Cortical and brainstem LTP-like plasticity in Huntington's disease

Brain Research Bulletin 75 (2008) 107–114

Research report

Cortical and brainstem LTP-like plasticity in Huntington’s disease Domenica Crupi a,b , Maria Felice Ghilardi a,b , Clara Mosiello a,b , Alessandro Di Rocco b , Angelo Quartarone c , Fortunato Battaglia a,b,∗ a

CUNY School of Medicine, Department of Physiology and Pharmacology, New York, NY, United States b NYU School of Medicine, Department of Neurology, New York, NY, United States c Institute of Neurosciences, Psychiatric and Anaesthesiological Sciences, University of Messina, Italy Received 4 June 2007; received in revised form 27 July 2007; accepted 27 July 2007 Available online 22 August 2007

Abstract Recent studies have reported abnormalities in short-term plasticity in patients with Huntington’s disease (HD). However, is not known whether long-term potentiation (LTP)-like plasticity is also affected in these patients. We tested cortical and brainstem LTP-like plasticity in eight symptomatic HD patients and in 10 healthy age-matched controls. To probe motor cortex LTP-like plasticity we used paired associative stimulation (PAS), a technique that combines repetitive electric stimulation of the median nerve with subsequent transcranial magnetic stimulation (TMS) of the contralateral motor cortex at 25 ms. To investigate brainstem plasticity, we induced LTP-like phenomena in the trigeminal wide dynamic range neurons (WDR) of the blink reflex circuit by pairing an high-frequency train of electrical stimuli (HFS) over the right supraorbital nerve (SO) coincident with the R2 response elicited by a preceding SO stimulus. Our results demonstrate impairment of both cortical and brainstem LTP-like plasticity in symptomatic HD patients which is similar to LTP deficits previously reported in HD animal models. These findings might well represent the neurophysiological correlates of memory deficits often present in HD. © 2007 Elsevier Inc. All rights reserved. Keywords: Transcranial magnetic stimulation; LTP; Huntington’s disease; Blink reflex

1. Introduction Huntington’s disease (HD) is a neurodegenerative disorder characterized by striatal cell loss and deposition of pathological aggregates in several cortical and subcortical areas. Symptomatic patients exhibit a combination of behavioral, cognitive and motor alterations [37]. Interestingly, memory deficits and blink reflex abnormalities are present in both presymptomatic and symptomatic patients. The mechanisms underlying these early manifestations are poorly understood [22,54]. Long-term potentiation (LTP), the most widely studied form of neuroplasticity in animal preparations, represents the cellular mechanism for learning processes [25]. Studies in genetic and phenotypic animal models of HD have shown the presence of LTP alterations [39,50]. Despite the few reports of abnormal short-term plastic-

∗ Corresponding author at: 138th Street & Convent Avenue, Room D-210, New York, NY 10031, United States. Tel.: +1 212 650 7964; fax: +1 212 650 7726. E-mail address: [email protected] (F. Battaglia).

0361-9230/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2007.07.029

ity in HD [4,51,30], there are no studies addressing the integrity of long-lasting forms of neural plasticity in HD patients. It is now possible to induce LTP-like changes in the human motor cortex (M1), by using a paired associative stimulation (PAS) paradigm [46]. Changes in motor cortex excitability induced by PAS are long-lasting, follow Hebbian rules and are blocked by dextromethorphan, a NMDA receptor antagonist [46,45]. Mao and Evinger [31] recently described a new induction protocol to investigate brainstem LTP-like plasticity in the wide dynamic range neurons (WDR) of the blink reflex circuit [31]. Such a protocol combines repetitive bursts of high-frequency stimulation (HFS) appropriately paired with movement feedback from the eyelid. With this appropriate timing (coincidence of HFS with the blink reflex), the HFS to the supraorbital nerve (SO) can induce a long-lasting facilitation of the R2 response of the blink reflex. In the present study, we employed PAS to examine M1 synaptic plasticity and the HFS protocol to study LTP-like plasticity of the blink reflex in symptomatic patients with HD. We hypothesize that the neurodegenerative processes in HD patients induce

