sensitive to detect this and may reflect activation of slightly different corticospinal neurones to those used in relation to voluntary movement. In seven subjects we therefore also investigated whether peripheral nerve stimulation induced any changes in the FCR and ECR EMG activity at an interval corresponding to the changes in ICI and ICF. Figure 7 illustrates data obtained from the ECR muscle in the subject who was also used for the illustration in Fig. 5. As shown in Fig. 5 the changes in ICI and ICF induced by median nerve stimulation were found at an interval between nerve stimulation and test TMS of 35 to 45 ms in this subject. The latency of the test MEP was 16 ms (Fig. 7A, 11.5 ± 1.2 % of M max ), which gives an estimate of the fastest conduction time from the motor cortex to the muscle. If the changes in ICI and ICF did result in changes of the output from the motor cortex, a facilitation would be expected at 51 ms ( ms) after the median nerve stimulation. Indeed, at an interval from 51 to 60 ms, a short-lasting facilitation of the rectified extensor EMG activity was observed (Fig. 7B). The facilitation was preceded and followed by inhibition. Despite the effects on the mean rectified extensor EMG activity, the median nerve did not significantly alter the size of the test MEP (Fig. 7C). In the six other subjects, Figure 7. Effect of median nerve stimulation on the ECR EMG activity The data are from the same subject as in Fig. 5. A, extensor MEP evoked by TMS (1.2 w MEP threshold). The trace is the mean of 10 stimuli. B, rectified extensor EMG activity following median nerve stimulation. The trace is the mean of 100 stimuli. C, the effects of the same median nerve stimulation on the extensor MEP. The latency of the MEP has been added to the interval between median nerve stimulation and the test TMS, so that the intervals correspond to the effects of the stimulation in the EMG (B). The size of the MEP conditioned by median nerve stimulation is expressed as a percentage of the control MEP size. Each error bar is one standard error of the mean.

10 900 J.-M. Aimonetti and J. B. Nielsen J. Physiol median nerve stimulation also induced a similar facilitation of the extensor EMG activity at a latency corresponding to that of the changes in ICI and ICF. Radial nerve stimulation also induced a similar facilitation of the flexor EMG activity in all seven subjects. The latency of this facilitation corresponded to the changes in ICI and ICF induced by radial nerve stimulation when adding the latency of the MEP. We also investigated the effect of homonymous nerve stimulation on the voluntary EMG activity in the FCR and ECR muscles. In all subjects only a short latency reflex burst (H-reflex) followed by a silent period was observed. Whether the changes in ICI and ICF induced by homonymous nerve stimulation had any effect on the EMG activities therefore could not be evaluated. DISCUSSION In the present study we investigated the effect of peripheral nerve stimulation on cortical excitability in the areas that control the wrist extensor and flexor radialis muscles. Methodological considerations The arguments suggesting that the depression of a test MEP by previous subthreshold TMS at a short interval reflects activation of a population of cortical interneurones that exerts intracortical inhibition on the corticospinal cells, whereas the facilitation seen at longer latencies reflects activation of a separate population of interneurones with excitatory effects on the cortical output neurones, have been presented previously (Kujirai et al. 1993; Ridding et al. 1995; Di Lazzaro et al. 1998; Abbruzzese et al. 1999; Ridding & Rothwell, 1999) and will not be discussed further here. However, one methodological question that needs to be addressed is whether the changes in ICI and ICF that we observed following the peripheral nerve stimuli were caused by changes in the excitability of the interneurones responsible for ICI and ICF or whether some other factor related to the way that the inhibition and facilitation were evaluated could be involved. Little is known about methodological problems related to the use of the MEP to test the effect of conditioning stimuli, but it may be assumed that the problems that are related to the use of the H-reflex also apply to the MEP. For the H-reflex it has been demonstrated that inhibitory and facilitatory effects are sensitive to changes in the size of the control H-reflex (Crone et al. 1990). This is explained by intrinsic properties in the motoneuronal pool and the distribution of the synaptic input. The implication is that it is necessary to maintain a constant size of the H-reflex when comparing the size of inhibitory or facilitatory effects in two different situations (Crone et al. 1990). In all likelihood this also applies to the MEP and it was therefore of importance that the peripheral nerve stimuli had no effect on the MEP at the intervals investigated. Changes in the MEP size induced by peripheral nerve stimulation therefore could not explain the changes in ICI and ICF. Mechanism for the decrease of ICI and increase of ICF induced by antagonist nerve stimulation Bertolasi et al. (1998) reported that median nerve stimulation depressed the ECR MEP at an interval of ms and suggested that this reflected reciprocal