Lack of LTP-like plasticity in primary motor cortex in Parkinson's disease

Experimental Neurology 227 (2011) 296–301

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Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r

Lack of LTP-like plasticity in primary motor cortex in Parkinson's disease A. Suppa a,b, L. Marsili a, D. Belvisi a, A. Conte a,b, E. Iezzi b, N. Modugno b, G. Fabbrini a,b, A. Berardelli a,b,⁎ a b

Department of Neurology and Psychiatry, Sapienza, University of Rome, Rome, Italy Neuromed Institute, Sapienza, University of Rome, Rome, Italy

a r t i c l e

i n f o

Article history: Received 14 September 2010 Revised 8 November 2010 Accepted 29 November 2010 Available online 9 December 2010 Keywords: Parkinson's disease Dyskinesias Primary motor cortex Plasticity TMS

a b s t r a c t In this study in patients with Parkinson's disease (PD), off and on dopaminergic therapy, with and without L-dopa-induced dyskinesias (LIDs), we tested intermittent theta-burst stimulation (iTBS), a technique currently used for non-invasively inducing long-term potentiation (LTP)-like plasticity in primary motor cortex (M1). The study group comprised 20 PD patients on and off dopaminergic therapy (11 patients without and 9 patients with LIDs), and 14 age-matched healthy subjects. Patients had mild-to-moderate PD, and no additional neuropsychiatric disorders. We clinically evaluated patients using the Unified Parkinson's Disease Rating Scale (UPDRS) and the Unified Dyskinesia Rating Scale (UDysRS). The left M1 was conditioned with iTBS at 80% active motor threshold intensity. Twenty motor evoked potentials (MEPs) were recorded from right first interosseous muscle before and at 5, 15 and 30 min after iTBS. Between-group analysis of variance (ANOVA) testing healthy subjects versus patients with and without LIDs, on and off therapy showed a significant interaction between factors “Group” and “Time”. After iTBS, MEP amplitudes in healthy subjects increased significantly at 5, 15 and 30 min (p b 0.01 at all time-points) but in PD patients with and without LIDs, on and off therapy, remained unchanged. In PD patients with and without LIDs, on and off therapy iTBS fails to increase MEP responses. This finding suggests lack of iTBS-induced LTP-like plasticity in M1 in PD regardless of patients' clinical features. © 2010 Elsevier Inc. All rights reserved.

Introduction Plasticity of the primary motor cortex (M1) can be studied in humans with the technique of repetitive transcranial magnetic stimulation (rTMS). In rTMS studies, plasticity refers to changes in motor evoked potential (MEP) amplitudes outlasting brain stimulation by seconds or minutes and reflecting activity-dependent changes in the effectiveness of synaptic transmission in M1 (Stefan et al., 2000; Ziemann et al., 2008; Siebner and Rothwell, 2003; Siebner et al., 2009). An rTMS approach increasingly used in healthy subjects to explore mechanisms of long-term potentiation (LTP)-like and long-term depression (LTD)-like plasticity in M1, consists in delivering paired Abbreviations: ANOVA, analysis of variance; AMT, active motor threshold; cTBS, continuous theta burst stimulation; EMG, electromyography; FDI, first dorsal interosseus; H&Y, Hoehn & Yahr scale; iTBS, intermittent theta burst stimulation; LIDs, L-dopa-induced dyskinesias; LTP, long-term potentiation; LTD, long-term depression; M1, primary motor cortex; MEPs, motor-evoked potentials; MVC, maximum voluntary contraction; NMDA, N-methyl-D-aspartate; PAS, paired associative stimulation; PD, Parkinson's disease; RMT, resting motor threshold; rTMS, repetitive transcranial magnetic stimulation; SNr, substantia nigra pars reticulata; STP, short-term plasticity; TBS, theta burst stimulation; TDCS, transcranial direct current stimulation; TMS, transcranial magnetic stimulation; UDysRS, Unified Dyskinesia Rating Scale; UPDRS, Unified Parkinson's Disease Rating Scale. ⁎ Corresponding author. Department of Neurology and Psychiatry, and Neuromed Institute, Sapienza University of Rome, Viale dell'Università, 30, 00185 Rome, Italy. Fax: + 39 06 49914700. E-mail address: [email protected] (A. Berardelli). 0014-4886/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2010.11.020

