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Changes in motor cortical excitability induced by paired associative stimulation
Clinical Neurophysiology 114 (2003) 1437–1444 www.elsevier.com/locate/clinph
Changes in motor cortical excitability induced by paired associative stimulation M.C. Ridding*, J. Uy Department of Physiology, University of Adelaide, Adelaide 5005, Australia Accepted 27 March 2003
Abstract Objective: Changes in afferent input have been shown to be capable of inducing reorganisations of motor cortex in humans. Using TCMS we examined the efficacy of a new associative afferent stimulation paradigm in inducing motor cortical reorganisation in humans. Methods: Using TCMS, stimulus response curves were constructed before and following a 1 h period of associative stimulation of two muscles motor points. The effect of an asynchronous peripheral stimulation paradigm was investigated in a separate series of control experiments. Results: One hour of associative stimulation of two muscles motor points resulted in a significant increase in the excitability of the corticospinal projection to those stimulated muscles. The increase in excitability peaked 1 h following the stimulation period. This increase in excitability did not generalise to either adjacent or more remote muscles. The control stimulation paradigm produced no significant change in corticospinal excitability. Conclusions: These results confirm the importance of associative input for the induction of plasticity in the human motor cortex. Significance: The findings reported here further elucidate the role of afferent input in motor cortical reorganisation. These findings have implications for our understanding of the mechanisms of motor learning and may also be relevant to the design of new afferent stimulation therapies. q 2003 International Federation of Clinical Neurophysiology. Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Afferent stimulation; Magnetic stimulation; Plasticity; Associative input; Human
1. Introduction Recently it has been demonstrated that stimulation of either peripheral nerves (Ridding et al., 2001), peripheral nerve and motor cortex (Stefan et al., 2000), or muscle motor point and motor cortex (McKay et al., 2002a) is capable of inducing reorganisations within the human motor cortex. The reorganisations are manifested as an increase in the excitability of the corticospinal projection to stimulated muscles (Ridding et al., 2000) and an increase in the cortical representational of stimulated muscles (McKay et al., 2002a; Ridding et al., 2001). These studies demonstrate the dynamic nature of the motor cortex organisation and the crucial role that afferent input has in modulating the organisation. * Corresponding author. Tel.: þ 61-8-8303-6433; fax: þ61-8-8303-3356. E-mail address: [email protected] (M.C. Ridding).
It is thought that the organisational changes induced by modifying afferent input are similar in nature to those seen during motor learning and there are several recent reports of afferent stimulation paradigms being employed to induce therapeutically relevant plasticity (Conforto et al., 2002; Fraser et al., 2002). Given the obvious therapeutic potential of this approach, optimisation of the afferent stimulation techniques is of great importance. All of these previous techniques apply continuous or short trains of regular stimuli to a variety of peripheral and central sites. Evidence exists to suggest further refinement of the afferent stimulation techniques may be possible. For example, it has been demonstrated that correlated inputs are very important for the induction of plasticity (Clark et al., 1988; Godde et al., 1996). Also, it may be important to present stimuli with a randomised timing between successive pairs to minimise habituation and adaptation effects (Godde et al., 1996). In the present study we investigated the efficacy of a modified
1388-2457/03/$30.00 q 2003 International Federation of Clinical Neurophysiology. Published by Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S1388-2457(03)00115-9
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afferent stimulation paradigm, designed to address these issues, in inducing motor cortex plasticity. This protocol was based on that reported by Godde et al. (1996).
2. Methods Fourteen normal subjects (3 female, 11 male; age range 20 –39 years) volunteered for the main experiments. All subjects were right-handed (assessed by the Edinburgh Handedness Inventory). The experimental protocols were approved by the local Human Research Ethics Committee and subjects gave written, informed consent. 2.1. Electromyographic recording The surface electromyogram was recorded from the following muscles in the right upper limb: first dorsal interosseus (FDI), abductor pollicis brevis (APB), abductor digiti minimus (ADM) and extensor carpi radialis (ECR). For FDI, the reference electrode was over the metacarpophalangeal joint of the index finger and the active electrode over the motor point; for APB the reference was over the metacarpophalangeal joint of the thumb and the active electrode over the motor point; for ADM the reference electrode was over the metacarpophalangeal joint of the little finger and the active electrode over the muscle belly; and for ECR the active electrode was placed over the muscle belly and the reference electrode was placed approximately 3 cm distal to this. Recordings were made with silver/silver chloride surface electrodes. The signals were amplified in the bandwidth 20 Hz to 1 kHz, sampled at 5 kHz and fed to a computer for off-line analysis. 2.2. Transcranial magnetic stimulation Transcranial magnetic stimulation (TMS) was delivered with a Magstim 200 magnetic stimulator (Magstim Co., Dyfed, UK). A round coil with an external diameter of 13 cm was used. The optimal position for evoking responses in the right FDI muscle was established and marked on the scalp to ensure reliable coil placement between trials. This coil placement was employed for all recordings. The coil was used with the ‘A’ side up. This is the optimal orientation for evoking responses in the right hand and results in a posterior to anterior current flow in the underlying cortex.