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alteration of cortical and brainstem circuits that might impair synaptic LTP-like neuronal plasticity. 2. Methods 2.1. Subjects Nineteen patients with HD were assessed for inclusion in the study. Taking into account that PAS LTP-like plasticity depend upon frontal somatosensory processing [52] and that HD patients often have somatosensory evoked potentials (SEP) abnormalities [3,48,9], we first recorded median nerve-SEP in 19 HD patient and included only patients with N20 and N30 amplitudes bigger than the lowest level obtained in 10 clinically normal, mutation-negative relatives (1.08 ␮V for N20 and 1.2 ␮V for N30) (Table 1). Only eight patients met our inclusion criteria. Regarding the brainstem LTP-like plasticity experiment, we included in the study patients with BR R2 latency shorter that the upper limit obtained in 10 clinically normal, mutation-negative relatives (average ipsilateral and contralateral R2 obtained after right SO stimulation, 39.5 ms). The same eight patients tested with TMS met the inclusion criteria for the brainstem LTP-like plasticity experiment. We further studied eight, sex-age matched, control subjects. Subjects’ demographic and clinical characteristics are reported in Table 1. One patient was treated with neuroleptic drugs that were discontinued for 72 h prior to the study. All subjects were right handed [38]. All participants gave written informed consent according to the declaration of Helsinki. The experimental protocol was approved by the institutional ethics committee. Subjects were tested in three different days (first day: clinical evaluation, inclusion criteria, MEP and BR I-O curves; second day PAS, third day BR LTP-like plasticity).

2.2. Somatosensory evoked potential recording During recording sessions subjects were seated in a reclining chair in semidarked room. The electrical stimuli (square wave pulse of 0.2 ms duration) were applied transcutaneously to the right median nerve at the wrist at intensity set between 1.5 and 2 times the sensory threshold, sufficient to produce a visible thumb twitch. The recording electrodes were placed at the posterior midline of the neck at C7 level (with an anterior cervical reference placed above the process of the thyroid cartilage) and on the scalp, over Fz, the contralateral hand field (2 cm posterior and 1 cm lateral to C3 according to the 10–20 International system) for the median nerve. For all scalp recordings, an ipsilateral earlobe reference was used. The impedance was < 5 k. The signals were fed into amplifiers (Nihon Kohden Neuropack) and bandpass filtered (5–3000 Hz). The sweep time was 100 ms: 1000 sweeps were averaged. Central conduction time (CCT) was determined as the difference between the peak latencies of parietal N20 and cervical N13. Peak-to-peak amplitudes for the median nerve at the frontal channel (P20–N30) and at the parietal channel (P14–N20) were determined.

2.3. M1 LTP-like plasticity All subjects were seated in a comfortable reclining chair. EMG activity was recorded with two surface electrodes (Ag/AgCl) placed over the right abductor Table 1 Subjects demographic and clinical characteristics

Mean age (years) Gender (female/male) Time since HD diagnosis (years) Mean UHDRS CAG repeats Neuroleptic treatment (n)

HD

Controls

46.6 ± 7.1 3/5 2.2 ± 0.67 31.2 ± 5.1 42.6 ± 4.1 1

44.4 ± 6.1 3/5

Values are expressed as mean ± S.D. UHDRS: unified Huntington’s disease rating scale motor score.

pollicis brevis (APB) muscle using a belly-tendon montage. EMG signals were amplified and filtered (bandwidth 5–1 kHz, Neurolog System, Digitimer Ltd., Welwyn Garden City, Herts, UK). Signals were acquired at a rate of 5 kHz (CED 1401) laboratory interface, Cambridge Electronic Design, Cambridge, UK) on a personal computer for off-line analysis. During the experiments EMG activity was continuously monitored with visual (oscilloscope) and auditory (speakers) feedback to ensure complete muscle relaxation TMS was performed using a high-power Magstim 200 stimulator (Magstim, Whitland, Dyfed) and a standard figure-of-eight coil, with external loop diameters of 9 cm. The coil was held tangentially to the skull with the handle pointing backwards and laterally at an angle of 45◦ to the sagittal plane. This orientation of the induced electrical field is thought to produce predominantly a trans-synaptic activation of the corticospinal neurons [44]. The site at which stimuli of slightly suprathreshold intensity consistently produced the largest MEPs in the relaxed right APB muscle was marked with a pen as the “motor hot spot” and used for TMS of the motor cortex. We first determined resting motor threshold (RMT), defined as the minimal stimulus intensity required to produce MEPs > 50 ␮V in at least 5 out of 10 consecutive trials; then we studied MEP input–output curve at stimulus intensities ranging from 100 to 150% of the RMT (10% steps). Stimulus intensities were delivered in random order. For each stimulation intensity, 20 MEP were collected and their peak-to-peak amplitudes were measured offline. M1 plasticity was tested by using the protocol of associative stimulation described by Stefan et al. [46] (Fig. 1A). Associative stimulation consisted of suprathreshold electrical stimulation of the right median nerve combined with a suprathreshold magnetic pulse applied over the left primary motor hand area 25 ms after peripheral nerve stimulation. Ninety pairs of stimuli were given every 20 s. The intensity of TMS was adjusted to evoke MEPs of peak-to peak amplitude of about 1 mV in the relaxed APB muscle. Electrical stimulation of the right median nerve was performed at the wrist through a bipolar electrode (cathode proximal), using constant current square wave pulses (duration, 1 ms) at an intensity of three times the perceptual threshold (Digitimer, Welwyn Garden City, Herts, UK). MEP amplitude measurements (average of 20 responses) were performed at baseline (B) and every 15 min for 60 min (T15, T30, T45 and T60) after the associative stimulation. Since the cortical plasticity is attention-dependant [47], we employed a paradigm previously described to monitor attention level during PAS [47]. During conditioning the subjects were asked to detect and recall the number of weak electric stimuli randomly applied to target region of PAS. Briefly, four to seven stimuli (stimulus width 200 ␮s, two times of the perceptual threshold) were delivered to either thumb (left and right) via ring electrodes. Electric pulses to the digit were given asynchronously with the paired associative stimuli. Subjects were asked to look at and to pay attention on their right hand throughout PAS. After PAS, subjects were asked to report the count of the stimuli they had identified. Subjects were not provided with any feedback as to the accuracy of the number reported.