associative stimulation (PAS), which combines cortical stimulation with peripheral nerve electrical stimulation (heterotopic plasticity) (Stefan et al., 2000; Wolters et al., 2003). Investigators using the PAS technique in Parkinson's disease (PD), have reported conflicting results in PD patients off therapy. Some described an enhancement (Bagnato et al., 2006), whereas others described a reduction in PAS-induced LTP-like plasticity (Ueki et al., 2006; Morgante et al., 2006). Only one study investigated PD patients with LIDs and found that they lacked PAS-induced after effects (Morgante et al., 2006). Whether the different responses to PAS in patients with and without LIDs depend on plasticity changes strictly confined to M1, or reflect additional changes in the sensory afferent input activated by PAS remains unclear. Another rTMS technique for investigating plasticity not requiring sensory inputs is theta-burst stimulation (TBS), which consists in applying high-frequency bursts at theta frequencies over M1 (homotopic plasticity). In healthy subjects, continuous TBS (cTBS) leads to decreasedamplitude MEPs reflecting LTD-like plasticity, whereas intermittent TBS (iTBS) leads to increased-amplitude MEPs reflecting LTP-like effects (Huang et al., 2005, 2007). Although in a recent study in PD patients off therapy, Eggers et al. (2010) have shown that cTBS over M1 does not elicit the normal MEP inhibitory pattern, suggesting impaired LTD-like M1 plasticity in PD, no studies have investigated iTBS-induced plasticity in M1 in PD under different experimental conditions including the presence of dopaminergic treatment and LIDs. Finding out how PD patients respond to iTBS when off and on dopaminergic therapy and with and without LIDs would provide

A. Suppa et al. / Experimental Neurology 227 (2011) 296–301

major insight into our current knowledge of the cortical plasticity changes possibly involved in the pathophysiology of PD. In this study, we investigated iTBS-induced plasticity in patients with PD. To verify the possible effect of dopamine on iTBS-induced changes in MEP amplitudes reflecting M1 LTP-like plasticity, we also tested patients in separate sessions while they were off and on dopaminergic therapy and also with and without LIDs. Finally, we also tested the possible correlation between iTBS-induced changes in MEP amplitudes and patients' clinical features under the four experimental conditions. Materials and methods Subjects Twenty PD patients (14 men and 6 women, mean age ± SD: 62.4 ± 7.38, range 48–76 years) and 14 age-matched healthy subjects (11 men and 3 women, mean age ± SD: 60 ± 11.28, range 49–81 years) participated. In the study we included 11 patients without LIDs (7 men and 4 women, mean age ± SD: 62.2 ± 8.13, range 48–72 years) and 9 patients with LIDs (7 men and 2 women, mean age ± SD: 62.6 ± 6.61, range 57–76 years). All participants were right-handed. The diagnosis of idiopathic PD was made using the criteria of the United Kingdom Brain Bank Criteria (Gibb and Lees, 1988). PD patients had a predominantly akinetic-rigid syndrome without dementia. Patients were recruited from the movement disorder outpatient clinic of the Department of Neurology and Psychiatry, Sapienza, University of Rome. Patients were studied while they were under their usual dopaminergic treatment (on) and after drugs had been withdrawn for at least 12 h (off). None of the patients received other neuropsychiatric medications. PD patients were clinically evaluated before starting each experimental session. Motor signs were scored using the motor section of the Unified Parkinson's Disease Rating Scale (UPDRS) and the Hoehn & Yahr (H&Y) scale. Peak dose LIDs were also evaluated with the Unified Dyskinesia Rating Scale (UDysRS). Demographic and clinical features of parkinsonian patients with and without LIDs are summarized in Tables 1 and 2. All subjects gave their informed consent, and the study was approved by the institutional review board and conformed with the Declaration of Helsinki. Stimulation techniques Subjects were comfortably seated in an armchair and the right arm was maintained relaxed in the same position throughout the experiment. Subjects were asked to fully relax and keep their eyes open without looking at the stimulated hand (Suppa et al., 2010). Single-pulse TMS was delivered through a monophasic Magstim 200 stimulator (The Magstim Company Ltd, Whitland, Dyfeld, UK) connected to a figure-of-eight coil placed tangentially over the left M1 in the optimal position (hot spot) for eliciting MEPs in the right first dorsal interosseous muscle (FDI). The hot spot was marked on the scalp with a soft-tipped pen. Resting motor threshold (RMT) was calculated as the lowest intensity evoking five MEPs of at least 50 μV in ten consecutive trials. Test TMS consisted in 20 single pulses delivered at the intensity able to evoke at baseline MEPs at about 1 mV peak-to-peak in amplitude. The same intensity was used for testing MEP amplitudes throughout the experiment. Conditioning TBS was delivered through a high-frequency biphasic magnetic stimulator (Magstim SuperRapid) connected to a figure-ofeight coil placed over the left M1. Active motor threshold (AMT) was calculated during a mild tonic contraction (20% of maximal voluntary contraction — MVC) as the lowest intensity evoking five MEPs of at least 200 μV in ten consecutive trials. Conditioning stimulation was delivered using iTBS (Huang et al., 2005) in bursts of three pulses at high frequency, 50 Hz, repeated at intervals of 200 ms, delivered in short trains lasting 2 s, with an 8 s pause between consecutive trains, for a total number of 600 pulses. The stimulation intensity for iTBS was set at 80% AMT.