Signal software (Cambridge Electronic Design Ltd, Cambridge, UK) the interval between successive TMS pulses was randomly varied in the range 5 –6 s. The motor evoked potential (MEP) was recorded for each of the muscles following stimulation and the average peak-to-peak amplitude calculated for each intensity step. MEPs were recorded with all of the muscles relaxed. Any trials in which background EMG activity was present were excluded from analysis. Muscle relaxation was monitored by giving subjects visual feedback of their EMG on a high gain oscilloscope. 2.4. Test afferent stimulation paradigm The afferent stimulation paradigm employed was based on that described by Godde et al. (1996). Using surface electrodes, square-wave electrical stimuli of 1 ms duration were applied to the FDI and APB muscles simultaneously (Digitimer DS7A stimulators, Digitimer Ltd, Welwyn Garden City, UK). The timing between successive pairs of stimuli was randomised between 0.15 and 2.85 s in 8 steps (range 0.35 – 6.7 Hz). The timing of the inter-pulse intervals was performed using Signal software (Cambridge Electronic Design Ltd, UK). The intensity of stimulation was adjusted for each muscle and set at a level just sufficient to evoke a minimal visible motor response (range 10 –30 mA). This intensity of stimulation was non-painful for all subjects. The afferent stimulation paradigm was applied for a period of 1 h. This stimulation paradigm will be described as associative stimulation in the following text. 2.5. Control afferent stimulation paradigm A control afferent stimulation paradigm was also employed. This was similar to the associative stimulation paradigm. However in this control paradigm stimuli were never presented to the two stimulated muscles at the same time. Therefore, the two muscles received the same number of stimuli at the same rate as in the associative stimulation paradigm but the muscles never received stimuli synchronously. Therefore, the control paradigm was designed to test the relative importance of associating the two afferent inputs in the test condition. This stimulation paradigm will be described as non-associative stimulation in the following text. 2.6. Experimental design
2.3. Stimulus response curves Stimulus response curves were recorded (Ridding and Rothwell, 1997) for the 4 muscles. The intensity range investigated was from 20 to 80% of maximum stimulator output in steps of 5% (of stimulator output). In all subjects this stimulus range included intensity values from below motor threshold to suprathreshold intensities. Five stimuli were applied at each intensity step in this range. Using
Thirteen subjects were tested with both the associative and non-associative afferent stimulation paradigms. The order in which subjects received the two paradigms was randomised, and subjects were unaware of the experimental hypothesis. For all subjects testing sessions were separated by at least 1 week. Baseline stimulus response curves were recorded as described above. Subjects were then given either a 1 h period of the associative or non-associative
M.C. Ridding, J. Uy / Clinical Neurophysiology 114 (2003) 1437–1444
stimulation. Immediately following the stimulation period stimulus response curves were recorded again and then repeated at 15 min, 1 and 2 h (only 7 subjects had stimulus response curves recorded at the 2 h timing). In an attempt to control for the influence of cognitive/attentional factors in the induction of plasticity we (1) performed the experiments in a quiet environment away from distractions, and (2) repeatedly encouraged our subjects ‘pay attention to the stimulus’. 2.7. Site of excitability change – control experiment In order to investigate the site at which any excitability changes took place a further subject (right-handed male aged 35 years) was studied using both TMS and transcranial electrical stimulation (TES) before and following a period of associative stimulation. TES was applied using silver/ silver chloride electrodes attached to the scalp. The anode was placed over the hand area of the left motor cortex (6 cm lateral to the vertex) and the cathode placed over the vertex. In the baseline condition the intensity of both the TES and TMS was adjusted to produce MEPs of approximately 1 mV (peak-to-peak amplitude). MEPs following 10 magnetic stimuli and 5 electrical stimuli were then recorded. The order of presentation of electrical and magnetic stimuli was randomised during the trial so that the subject did not know what type of stimulus to expect. Following the baseline measurements a 1 h period of associative stimulation was applied to the subject (as described above). Immediately following the associative stimulation period MEPs were once again recorded following TES and TMS. 2.8. Analysis Data for the associative and non-associative paradigm experiments were analysed separately but in a similar manner. A repeated measures analysis of variance (ANOVA) was performed with main factors of muscle (4 levels), time (5 levels) and intensity (13 levels). Where there were significant muscle effects, further ANOVAs were performed for each muscle, with factors of time (5 levels) and intensity (13 levels). Where there were significant time – intensity interactions a further planned analysis was performed to determine which response curves were significantly different to baseline; where appropriate corrections for multiple comparisons were made. For the site of excitability control experiment, responses to TMS and TES were compared using paired sample t tests.
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were no significant differences between the muscles (muscle, P . 0:05). Analysis of individual muscle data revealed that, for all muscles, post-intervention response curves were not significantly different from baseline curves (ANOVA; time and time –intensity interaction, P . 0:05 for all muscles; see Fig. 1). 3.2. Associative stimulation paradigm There were significant differences between muscles (ANOVA; effect of muscle, P , 0:05) and therefore the results for individual muscles were explored further using ANOVA. 3.3. First dorsal interosseus There was a significant intensity effect for FDI (ANOVA, F ¼ 49:88, P , 0:001). There was also a significant time effect (ANOVA, F ¼ 6:168, P , 0:05), and a significant time– intensity interaction (ANOVA, F ¼ 4:696, P , 0:05). Further analysis revealed that the intensity curves recorded both immediately following (ANOVA, F ¼ 4:836, P , 0:05), and 1 h following (ANOVA, F ¼ 6:703, P , 0:05) the afferent stimulation period were significantly different from the baseline intensity curve (see Fig. 2). The intensity curves at 15 min following, and 2 h following, were above the baseline curve but this change did not reach significance. 3.4. Abductor pollicis brevis There was a significant intensity effect for APB (ANOVA, F ¼ 87:227, P , 0:001) as well as significant time (ANOVA, F ¼ 4:993, P , 0:05), and a significant time – intensity interaction (ANOVA, F ¼ 6:498, P , 0:05) effects. Further analysis revealed that the intensity curves recorded both 15 min following (ANOVA, F ¼ 4:724, P , 0:05), and 1 h following (ANOVA, F ¼ 4:389, P , 0:05) the afferent stimulation period were significantly different to the baseline intensity curve. The intensity curves immediately following, and 2 h following, were above the baseline curve but this change was not significant (see Fig. 2). 3.5. Abductor digiti minimus and extensor carpi radialis Again, as expected, there was a significant intensity effect for both muscles (ADM F ¼ 39:373, P , 0:05; ECR F ¼ 18:041, P , 0:05). However, there were no significant time effects or time –intensity interactions (see Fig. 2).