inhibition at a cortical level similar to the well-known spinal reciprocal inhibition (Crone & Nielsen, 1994). Our initial expectation was therefore that stimulation of the median nerve would increase ICI and decrease ICF at a short latency, as this would provide a possible explanation of the findings by Bertolasi et al. (1998). However, we saw the opposite: median nerve stimulation decreased ICI and increased ICF measured by the ECR MEP. Radial nerve stimulation likewise decreased ICI and increased ICF measured by the FCR MEP. Instead of changes in the excitability of the interneuronal populations responsible for ICI and ICF, which would fit with reciprocal inhibition, we observed changes that would fit better with reciprocal excitation. Furthermore, the latency of these changes was much greater than that of the changes in the ECR MEP described by Bertolasi et al. (1998). The changes that they observed had a latency only slightly greater than the latency of the somatosensory evoked potential induced by median nerve stimulation (around 16 ms), suggesting that a fast and rather direct pathway was responsible for the effects that they observed. We also tested such short intervals as part of the pilot experiments performed at the beginning of the present study, but did not observe any changes in ICI or ICF. The earliest changes in ICI and ICF that we could consistently evoke by peripheral nerve stimulation had latencies of around 33 ms, i.e. around 17 ms later than the arrival of the fastest conducting afferent volley at a cortical level. This suggests that the changes in ICI and ICF that we have observed must be caused by a slow conducting afferent pathway and/or transmission through several interneuronal relays before reaching the two populations of cortical interneurones. The latter of these two possibilities seems the most likely, since the effects on ICI and ICF by the peripheral nerve stimuli had a low threshold, which suggests that large diameter afferents with high conduction velocity were involved. At the present time we have no way of saying which cortical circuits are involved in this long processing time, but it does suggest that the effects that we have observed do not reflect activation of a simple reflex system where the sensory input is relatively closely linked to the output. Our observation that cutaneous stimulation had no effect on ICI or ICF, whereas stimulation of low-threshold muscular afferents (probably group I afferents) did, is of interest in relation to the study by Ridding & Rothwell (1999). They demonstrated that stimulation of cutaneous digital nerves decreased ICI and increased ICF measured

11 J. Physiol Human cortical excitability and sensory input 901 by the MEP in the first dorsal interosseous muscle. The latency of this effect was in the same range as the effects observed in our study. The most reasonable explanation for this is that it reflects a difference in the cortical control of the wrist muscles investigated in our study and the distal finger muscle investigated by Ridding & Rothwell (1999). However, confirmation of this is necessary in a separate study where the effects of muscular and cutaneous afferents on ICI and ICF in the cortical representation of proximal and distal muscles are compared. Functional considerations The lack of effect of peripheral nerve stimulation on the MEP at the investigated intervals is on the one hand a methodological advantage as already discussed, but on the other hand it also raises some questions in relation to the functional significance of the findings. If the changes in ICI and ICF do not produce any changes in the cortical output as measured from the MEP, do they then play any role in the cortical control of the muscles? We did find a facilitation of EMG activity following antagonist nerve stimulation, which would fit in latency with the decrease of ICI and increase of ICF. This may suggest that the changes in ICI and ICF, despite the lack of changes in MEP, nevertheless influenced the output from the motor cortex. The reason why this was not observed as a change in the MEP size may be that the MEP reflects partly different corticospinal cells to those activated in relation to voluntary contraction of the wrist muscles. Alternatively, the sensitivity of the MEP may be insufficient to detect changes in motor cortical output demonstrated by averaging of the background EMG activity. There is thus a possibility that the changes in ICI and ICF could have produced direct changes in the motor cortical output and that they could thus be part of the mechanism underlying transcortical reflexes (Marsden et al. 1983; Cheney & Fetz, 1984; Deuschl et al. 1991; Christensen et al. 2000). In relation to this it is of importance that Cheney & Fetz (1984) observed in the monkey that corticospinal cells could be activated by stretching of the wrist muscles in both the extension and flexion direction and that (transcortical) reflex bursts were seen in both agonist and antagonist muscles. They suggested that this could be of importance for regulation of stiffness across the ankle joint by eliciting cocontraction of the antagonist muscles. The reciprocal excitatory pattern that we observed in the present study may serve a similar purpose. 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