297

Table 1 Clinical features of the patients with Parkinson's disease without L-dopa-induced dyskinesias (LIDs) and with LIDs who participated in the study. UPDRS: Unified Parkinson's Disease Rating Scale (motor score) “on” and “off” dopaminergic therapy. L-dopa equivalent dose (mg) was calculated for each patient according to the criteria of Hobson et al. (2002). AV: average. SD: standard deviation. Case Age (years)

PD patients 1 without LIDS 2 3 4 5 6 7 8 9 10 11 AV. SD. PD patients 1 with LIDS 2 3 4 5 6 7 8 9 AV. SD.

48 69 60 72 49 59 61 62 67 72 65 62 8.1 65 56 53 64 65 62 66 76 57 63 6.8

Hoehn Disease Treatment & Yahr duration

Sex UPDRS

F M M M M M F M

M M M F M M M F M

ON

OFF

10 24 14 21 20 15 10 14 21 16 15 16 4.6 9 28 15 27 13 13 14 22 27 18 7.3

17 29 20 30 48 24 27 22 26 18 23 26 8.5 38 33 23 40 16 17 30 35 33 29 8.8

3 2 2 2.5 2 2 2 2 2 2 2.5

3 2.5 1.5 2 1.5 1.5 2 2.5 3

(years)

(L-dopa equivalent dose — mg)

2 2 2 8 10 3 10 5 4 4 12 5 3.7 7 15 13 6 5 5 6 11 19 9 5.1

667 300 267 300 825 300 250 333 236 300 336 374 189.8 683 241 493 940 346 567 850 1000 1250 708 329.5

Recording techniques and measurements The EMG activity from the right FDI muscle was recorded using a pair of silver chloride disc surface electrodes, in a belly-tendon montage. The EMG raw signals were amplified and band-pass filtered (20 Hz to 1 kHz) by a Digitimer D360 amplifier (Digitimer Ltd, Welwyn Garden City, Herts, UK), digitized at a sampling rate of 5 kHz (CED 1401 laboratory interface; Cambridge Electronic Design, Cambridge, UK) and stored on a laboratory computer for on-line visual display and later off-line analysis using a dedicated software (Signal software; Cambridge Electronic Design). The level of baseline EMG activity was controlled by visual-feedback through an oscilloscope screen and auditory feedback through a loudspeaker. We rejected trials with involuntary EMG activity from FDI muscle greater than

Table 2 Clinical evaluation of peak dose dyskinesias in patients with Parkinson's disease with LIDs. UDysRS: Unified Dyskinesias Rating Scale (total score). AV: average. SD: standard deviation. Case

PD patients with LIDS

1 2 3 4 5 6 7 8 9 AV. SD.