3. Results
3.6. Site of excitability change – control experiment
3.1. Non-associative stimulation paradigm
Following the associative stimulation period the amplitude of MEPs evoked by TES was not significantly different to baseline (baseline 1.08 ^ 0.19 mV, immediately postintervention 1.27 ^ 0.43 mV; paired sample t test,
As expected, for all 4 muscles studied there were significant intensity effects (ANOVA, P , 0:05). There
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Fig. 1. Stimulus response curves for all 4 muscles following a 1 h period of afferent stimulation using the control paradigm of asynchronous electrical stimulation of the motor points of FDI and APB muscles. There are no significant differences between baseline and post-stimulation stimulus response curves for any of the muscles investigated.
P . 0:05). However, the amplitude of MEPs evoked following TMS was significantly larger following associative stimulation (baseline 0.94 ^ 0.44 mV, immediately post-intervention 1.58 ^ 0.53 mV; paired sample t test, P , 0:05; see Figs. 3 and 4).
4. Discussion The novel finding reported here is that a 1 h period of
paired stimulation of the motor points of the FDI and APB muscles results in a significant increase in the excitability of the corticospinal projection to the stimulated muscles that is maximal approximately 1 h following the stimulation period. This increase in excitability does not generalise to neighbouring (ADM) or more remote (ECR) muscles. A control paradigm employing independent stimulation of the two target muscles induced no significant change in the level of excitability of the corticospinal projection to any of the muscles investigated.
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Fig. 2. Stimulus response curves for all 4 muscles following a 1 h period of paired associative stimulation to the motor points of the FDI and APB muscles. There is a significant facilitation of the response curve for FDI (immediately post-intervention and 1 h post-intervention) and APB (15 min post-intervention and 1 h post-intervention). No significant differences are seen in the response curves of either ADM or ECR.
4.1. Rationale for use of an associative stimulation paradigm There is good evidence that correlated inputs are important for inducing plastic changes in the cortex. For example, surgically connecting the skin surfaces of two fingers (the formation of syndactyly) increases the correlation of inputs from skin surfaces of adjacent fingers and results in dramatic reorganisation of the sensory cortex (Clark et al., 1988). Given this finding, Godde et al. (1996)
designed an associative tactile stimulation paradigm that was capable of inducing significant reorganisation of the sensory cortex in rats. Following 6 –15 h periods of paired associative tactile stimulation, receptive fields of cells in the sensory cortex increased in size. Evidence was presented that such changes were accompanied by improvements in the performance of a tactile discrimination task in humans. The importance of associative pairing of inputs in driving cortical plasticity has also been demonstrated in the motor cortex (Baranyi et al., 1991). The importance of
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Fig. 3. Graph to illustrate the change in MEP amplitudes, evoked at an intensity of 50% of maximal stimulator output, for each muscle investigated following a 1 h period of associative stimulation (of the FDI and APB muscles). In each case bars indicate means þ 1 SE.
synchronised inputs in inducing cortical plasticity has also been demonstrated in human subjects. Liepert et al. (1999) had subjects perform synchronised voluntary movements of the thumb and foot. They used TCMS to map the scalp representation of the APB before and following the synchronised movements. The authors reported that the centre of gravity of the map shifted in the direction of the foot representation, and suggested that this change represented
short-term plasticity of the motor cortex. In a control experiment using asynchronous movements of the thumb and foot they found no such change in the centre of gravity. The stimulation paradigm employed in the current study was a modification of that described by Godde et al. (1996), and consisted of paired stimuli applied to the motor points of two hand muscles. Motor point stimulation is as effective as peripheral nerve stimulation in inducing plasticity (McKay et al., 2002a) and has the advantage that stimulation can be localised to individual target muscles. 4.2. Time course of excitability change
Fig. 4. Raw data traces from FDI for the ‘site of excitability change control experiment’. The top two superimposed traces are average MEPs (10 responses) evoked by TMS prior to (solid line) and following (dashed line) a 1 h period of associative stimulation. The lower two superimposed traces are averages (5 responses) of MEPS evoked by TES prior to (solid line) and following (dashed line) a 1 h period of associative stimulation. The MEPs evoked following TMS are significantly facilitated after associative stimulation, while the MEPs evoked by TES are not.