2.4. Blink reflex LTP-like plasticity LTP-like plasticity of the R2 component of the human BR was induced by using the protocol developed by Mao and Evinger [31]. The conditioning stimulation (HSF, high-frequency stimulation) necessary to produce LTP-like effect consisted of electrical stimulation (square wave pulse, width of 200 ␮s) of the SO nerve performed using silver chloride disc surface electrodes (Fig. 1B). We first determined the R2 threshold (the minimum intensity required to evoke a reliable R2 response with amplitude of at least 50 ␮V) and then, we evoked R2 responses by setting the stimulus intensity at two and three times the threshold (Tr2 and Tr3). We investigated basal synaptic transmission of the blink reflex circuit by plotting the stimulation intensity as a multiple of threshold (Tr2 and Tr3) against area of the R2 response to generate input–output relationships. Tr2 intensity of stimulation was used to induce BR plasticity. LTP-like plasticity was induced with a conditioning protocol, HFS, administered at the onset of the R2 response elicited by the conditioning stimulus (see Fig. 1B). The induction protocol consisted of three blocks of trains high-frequency stimulation (HFS) separated by a 5 min inter-block interval. Each block consisted of four trains of HFS (nine stimuli, 400 Hz) given every 10 s. The area of the R2 component of the blink reflex was assessed before and up to 1 h after the conditioning protocol (Fig. 1C). The primary outcome measure was the EMG area of the R2

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Fig. 1. M1 and brainstem LTP-like plasticity. (A) PAS recording, setting overview: TMS pulse is applied in close temporal relation to an electrical stimulus of the median nerve at the wrist. If the stimuli are timed with an interstimulus interval of 25 ms, the sensory afferent input reaches the motor cortex just before the TMS is given. Repeated pairings (90 given every 3 s) lead to long-lasting LTP-like plasticity of the corticospinal system. MEP amplitudes were assessed at baseline and up to 1 h after PAS. (B) Blink reflex recording, setting overview. LTP-like plasticity of the R2 component of the blink reflex was induced with a tetanic stimulation delivered to the right SO nerve at an intensity of two times threshold (2T) for producing an R2 response and coincided with the R2 response elicited by a preceding SO stimulus. (C) The induction protocol consisted of three blocks of trains high-frequency stimulation (HFS) separated by a 5 min inter-block interval. Each block consisted of four trains of HFS (nine stimuli, 400 Hz) given every 10 s. The area of the R2 component of the blink reflex was assessed before and up to 1 h after the conditioning protocol (baseline, T0, T30 and T60). SO: supraorbital nerve. response in right and left orbicularis oculi muscles (OO), recorded with pairs of silver chloride disc surface electrodes and measured immediately before HFS conditioning (i.e. baseline; B) as well as at T0, T30 and T60 after HFS conditioning. Twenty trials were collected and averaged for each block of measurements. Trials with movement artifacts were rejected. The electromyographic (EMG) signal was amplified and bandpass filtered (20 Hz to 3 KHz; D150 amplifier, Digitimer Ltd., Welwyn Garden City, Herts, UK) and stored at a sampling rate of 5 KHz on a personal computer for off-line analysis (SigAvg Software, Cambridge Electronic Design, Cambridge, UK). The area of the R2 response was calculated for each block by integrating the rectified EMG activity of the OO muscles using NuCursor software (Sobell Research Department of Motor Neuroscience and Movement Disorders, Institute of Neurology, University College of London, United Kingdom). The onset and offset of the R2 response were estimated visually from averaged rectified EMG measures.