UDysRS

UDysRS

(Total score)

(Items) Face

Neck

Right arm

Left arm

Trunk

Right leg

Left leg

27 21 32 43 18 17 34 24 56 30 12.8

0 0 0 2 0 0 0 1 1

0 0 0 2 0 2 0 0 0

0 0 1 2 0 0 1 1 0

1 0 3 2 0 0 0 0 0

1 1 3 2 0 1 0 2 1

1 0 2 2 1 1 1 2 2

1 0 3 2 0 2 0 2 0

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50 μV in a time window of 500 ms preceding MEPs. MEPs were measured peak-to-peak and then averaged. Experimental paradigm To test LTP-like plasticity in M1 in PD patients with and without LIDs we used conditioning-test TMS. Conditioning stimulation consisted of iTBS whereas test stimulation consisted of single pulse TMS. As measure for LTP-like M1 plasticity we collected 20 MEPs before (T0) and 5 (T1), 15 (T2) and 30 min (T3) after iTBS. PD patients were randomly assigned to participate in two separate sessions, on and off dopaminergic therapy. All experimental sessions took place at comparable daytime and with comparable intervals between L-dopa challenge and iTBS, and at least one week elapsed between each experimental session (Fig. 1). Statistical analysis Data collected after iTBS were all expressed as absolute values (mV). Unpaired Student's t test was used to compare RMT, AMT values, the intensity used for evoking MEPs and for conditioning iTBS in healthy subjects and PD patients with and without LIDs, on and off therapy. Paired t test was used to test the effect of therapy on the same TMS measures in patients. To test the effect of iTBS on MEP amplitudes in healthy subjects and PD patients without LIDs, on and off therapy, we used separate repeated measures analysis of variance (ANOVA) with “Time” (T0 versus T1, T2 and T3) as within-subjects factor, and “Group” (fourteen healthy subjects versus eleven PD patients without LIDs on therapy, fourteen healthy subjects versus eleven PD patients without LIDs off therapy) as between-subjects factor. Similarly, to test the effect of iTBS on MEP amplitudes in healthy subjects and PD patients with LIDs, on and off therapy, we used separate between-group ANOVA with “Time” (T0 versus T1, T2 and T3), and “Group” (fourteen healthy subjects versus nine PD patients with LIDs on therapy, fourteen healthy subjects versus nine PD patients with LIDs off therapy) as main factors of analysis. To test the effect of dopaminergic therapy on iTBS-induced changes in MEP amplitudes in PD patients with and without LIDs we also used separate two way repeated-measures ANOVA with “Dopaminergic therapy” (on versus off) and “Time” (T0 versus T1, T2 and T3) as main factors of analysis. To test possible effect of LIDs on iTBS-induced changes in MEP amplitudes we used separate between-group ANOVA with “Time” (T0 versus T1, T2 and T3) as within-subjects factor, and “Group” (patients without LIDs on therapy versus patients with LIDs on therapy, patients without LIDs off therapy versus patients with LIDs off therapy) as between-subjects