The increase in excitability of the cortical projection to the stimulated muscles was apparent immediately following the stimulation period. However, this change was even greater 1 h following the afferent stimulation period (see Fig. 2). Two hours following the afferent stimulation period an increase in excitability was still evident for the stimulated muscles, although this increase was non-significant. We have previously reported that following a 2 h period of ulnar nerve stimulation, cortical excitability to ulnar-innervated muscles is increased, and this increase is greater 15 min following the end of the stimulation period than immediately following the stimulation period (Ridding et al., 2000). The time course of excitability change reported in the present study is similar to that described by Fraser et al. (2002). These authors electrically stimulated the pharynx and then studied the excitability of the corticobulbar projection to the stimulated pharynx. With optimum stimulus parameters they reported an increase in excitability that peaked at 60 –90 min following the period of afferent stimulation. Interestingly, shorter periods (10 min) of afferent
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stimulation appear capable of inducing plasticity in the cortical projection to the pharynx than those reported for hand muscles (McKay et al., 2002b). 4.3. Site of excitability change Previously it has been demonstrated that following a variety of afferent stimulation paradigms an increase in corticospinal excitability is seen, and that the site of increase is likely to be within the motor cortex. For example, following peripheral nerve stimulation, MEPs are facilitated while F-waves are unchanged (Ridding et al., 2000). It has also been shown that, following dual peripheral and central stimulation, F-waves and responses to electrical stimulation of the brainstem are unaffected while MEPs evoked by TMS are facilitated (Stefan et al., 2000). Finally, following pharyngeal electrical stimulation, the MEPs elicited by TMS are facilitated but brainstem reflexes are unaffected (Fraser et al., 2002). The results from our site of excitability control experiment lend further support to this line of evidence. Responses to TMS were significantly facilitated following a period of associative stimulation but responses to TES were not. It is probable that TMS activates pyramidal cells transynaptically while TES activates pyramidal cells directly (Day et al., 1989). Therefore the differential effect on responses to TMS and TES suggests that the site of excitability change lies within the motor cortex. 4.4. Potential significance The results described here support the hypothesis that an afferent stimulation paradigm designed to activate peripheral afferents from two peripheral sites synchronously but at unpredictable times (associative stimulation) is highly effective for inducing motor cortex reorganisation. The importance of the associative nature of the stimulation is highlighted by the results from the control non-associative stimulation paradigm. Here, the target muscles were stimulated in a manner very similar to that applied in the test paradigm except that the two muscles were never stimulated concurrently. Stimulating the muscles in this manner produced no significant change in the excitability of the corticospinal projection to the stimulated muscles. Therefore, the associative nature of the test paradigm seems to be critically important for the induction of plasticity. These results extend the observations made in animal models regarding the importance of associative input for induction of cortical plasticity to humans. Afferent stimulation is capable of inducing reorganisation within the motor cortex (Hamdy et al., 1998; Ridding et al., 2000; Ridding et al., 2001; Stefan et al., 2000). With repeated sessions of afferent stimulation, the reorganisation can be made to persist for at least a few days (McKay et al., 2002a). It has been proposed that the reorganisation induced by afferent stimulation may be similar to that seen during motor learning and may have a potential therapeutic
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application for the rehabilitation of brain-injured patients; encouraging preliminary results have been reported recently (Conforto et al., 2002). Given this potential for rehabilitation, it is critical to determine the optimal paradigm for inducing plasticity. The present study demonstrates that 1 h of associative afferent stimulation is capable of inducing significant plasticity of the motor cortex that is of similar magnitude to that induced by 2 h of repetitive peripheral nerve stimulation (Ridding et al., 2000). Therefore, associative afferent stimulation may offer a more efficient therapeutic option. In summary, we have demonstrated that an afferent stimulation paradigm designed to provide associative stimulation to two peripheral muscles is capable of inducing a significant increase in the excitability of the corticospinal projection to the stimulated muscles. This effect is localised to stimulated muscles and does not generalise to adjacent or more remote muscles. The increase in excitability is similar in magnitude to that reported following longer periods of repetitive peripheral nerve stimulation and lasts for at least 1 h following the end of the afferent stimulation period. This new paradigm may offer a useful therapeutic option for motor rehabilitation of brain-injured patients.
Acknowledgements This work was supported by a grant from the Australian Research Council. M.C. Ridding was supported by a University of Adelaide, Faculty of Health Sciences Research Associateship. This work is funded by a grant from the Australian Research Council.
References Baranyi A, Szente MB, Woody CD. Properties of associative long-lasting potentiation induced by cellular conditioning in the motor cortex of conscious cats. Neuroscience 1991;42:321–34. Clark SA, Allard T, Jenkins WM, Merzenich MM. Receptive fields in the body-surface map in adult cortex defined by temporally correlated inputs. Nature 1988;332:444–5. Conforto AB, Kaelin L, Cohen LG. Increase in hand muscle strength of stroke patients after somatosensory stimulation. Ann Neurol 2002;51: 122– 5. Day BL, Dressler D, Maertens de Noordhout A, Marsden CD, Nakashima K, Rothwell JC, Thompson PD. Electric and magnetic stimulation of human motor cortex: surface EMG and single motor unit responses. J Physiol (Lond) 1989;412:449–73. Fraser C, Power M, Hamdy S, Rothwell J, Hobday D, Hollander I, Tyrell P, Hobson A, Williams S, Thompson D. Driving plasticity in human adult motor cortex is associated with improved motor function after brain injury. Neuron 2002;34:5– 40. Godde B, Spengler F, Dinse HR. Associative pairing of tactile stimulation induces somatosensory cortical reorganisation in rats and humans. Neuroreport 1996;8:281–5. Hamdy S, Rothwell JC, Aziz Q, Singh KD, Thompson DG. Long-term reorganization of human motor cortex driven by short-term sensory stimulation. Nat Neurosci 1998;1:64–8.