3. Data analysis Median nerve SEP: Differences in CCT, P14–N20 and P20–N30 amplitudes between patients and controls were analyzed with unpaired t-test. M1 LTP-like plasticity: RMT comparisons between controls and HD patients were performed with unpaired t-test and input–output curves comparisons with ANOVA. Baseline MEP responses were analyzes with unpaired t-test. MEP amplitudes collected after PAS (T15, T30, T45 and T60) were expressed as a percentage of the responses obtained at baseline. Comparisons between the two groups were performed with ANOVA (time: T15, T30, T45 and T60; group: controls, HD patients). Blink reflex LTP-like plasticity: We analyzed differences in R2 latency between HD patients and controls by using an

unpaired t-test. In the blink reflex LTP-like experiment we used the area of the R2 response collected in the right and left OO muscles as dependent variable in the ANOVA design. The responses collected after HFS (T0, T30 and T60) then were expressed as a percentage of the responses obtained at baseline (prior HFS). We analyzed changes in R2 area in time (three levels; T0, T30 and T60), muscle (two levels; right OO muscle versus left OO muscle) and group (two levels; HD patients and controls) with ANOVA. Conditional on a significant F value, post hoc paired t-tests with correction for multiple comparisons WERE performed. The Spearman rank correlation test was used to assess correlations between the UHDRS score, disease duration, CAG repeats and the amount of potentiation obtained 60 min after the induction protocols. In addition, we correlated the amount of potentiation obtained in motor cortex and in brainstem. 4. Results 4.1. SEP Patients included in the study had normal CCT, P14–N20 and P20–N30 amplitudes (Table 2). 4.2. M1 LTP-like plasticity The attention, as assessed by the number of errors in detecting a weak electrical stimulus delivered to the target finger, was

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Table 2 Mean ± S.D. of central conduction time (CCT) and peak-to-peak amplitude of the early components of the median nerve SSEP in HD patients and in control subjects

CCT N13-N20 (ms) P14-N20 (␮V) P20–N30 (␮V)

HD

Controls

P values (ns)

5.5 ± 1 2.3 ± 0.96 1.9 ± 0.6

5.63 ± 0.9 3.1 ± 1.4 2.5 ± 1

0.9 0.07 0.08

comparable between HD patients and control subjects (numbers of errors, % of test stimuli: patients: 12.9 ± 3.9, controls: 10.1 ± 4.1; p > 0.05). RMT was similar in HD patients and healthy controls (mean ± S.E., controls 37.1 ± 2.5%, HD patients 35.7 ± 2.4%; p > 0.05). HD patients and controls had also similar MEP amplitude as tested with input–output curve. There was a main effect stimulation intensity (F(1,5) = 74.7, p < 0.0001) with no main effect of group (F(1,96) = 0.9, p = 0.3) indicating that in patients and in controls increased stimulation intensities induced equally increase in MEP size (Fig. 2). Baseline MEP amplitudes were not different between patients and controls (mean ± S.E., controls 0.97 ± 0.0061 mV, HD patients 1.01 ± 0.0057 mV; p > 0.05). PAS induced a significant increase in MEP amplitudes in control subjects while HD patients showed lack of potentiation (Fig. 3) (ANOVA: group, F(1,64) = 62.4, P < 0.0001; time, F(3,64) = 0.7 P = 0.8; time × group, F(3,64) = 0.09, P = 0.9). Post hoc analysis revealed that the differences between controls and patients were present at each time point after PAS (T15, T30, T45 and T60; all p < 0.0001). As we further explored the effects of PAS, we found that in controls, PAS induced significant potentiation of MEP amplitude at all time points (baseline versus: T15: t = −8.06, p < 0.0001; T30: t = −5.07, p = 0.0002; T45: t = −4.3, p = 0.0001; T60: t = −4.1, p = 0.0001) while in HD patients, PAS induced did not induces significant potentiation (baseline versus: T15: t = −0.7, p = 0.4; T30: t = −0.73, p = 0.48; T45: t = −0.4, p = 0.6; T60, t = −0.39, p = 0.42).

Fig. 3. M1 LTP-like plasticity. Mean MEP amplitudes (±S.E.) at baseline and up to 1 h after PAS in HD patients (black filled circles) and controls (empty circles).