factor. Finally, to test the effect of LIDs in the target hand on iTBS-induced changes in MEP amplitudes we used between-group ANOVA with “Time” (T0 versus T1, T2 and T3) as within-subjects factor, and “Body regions with LIDs” (patients on therapy with LIDs in the right hand versus patients on therapy with LIDs in other body regions) as between-subjects factor. Tukey Honestly Significant Difference test was used for all post hoc analyses. The Mann–Whitney U test was used to compare patients' clinical features (UPDRS on and off therapy, H&Y, disease duration and treatment), in PD patients with and without LIDs. Spearman rank correlation test was also used to assess a possible correlation between patients' clinical features in patients with and without LIDs (UPDRS on and off therapy, H&Y, UDysRS, disease duration and treatment), and iTBS-induced changes in MEP amplitudes at all time points. P values less than 0.05 were considered to indicate statistical significance. All values are expressed as mean ± SE. Results None of the subjects experienced any adverse effects during or after iTBS. None of the patients reported adverse effects when drugs were withdrawn. Unpaired t test showed comparable RMT and AMT values, intensity for eliciting baseline MEPs and for conditioning iTBS in healthy subjects and in PD patients with and without LIDs, on and off therapy (non-significant p values for all comparisons). Paired t test also showed that RMT and AMT values, intensity for eliciting baseline MEPs and for conditioning iTBS were all similar in PD patients on and off therapy (Table 3). Effect of iTBS in PD patients without LIDs, on and off therapy ANOVA showed that, after conditioning iTBS, MEP amplitudes measured at T1, T2 and T3 in healthy subjects increased but in PD patients without LIDs, on and off therapy, remained unchanged. iTBSinduced changes in MEP amplitudes differed in healthy subjects and PD patients without LIDs off therapy. Between-group ANOVA showed a significant interaction between factors “Group” and “Time” (F3.69 = 6.4; p b 0.01). In healthy subjects, post hoc one-way ANOVA showed a significant effect of factor “Time” (F3.39 = 26.12; p b 0.01); after conditioning iTBS, MEP amplitudes increased significantly at T1 (p= 0.03), T2 (pb 0.01) and T3 (p b 0.01). Conversely, in patients without LIDs off therapy, the factor “Time” had a non-significant effect (Fig. 2). When we compared healthy subjects and patients without LIDs on therapy between group ANOVA showed a significant interaction between factors “Group” and “Time” (F3.69 = 5.79; p b 0.01). Differently

Fig. 1. Experimental protocol used for testing primary motor cortex (M1) long-term potentiation (LTP)-like plasticity. We tested motor evoked potential (MEP) amplitudes before (T0) and after intermittent theta-burst stimulation (iTBS) at 5 (T1), 15 (T2) and 30 min (T3).

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299

Table 3 Transcranial magnetic stimulation (TMS) data in the experiment testing primary motor cortex (M1) plasticity in healthy subjects, in patients with Parkinson's disease without L-dopa-induced dyskinesias (LIDs), and in patients with Parkinson's disease with LIDs. iTBS: intermittent theta-burst stimulation; RMT: resting motor threshold; AMT: active motor threshold; Intensity for MEPs: TMS intensity used for evoking motor evoked potential (MEP) amplitudes of about 1 mV at baseline; Intensity for iTBS: intensity used for delivering iTBS; RMT, AMT, 1 mV and TBS are expressed as percentage of the maximum stimulator output (average±SD). iTBS

PD patients

Without LIDs

Off On Off On

With LIDs Healthy subjects

RMT (%)

AMT (%)

Intensity for MEPs (%)

Intensity for iTBS (%)