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Liepert J, Terborg C, Weiller C. Motor plasticity induced by synchronized thumb and foot movements. Exp Brain Res 1999;125:435–9. McKay DR, Ridding MC, Thompson PD, Miles TS. Induction of persistent changes in the organisation of the human motor cortex. Exp Brain Res 2002a;143:342 –9. McKay DR, Brooker R, Giacomin P, Ridding MC, Miles TS. Time course in the induction of increased human motor cortical excitability induced by peripheral nerve stimulation. Neuroreport 2002b;13:1271–3. Ridding MC, Rothwell JC. Stimulus/response curves as a method of measuring motor cortical excitability in man. Electroenceph clin Neurophysiol 1997;105:340 –4.
Ridding MC, Brouwer B, Miles TS, Pitcher JB, Thompson PD. Changes in muscle responses to stimulation of the motor cortex induced by peripheral nerve stimulation in human subjects. Exp Brain Res 2000; 131:135– 43. Ridding MC, McKay DR, Thompson PD, Miles TS. Changes in corticomotor representations induced by prolonged peripheral nerve stimulation in humans. Clin Neurophysiol 2001;112:1461–9. Stefan K, Kunesch E, Cohen LG, Benecke R, Classen J. Induction of plasticity in the human motor cortex by paired associative stimulation. Brain 2000;123(Pt 3):572–84.
Changes in motor cortical excitability induced by paired associative stimulation M.C. Ridding*, J. Uy Department of Physiology, University of Adelaide, Adelaide 5005, Australia Accepted 27 March 2003
Abstract Objective: Changes in afferent input have been shown to be capable of inducing reorganisations of motor cortex in humans. Using TCMS we examined the efficacy of a new associative afferent stimulation paradigm in inducing motor cortical reorganisation in humans. Methods: Using TCMS, stimulus response curves were constructed before and following a 1 h period of associative stimulation of two muscles motor points. The effect of an asynchronous peripheral stimulation paradigm was investigated in a separate series of control experiments. Results: One hour of associative stimulation of two muscles motor points resulted in a significant increase in the excitability of the corticospinal projection to those stimulated muscles. The increase in excitability peaked 1 h following the stimulation period. This increase in excitability did not generalise to either adjacent or more remote muscles. The control stimulation paradigm produced no significant change in corticospinal excitability. Conclusions: These results confirm the importance of associative input for the induction of plasticity in the human motor cortex. Significance: The findings reported here further elucidate the role of afferent input in motor cortical reorganisation. These findings have implications for our understanding of the mechanisms of motor learning and may also be relevant to the design of new afferent stimulation therapies. q 2003 International Federation of Clinical Neurophysiology. Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Afferent stimulation; Magnetic stimulation; Plasticity; Associative input; Human
1. Introduction Recently it has been demonstrated that stimulation of either peripheral nerves (Ridding et al., 2001), peripheral nerve and motor cortex (Stefan et al., 2000), or muscle motor point and motor cortex (McKay et al., 2002a) is capable of inducing reorganisations within the human motor cortex. The reorganisations are manifested as an increase in the excitability of the corticospinal projection to stimulated muscles (Ridding et al., 2000) and an increase in the cortical representational of stimulated muscles (McKay et al., 2002a; Ridding et al., 2001). These studies demonstrate the dynamic nature of the motor cortex organisation and the crucial role that afferent input has in modulating the organisation. * Corresponding author. Tel.: þ 61-8-8303-6433; fax: þ61-8-8303-3356. E-mail address: [email protected] (M.C. Ridding).
It is thought that the organisational changes induced by modifying afferent input are similar in nature to those seen during motor learning and there are several recent reports of afferent stimulation paradigms being employed to induce therapeutically relevant plasticity (Conforto et al., 2002; Fraser et al., 2002). Given the obvious therapeutic potential of this approach, optimisation of the afferent stimulation techniques is of great importance. All of these previous techniques apply continuous or short trains of regular stimuli to a variety of peripheral and central sites. Evidence exists to suggest further refinement of the afferent stimulation techniques may be possible. For example, it has been demonstrated that correlated inputs are very important for the induction of plasticity (Clark et al., 1988; Godde et al., 1996). Also, it may be important to present stimuli with a randomised timing between successive pairs to minimise habituation and adaptation effects (Godde et al., 1996). In the present study we investigated the efficacy of a modified
1388-2457/03/$30.00 q 2003 International Federation of Clinical Neurophysiology. Published by Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S1388-2457(03)00115-9
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afferent stimulation paradigm, designed to address these issues, in inducing motor cortex plasticity. This protocol was based on that reported by Godde et al. (1996).
2. Methods Fourteen normal subjects (3 female, 11 male; age range 20 –39 years) volunteered for the main experiments. All subjects were right-handed (assessed by the Edinburgh Handedness Inventory). The experimental protocols were approved by the local Human Research Ethics Committee and subjects gave written, informed consent. 2.1. Electromyographic recording The surface electromyogram was recorded from the following muscles in the right upper limb: first dorsal interosseus (FDI), abductor pollicis brevis (APB), abductor digiti minimus (ADM) and extensor carpi radialis (ECR). For FDI, the reference electrode was over the metacarpophalangeal joint of the index finger and the active electrode over the motor point; for APB the reference was over the metacarpophalangeal joint of the thumb and the active electrode over the motor point; for ADM the reference electrode was over the metacarpophalangeal joint of the little finger and the active electrode over the muscle belly; and for ECR the active electrode was placed over the muscle belly and the reference electrode was placed approximately 3 cm distal to this. Recordings were made with silver/silver chloride surface electrodes. The signals were amplified in the bandwidth 20 Hz to 1 kHz, sampled at 5 kHz and fed to a computer for off-line analysis. 2.2. Transcranial magnetic stimulation Transcranial magnetic stimulation (TMS) was delivered with a Magstim 200 magnetic stimulator (Magstim Co., Dyfed, UK). A round coil with an external diameter of 13 cm was used. The optimal position for evoking responses in the right FDI muscle was established and marked on the scalp to ensure reliable coil placement between trials. This coil placement was employed for all recordings. The coil was used with the ‘A’ side up. This is the optimal orientation for evoking responses in the right hand and results in a posterior to anterior current flow in the underlying cortex.
Signal software (Cambridge Electronic Design Ltd, Cambridge, UK) the interval between successive TMS pulses was randomly varied in the range 5 –6 s. The motor evoked potential (MEP) was recorded for each of the muscles following stimulation and the average peak-to-peak amplitude calculated for each intensity step. MEPs were recorded with all of the muscles relaxed. Any trials in which background EMG activity was present were excluded from analysis. Muscle relaxation was monitored by giving subjects visual feedback of their EMG on a high gain oscilloscope. 2.4. Test afferent stimulation paradigm The afferent stimulation paradigm employed was based on that described by Godde et al. (1996). Using surface electrodes, square-wave electrical stimuli of 1 ms duration were applied to the FDI and APB muscles simultaneously (Digitimer DS7A stimulators, Digitimer Ltd, Welwyn Garden City, UK). The timing between successive pairs of stimuli was randomised between 0.15 and 2.85 s in 8 steps (range 0.35 – 6.7 Hz). The timing of the inter-pulse intervals was performed using Signal software (Cambridge Electronic Design Ltd, UK). The intensity of stimulation was adjusted for each muscle and set at a level just sufficient to evoke a minimal visible motor response (range 10 –30 mA). This intensity of stimulation was non-painful for all subjects. The afferent stimulation paradigm was applied for a period of 1 h. This stimulation paradigm will be described as associative stimulation in the following text. 2.5. Control afferent stimulation paradigm A control afferent stimulation paradigm was also employed. This was similar to the associative stimulation paradigm. However in this control paradigm stimuli were never presented to the two stimulated muscles at the same time. Therefore, the two muscles received the same number of stimuli at the same rate as in the associative stimulation paradigm but the muscles never received stimuli synchronously. Therefore, the control paradigm was designed to test the relative importance of associating the two afferent inputs in the test condition. This stimulation paradigm will be described as non-associative stimulation in the following text. 2.6. Experimental design
2.3. Stimulus response curves Stimulus response curves were recorded (Ridding and Rothwell, 1997) for the 4 muscles. The intensity range investigated was from 20 to 80% of maximum stimulator output in steps of 5% (of stimulator output). In all subjects this stimulus range included intensity values from below motor threshold to suprathreshold intensities. Five stimuli were applied at each intensity step in this range. Using
Thirteen subjects were tested with both the associative and non-associative afferent stimulation paradigms. The order in which subjects received the two paradigms was randomised, and subjects were unaware of the experimental hypothesis. For all subjects testing sessions were separated by at least 1 week. Baseline stimulus response curves were recorded as described above. Subjects were then given either a 1 h period of the associative or non-associative
M.C. Ridding, J. Uy / Clinical Neurophysiology 114 (2003) 1437–1444
stimulation. Immediately following the stimulation period stimulus response curves were recorded again and then repeated at 15 min, 1 and 2 h (only 7 subjects had stimulus response curves recorded at the 2 h timing). In an attempt to control for the influence of cognitive/attentional factors in the induction of plasticity we (1) performed the experiments in a quiet environment away from distractions, and (2) repeatedly encouraged our subjects ‘pay attention to the stimulus’. 2.7. Site of excitability change – control experiment In order to investigate the site at which any excitability changes took place a further subject (right-handed male aged 35 years) was studied using both TMS and transcranial electrical stimulation (TES) before and following a period of associative stimulation. TES was applied using silver/ silver chloride electrodes attached to the scalp. The anode was placed over the hand area of the left motor cortex (6 cm lateral to the vertex) and the cathode placed over the vertex. In the baseline condition the intensity of both the TES and TMS was adjusted to produce MEPs of approximately 1 mV (peak-to-peak amplitude). MEPs following 10 magnetic stimuli and 5 electrical stimuli were then recorded. The order of presentation of electrical and magnetic stimuli was randomised during the trial so that the subject did not know what type of stimulus to expect. Following the baseline measurements a 1 h period of associative stimulation was applied to the subject (as described above). Immediately following the associative stimulation period MEPs were once again recorded following TES and TMS. 2.8. Analysis Data for the associative and non-associative paradigm experiments were analysed separately but in a similar manner. A repeated measures analysis of variance (ANOVA) was performed with main factors of muscle (4 levels), time (5 levels) and intensity (13 levels). Where there were significant muscle effects, further ANOVAs were performed for each muscle, with factors of time (5 levels) and intensity (13 levels). Where there were significant time – intensity interactions a further planned analysis was performed to determine which response curves were significantly different to baseline; where appropriate corrections for multiple comparisons were made. For the site of excitability control experiment, responses to TMS and TES were compared using paired sample t tests.