41.3 ± 5.7 43.3 ± 5.7 41.4 ± 7.2 43.2 ± 7.1 41.7 ± 7.7

47.2 ± 6.6 49.1 ± 11.2 48.4 ± 7.1 51.3 ± 9.2 45.8 ± 9.6

52.3 ± 8.8 54.9 ± 8.8 54.7 ± 11.4 55.7 ± 10.7 51.9 ± 9.6

38 ± 5.3 38.2 ± 7.5 39 ± 5.8 43.3 ± 8.3 36.8 ± 7.6

from healthy subjects, also in patients without LIDs on therapy, the factor “Time” had a non-significant effect (Fig. 2). Finally, when we tested the effect of dopaminergic therapy in patients without LIDs repeated measures ANOVA showed a non-significant effect of factors “Dopaminergic therapy” and “Time”. Effect of iTBS in PD patients with LIDs, on and off therapy ANOVA showed that, differently from healthy subjects, in PD patients with LIDs, on and off therapy, MEP amplitudes measured at T1, T2 and T3 remained unchanged. When we compared healthy subjects with patients with LIDs, off therapy, between-group ANOVA showed a significant interaction between factors “Group” and “Time” (F3.63 = 8.06; p b 0.01). Differently from healthy subjects (see above), patients with LIDs off therapy had a non-significant effect of factor “Time” (Fig. 3). When we compared healthy subjects and patients with LIDs on therapy again between group ANOVA showed a significant interaction between factors “Group” and “Time” (F3.63 = 6.93; p b 0.01). The factor “Time” had a non-significant effect also in PD patients with LIDs on therapy (Fig. 3). Finally, repeated measures ANOVA showed that factors “Dopaminergic therapy” and “Time” were not significant also in patients with LIDs. When we tested the influence of LIDs on iTBS-induced changes in MEP amplitudes between-group ANOVA failed to detect significant effect of factors “Group” and “Time” in PD patients on and off therapy. Between-group ANOVA failed to detect significant effect of factors “Body regions with LIDs” or “Time” in PD patients on therapy with LIDs. The Mann–Whitney U test showed that UPDRS on and off therapy and H&Y values were all comparable in patients with and without

LIDs. Conversely, patients with LIDs had significantly longer disease duration (P = 0.05), and significantly higher L-dopa daily doses (P = 0.01) than patients without LIDs. Spearman rank correlation test found no correlation between patients' clinical features (UPDRS on and off therapy, H&Y, UDysRS, disease duration and treatment), and iTBS-induced changes in MEP amplitudes at all time points. Discussion In this study, a new finding is that in patients with PD iTBS failed to elicit the normal MEP amplitude facilitation, the variable reflecting M1 LTP-like plasticity, regardless of whether patients receiving dopaminergic therapy. ITBS also failed to elicit the normal MEP facilitation in patients with LIDs. The observation that iTBS induced the expected changes in MEP amplitudes in all aged healthy subjects we studied confirms a previous report demonstrating non significant age-related decline in the degree of iTBS-induced changes in MEP amplitudes (Di Lazzaro et al., 2008). Although we did not deliver a sham stimulation, the observation that, differently from healthy subjects, all PD patients off and on therapy, with and without LIDs lacked iTBS-induced changes in MEP amplitudes suggests that the abnormal response to iTBS is a neurophysiological feature of PD. The lack of iTBS-induced changes in MEP amplitudes we found in PD patients may depend on several technical factors. Because we found similar RMT, AMT and MEP amplitudes, and similar absolute iTBS intensity in healthy subjects and in patients off and on therapy, with and without LIDs, we exclude differences in baseline measures of

iTBS in PD with LIDs

iTBS in PD without LIDs PD On

PD Off

Healthy Subjects

MEP Amplitude (%)

MEP Amplitude (%)

160 140 120 100

PD on

160

PD off

Healthy Subjects

140 120 100 80

80 baseline

5'

15'

30'

Time Fig. 2. Primary motor cortex (M1) plasticity. Motor evoked potentials (MEPs) elicited at baseline and 5, 15 and 30 min after intermittent theta-burst stimulation (iTBS) in Parkinson's disease patients without L-dopa-induced dyskinesias (LIDs), on and off dopaminergic therapy, and in healthy subjects. Each point corresponds to the mean MEP amplitude expressed as a percentage of the responses obtained at baseline; vertical bars denote SE. Note the significant difference in MEP amplitudes before and after iTBS in patients and in healthy subjects.

baseline

5'

15'

30'

Time Fig. 3. Primary motor cortex (M1) plasticity. Motor evoked potentials (MEPs) elicited at baseline and 5, 15 and 30 min after intermittent theta-burst stimulation (iTBS) in Parkinson's disease patients with L-dopa-induced dyskinesias (LIDs), on and off dopaminergic therapy, and in healthy subjects. Each point corresponds to the mean MEP amplitude expressed as a percentage of the responses obtained at baseline; vertical bars denote SE. Note the significant difference in MEP amplitudes before and after iTBS in patients and in healthy subjects.