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were no significant differences between the muscles (muscle, P . 0:05). Analysis of individual muscle data revealed that, for all muscles, post-intervention response curves were not significantly different from baseline curves (ANOVA; time and time –intensity interaction, P . 0:05 for all muscles; see Fig. 1). 3.2. Associative stimulation paradigm There were significant differences between muscles (ANOVA; effect of muscle, P , 0:05) and therefore the results for individual muscles were explored further using ANOVA. 3.3. First dorsal interosseus There was a significant intensity effect for FDI (ANOVA, F ¼ 49:88, P , 0:001). There was also a significant time effect (ANOVA, F ¼ 6:168, P , 0:05), and a significant time– intensity interaction (ANOVA, F ¼ 4:696, P , 0:05). Further analysis revealed that the intensity curves recorded both immediately following (ANOVA, F ¼ 4:836, P , 0:05), and 1 h following (ANOVA, F ¼ 6:703, P , 0:05) the afferent stimulation period were significantly different from the baseline intensity curve (see Fig. 2). The intensity curves at 15 min following, and 2 h following, were above the baseline curve but this change did not reach significance. 3.4. Abductor pollicis brevis There was a significant intensity effect for APB (ANOVA, F ¼ 87:227, P , 0:001) as well as significant time (ANOVA, F ¼ 4:993, P , 0:05), and a significant time – intensity interaction (ANOVA, F ¼ 6:498, P , 0:05) effects. Further analysis revealed that the intensity curves recorded both 15 min following (ANOVA, F ¼ 4:724, P , 0:05), and 1 h following (ANOVA, F ¼ 4:389, P , 0:05) the afferent stimulation period were significantly different to the baseline intensity curve. The intensity curves immediately following, and 2 h following, were above the baseline curve but this change was not significant (see Fig. 2). 3.5. Abductor digiti minimus and extensor carpi radialis Again, as expected, there was a significant intensity effect for both muscles (ADM F ¼ 39:373, P , 0:05; ECR F ¼ 18:041, P , 0:05). However, there were no significant time effects or time –intensity interactions (see Fig. 2).
3. Results
3.6. Site of excitability change – control experiment
3.1. Non-associative stimulation paradigm
Following the associative stimulation period the amplitude of MEPs evoked by TES was not significantly different to baseline (baseline 1.08 ^ 0.19 mV, immediately postintervention 1.27 ^ 0.43 mV; paired sample t test,
As expected, for all 4 muscles studied there were significant intensity effects (ANOVA, P , 0:05). There
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Fig. 1. Stimulus response curves for all 4 muscles following a 1 h period of afferent stimulation using the control paradigm of asynchronous electrical stimulation of the motor points of FDI and APB muscles. There are no significant differences between baseline and post-stimulation stimulus response curves for any of the muscles investigated.
P . 0:05). However, the amplitude of MEPs evoked following TMS was significantly larger following associative stimulation (baseline 0.94 ^ 0.44 mV, immediately post-intervention 1.58 ^ 0.53 mV; paired sample t test, P , 0:05; see Figs. 3 and 4).
4. Discussion The novel finding reported here is that a 1 h period of
paired stimulation of the motor points of the FDI and APB muscles results in a significant increase in the excitability of the corticospinal projection to the stimulated muscles that is maximal approximately 1 h following the stimulation period. This increase in excitability does not generalise to neighbouring (ADM) or more remote (ECR) muscles. A control paradigm employing independent stimulation of the two target muscles induced no significant change in the level of excitability of the corticospinal projection to any of the muscles investigated.
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Fig. 2. Stimulus response curves for all 4 muscles following a 1 h period of paired associative stimulation to the motor points of the FDI and APB muscles. There is a significant facilitation of the response curve for FDI (immediately post-intervention and 1 h post-intervention) and APB (15 min post-intervention and 1 h post-intervention). No significant differences are seen in the response curves of either ADM or ECR.
4.1. Rationale for use of an associative stimulation paradigm There is good evidence that correlated inputs are important for inducing plastic changes in the cortex. For example, surgically connecting the skin surfaces of two fingers (the formation of syndactyly) increases the correlation of inputs from skin surfaces of adjacent fingers and results in dramatic reorganisation of the sensory cortex (Clark et al., 1988). Given this finding, Godde et al. (1996)
designed an associative tactile stimulation paradigm that was capable of inducing significant reorganisation of the sensory cortex in rats. Following 6 –15 h periods of paired associative tactile stimulation, receptive fields of cells in the sensory cortex increased in size. Evidence was presented that such changes were accompanied by improvements in the performance of a tactile discrimination task in humans. The importance of associative pairing of inputs in driving cortical plasticity has also been demonstrated in the motor cortex (Baranyi et al., 1991). The importance of
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Fig. 3. Graph to illustrate the change in MEP amplitudes, evoked at an intensity of 50% of maximal stimulator output, for each muscle investigated following a 1 h period of associative stimulation (of the FDI and APB muscles). In each case bars indicate means þ 1 SE.