300

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cortical excitability. Although we cannot fully exclude the possibility that our sample of parkinsonian patients included patients with genetic variants of PD, such as polymorphism in brain-derived neurotrophic factor (BDNF) (Cheeran et al., 2008; Foltynie et al., 2009), the observation that all PD patients had a similar response to iTBS makes this possibility unlikely. The altered response to iTBS might in theory depend on patients' incomplete muscle relaxation or LIDs occurring immediately before, during, or soon after conditioning iTBS (Ziemann et al., 2008; Huang et al., 2008, 2010a,b; Gentner et al., 2008; Iezzi et al., 2008; Siebner et al., 2009). We consider this hypothesis unlikely for several reasons. We carefully checked EMG activity throughout the experiments and in none of the patients EMG activity was present in the FDI muscle immediately before, during or soon after iTBS. Finally, the altered response to iTBS occurred also in PD patients who had LIDs in body regions other than the right arm. Because repetitive application of plasticity-inducing protocols within a restricted time window may significantly affect the outcome measures of cortical plasticity through mechanisms of homeostatic or non-homeostatic metaplasticity (Bienenstock et al., 1982; Siebner et al., 2009; Todd et al., 2009; Huang et al., 2010a,b), our study design consisting of at least one week between the different experimental sessions, also excluded a possible interference of the first on the second experimental session. Given that neurophysiological and pharmacological studies in healthy subjects support the hypothesis that the iTBS-induced changes in MEP amplitudes arise through homotopic N-methyl-D-aspartate (NMDA)-dependent LTP-like plasticity in M1 (Huang et al., 2005, 2007, 2010a,b; Teo et al., 2007), our observation that iTBS failed to elicit the expected facilitatory response in parkinsonian patients off and on therapy, with and without LIDs suggests abnormal iTBS-induced LTP-like plasticity in M1 in PD. Because Spearman correlation test disclosed no significant correlation between iTBS-induced changes in MEP amplitudes and patients' clinical features, we suggest that altered M1 LTP-like plasticity in PD is unrelated to dopaminergic therapy, the severity of disease, and the presence of LIDs. Altered M1 plasticity in PD patients off therapy receives support also from the recent study of Eggers et al. (2010) who found that like iTBS, continuous TBS (cTBS) also failed to elicit the expected inhibitory changes in PD patients off therapy. Our new iTBS finding agrees with the previous PAS studies by Ueki et al. (2006) and Morgante et al. (2006) who also found reduced responses to PAS in patients off therapy. When we studied patients on therapy without LIDs, dopaminergic therapy did not restore the abnormal response to iTBS. This observation differs from previous studies showing that L-dopa promotes PAS-induced LTP-like plasticity in healthy subjects (Kuo et al., 2008; Monte-Silva et al., 2009), and restores abnormal plasticity in parkinsonian patients without LIDs (Bagnato et al., 2006; Ueki et al., 2006; Morgante et al., 2006). Differences between the findings obtained with PAS (Bagnato et al., 2006; Ueki et al., 2006; Morgante et al., 2006) and iTBS may depend on the fact that the mechanisms underlying PAS-induced changes in MEP amplitudes differ from those responsible for the iTBS-induced after effects. PAS is considered a form of heterotopic spike timing-dependent plasticity because it induces NMDA-dependent LTP-like and LTD-like phenomena in M1 by repetitively activating specific M1 sensorimotor circuits (Stefan et al., 2000; Wolters et al., 2003), whereas iTBS-induced changes in MEP amplitudes arise through homotopic NMDA-dependent LTP-like plasticity in M1 (Huang et al., 2005, 2007; Teo et al., 2007). Supporting the hypothesis that abnormal cortical plasticity might contribute to the pathophysiology of PD several studies have described altered LTP-like plasticity in PD. Experimental studies in animal models of PD have shown that dopaminergic denervation disrupts NMDA-dependent LTP plasticity at the level of corticostriatal transmission (Centonze et al., 1999; Calabresi et al., 2007, 2009; Di Filippo et al., 2009). In a recent study, with a new methodological