synchronised inputs in inducing cortical plasticity has also been demonstrated in human subjects. Liepert et al. (1999) had subjects perform synchronised voluntary movements of the thumb and foot. They used TCMS to map the scalp representation of the APB before and following the synchronised movements. The authors reported that the centre of gravity of the map shifted in the direction of the foot representation, and suggested that this change represented
short-term plasticity of the motor cortex. In a control experiment using asynchronous movements of the thumb and foot they found no such change in the centre of gravity. The stimulation paradigm employed in the current study was a modification of that described by Godde et al. (1996), and consisted of paired stimuli applied to the motor points of two hand muscles. Motor point stimulation is as effective as peripheral nerve stimulation in inducing plasticity (McKay et al., 2002a) and has the advantage that stimulation can be localised to individual target muscles. 4.2. Time course of excitability change
Fig. 4. Raw data traces from FDI for the ‘site of excitability change control experiment’. The top two superimposed traces are average MEPs (10 responses) evoked by TMS prior to (solid line) and following (dashed line) a 1 h period of associative stimulation. The lower two superimposed traces are averages (5 responses) of MEPS evoked by TES prior to (solid line) and following (dashed line) a 1 h period of associative stimulation. The MEPs evoked following TMS are significantly facilitated after associative stimulation, while the MEPs evoked by TES are not.
The increase in excitability of the cortical projection to the stimulated muscles was apparent immediately following the stimulation period. However, this change was even greater 1 h following the afferent stimulation period (see Fig. 2). Two hours following the afferent stimulation period an increase in excitability was still evident for the stimulated muscles, although this increase was non-significant. We have previously reported that following a 2 h period of ulnar nerve stimulation, cortical excitability to ulnar-innervated muscles is increased, and this increase is greater 15 min following the end of the stimulation period than immediately following the stimulation period (Ridding et al., 2000). The time course of excitability change reported in the present study is similar to that described by Fraser et al. (2002). These authors electrically stimulated the pharynx and then studied the excitability of the corticobulbar projection to the stimulated pharynx. With optimum stimulus parameters they reported an increase in excitability that peaked at 60 –90 min following the period of afferent stimulation. Interestingly, shorter periods (10 min) of afferent
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stimulation appear capable of inducing plasticity in the cortical projection to the pharynx than those reported for hand muscles (McKay et al., 2002b). 4.3. Site of excitability change Previously it has been demonstrated that following a variety of afferent stimulation paradigms an increase in corticospinal excitability is seen, and that the site of increase is likely to be within the motor cortex. For example, following peripheral nerve stimulation, MEPs are facilitated while F-waves are unchanged (Ridding et al., 2000). It has also been shown that, following dual peripheral and central stimulation, F-waves and responses to electrical stimulation of the brainstem are unaffected while MEPs evoked by TMS are facilitated (Stefan et al., 2000). Finally, following pharyngeal electrical stimulation, the MEPs elicited by TMS are facilitated but brainstem reflexes are unaffected (Fraser et al., 2002). The results from our site of excitability control experiment lend further support to this line of evidence. Responses to TMS were significantly facilitated following a period of associative stimulation but responses to TES were not. It is probable that TMS activates pyramidal cells transynaptically while TES activates pyramidal cells directly (Day et al., 1989). Therefore the differential effect on responses to TMS and TES suggests that the site of excitability change lies within the motor cortex. 4.4. Potential significance The results described here support the hypothesis that an afferent stimulation paradigm designed to activate peripheral afferents from two peripheral sites synchronously but at unpredictable times (associative stimulation) is highly effective for inducing motor cortex reorganisation. The importance of the associative nature of the stimulation is highlighted by the results from the control non-associative stimulation paradigm. Here, the target muscles were stimulated in a manner very similar to that applied in the test paradigm except that the two muscles were never stimulated concurrently. Stimulating the muscles in this manner produced no significant change in the excitability of the corticospinal projection to the stimulated muscles. Therefore, the associative nature of the test paradigm seems to be critically important for the induction of plasticity. These results extend the observations made in animal models regarding the importance of associative input for induction of cortical plasticity to humans. Afferent stimulation is capable of inducing reorganisation within the motor cortex (Hamdy et al., 1998; Ridding et al., 2000; Ridding et al., 2001; Stefan et al., 2000). With repeated sessions of afferent stimulation, the reorganisation can be made to persist for at least a few days (McKay et al., 2002a). It has been proposed that the reorganisation induced by afferent stimulation may be similar to that seen during motor learning and may have a potential therapeutic
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application for the rehabilitation of brain-injured patients; encouraging preliminary results have been reported recently (Conforto et al., 2002). Given this potential for rehabilitation, it is critical to determine the optimal paradigm for inducing plasticity. The present study demonstrates that 1 h of associative afferent stimulation is capable of inducing significant plasticity of the motor cortex that is of similar magnitude to that induced by 2 h of repetitive peripheral nerve stimulation (Ridding et al., 2000). Therefore, associative afferent stimulation may offer a more efficient therapeutic option. In summary, we have demonstrated that an afferent stimulation paradigm designed to provide associative stimulation to two peripheral muscles is capable of inducing a significant increase in the excitability of the corticospinal projection to the stimulated muscles. This effect is localised to stimulated muscles and does not generalise to adjacent or more remote muscles. The increase in excitability is similar in magnitude to that reported following longer periods of repetitive peripheral nerve stimulation and lasts for at least 1 h following the end of the afferent stimulation period. This new paradigm may offer a useful therapeutic option for motor rehabilitation of brain-injured patients.
Acknowledgements This work was supported by a grant from the Australian Research Council. M.C. Ridding was supported by a University of Adelaide, Faculty of Health Sciences Research Associateship. This work is funded by a grant from the Australian Research Council.
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