approach in parkinsonian patients undergoing stereotactic surgery for implantation of deep brain stimulation (DBS) electrodes, Prescott et al. (2009) found lack of LTP-like plasticity in the substantia nigra pars reticulata (SNr). The altered M1 LTP-like plasticity we found in PD fits in well with our previous studies investigating short-term plasticity (STP) in parkinsonian patients off and on therapy without LIDs (Gilio et al., 2002; Suppa et al., 2010). When short bursts of suprathreshold 5 Hz rTMS are delivered over M1 in healthy subjects, MEP amplitudes progressively increase during the train (Pascual-leone et al., 1994; Berardelli et al., 1998). This MEP facilitation is thought to reflect STP resembling short-term potentiation of synaptic connections described in animal experiments (Berardelli et al., 1998; Zucker, 1989; Castro-Alamancos and Connors, 1996). Conversely, in PD, during suprathreshold 5 Hz-rTMS, MEP size remains unchanged showing that STP is also abnormal in parkinsonian patients off and on therapy without LIDs (Gilio et al., 2002; Suppa et al., 2010). We therefore conclude that abnormal STP and LTP-like plasticity in M1 exists in PD, regardless of dopaminergic therapy. How dopamine denervation influences M1 LTP-like plasticity in PD remains conjectural. Although we cannot fully exclude the possibility that lack of M1 plasticity also depends on direct dopaminergic denervation at cortical level due to altered mesocortical projections (Wang and O'Donnell, 2001; Remy and Samson, 2003; Tseng and O'Donnell, 2004; Molina-Luna et al., 2009), changes in M1 LTP-like plasticity most probably reflect dopamine depletion at the basal ganglia (Wichmann and DeLong, 1996; DeLong and Wichmann, 2007; Rodriguez-Oroz et al., 2009). In PD patients off therapy, the reduced thalamo-cortical inputs might alter LTP-like plasticity in M1 cortical layers responsible for the iTBS-induced after-effects (Ziemann et al., 2008; Siebner and Rothwell, 2003; Di Lazzaro et al., 2008; Siebner et al., 2009). In the present study PD patients with LIDs had longer disease duration compared to patients without LIDs, whereas UPDRS values in patients off and on therapy were similar in the two study groups. It is known that after five years of L-dopa therapy, LIDs develop in 50% of parkinsonian patients, regardless of UPDRS values (Ahlskog and Muenter, 2001; Fabbrini et al., 2009). The similar UPDRS values in patients with and without LIDs, on and off therapy are important for concluding that the lack of iTBS-induced LTP-like plasticity is present in PD regardless of patients' clinical features. In this study, patients with LIDs had higher L-dopa daily doses compared to patients without LIDs. In healthy humans studies with PAS and also with transcranial direct current stimulation (TDCS) – a neurophysiological technique able to induce homotopic LTP-like and LTD-like plasticity in M1 – have demonstrated that L-dopa exerts a dose-dependent role in modulating mechanisms of PAS-induced and TDCS-induced LTP-like plasticity in M1 (Kuo et al., 2008; Monte-Silva et al., 2009, 2010). It might be therefore that the lack of iTBS-induced facilitatory responses we found in PD patients on therapy with LIDs is due to the high L-dopa daily doses that were present in this study group. We believe, however, that this possibility is unlikely because a similar response to iTBS was also present in patients without LIDs in whom L-dopa daily doses were significantly lower compared to patients with LIDs. Finally, no studies have investigated the effect of different L-dopa daily doses on iTBS-induced LTP-like plasticity in PD patients with and without LIDs. The lack of iTBS-induced changes in MEP amplitudes we found in PD patients on therapy with LIDs agrees with a previous study demonstrating lack of PAS-induced LTP-like plasticity in M1 in parkinsonian patients with LIDs (Morgante et al., 2006). Further supporting a role for abnormal plasticity in PD with LIDs, experimental studies in dyskinetic parkinsonian animals have demonstrated that a lowfrequency stimulation protocol fails to depotentiate or de-depress, as normally expected, previously induced cortico-striatal LTP and LTD plasticity (Picconi et al., 2003; Belujon et al., 2010). In patients with LIDs the lack of M1 LTP-like plasticity may depend on factors other than increased thalamo-cortical inputs as predicted by the hyperkinetic model, possibly including altered firing rates and abnormal oscillatory activity in

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