There were no significant correlations between the amount of potentiation obtained 60 min after the induction protocol and the CAG repeats, the disease duration and the UHDRS score (Table 3). 4.3. Blink reflex LTP-like plasticity R2 latencies were not statistically different between HD patients and control subjects (R OO: t = −1.2, p = 0.1; L OO: t = −1.3, p = 0.13). We first evaluated the input–output curve of the blink reflex by using two stimulation intensities (Tr2 and Tr3). Increased stimulation intensity resulted in larger response in both groups for both recording sides, without any difference between groups or sides (ANOVA: stimulation: F(1,68) = 52.8, p < 0.0001; group, F(1,68) = 0.006, p = 0.9: recording site, F(1,68) = 0.1, p = 0.6; group × muscle × stimulation, F(1,68) = 0.02, p = 0.6) (Fig. 4). In the LTP-like experiment, there was no between-group difference in the R2 responses at baseline (p > 0.05). Also, the mean intensity to evoke it did not differ between controls (10.3 ± 2.2 mA) and HD patients (10.9 ± 2.6 mA; p > 0.05). Table 3 Correlation between clinical and electrophysiological data in HD patients

Fig. 2. Motor-evoked potential (MEP) input–output curve. HD patients and normal subjects had similar MEP amplitudes. Error bars represent S.E.

Rho

p

CAG repeats PAS Right OO muscle Left OO muscle

−0.35 −0.14 −0.48

0.36 0.69 0.2

Disease duration PAS Right OO muscle Left OO muscle

−0.75 0.03 −0.24

0.06 0.9 0.51

UHDRS score PAS Right OO muscle Left OO muscle

−0.26 −0.2 −0.02

0.48 0.5 0.94

PAS: paired associative stimulation; OO muscle: orbicularis oculi muscle; UHDRS: unified Huntington’s disease rating scales.

D. Crupi et al. / Brain Research Bulletin 75 (2008) 107–114

Fig. 4. Blink reflex R2 input–output curve. R2 area increased at higher intensity (Tr2, Tr3) in both HD patients and controls. Each error bar equals standard error of the mean (S.E.M.). Controls, black columns; patients, shaded columns; OO: orbicularis oculi muscle; ** p < 0.01.

HFS stimulation induced a significant increase in R2 area only in healthy controls (Fig. 5A and B). In fact, ANOVA revealed a main effect of group (F(1,96) = 28.7; p < 0.0001) without significant effect of time (F(2,96) = 0.02; p = 0.9 0.02), and muscle (F(1,96) = 0.02; p = 0.9). Interaction of group × time × muscle was not significant (F(2,96) = 0.2, P = 0.2). The difference

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between healthy controls and HD patients was evident at each time point after HFS (T0, T30 and T60, p < 0.001). We then explored the effects of HFS conditioning for each group. In controls, HFS induced potentiation in both OO muscles at each time point, while in HD patients it did not induce any significant potentiation (controls: R OO: T0: t = −2.7, p = 0.01; T30: t = −2.3, p = 0.04; T60: t = −2.32, p = 0.03; L OO: T0: t = −3.2, p = 0.01; T30: t = −2.5, p = 0.03; T60: t = −2.3, p = 0.03. HD patients R OO: T0: t = −0.1 p = 0.8; T30: t = −0.3, p = 0.7; T60: t = −0.07, p = 0.9; L OO: T0: t = −0.06 p = 0.9; T30: t = −0.02, p = 0.8; T60: t = −0.2, p = 08). There were no significant correlations between the amount of potentiation obtained 60 min after the induction protocol in the CAG repeats, disease duration and UHDRS score (Table 3). Furthermore, there was no correlation between cortical and brainstem LTP-like plasticity (p > 0.5). 5. Discussion This is the first study addressing long-lasting activitydependent changes of synaptic activity in HD patients. The main result is that symptomatic HD patients present with decreased M1 associative plasticity assessed with PAS and lack of LTP-like plasticity of the trigeminal blink reflex. 5.1. M1 plasticity

Fig. 5. LTP-like plasticity of blink reflex. Mean R2 areas (±S.E.) at baseline and up to 1 h after PAS in HD patients (black filled circles) and controls (empty circles). (A) Recording from right OO; (B) recording from left OO. After HFS, control subjects showed a significant increase of the R2 area consistent with a long-term potentiation. HD patients showed a lack of potentiation.

Using a well-characterized PAS-protocol, we showed that the facilitatory effects on TMS-evoked MEPs are decreased in symptomatic HD patients. This PAS protocol induces plastic changes of excitability in the human motor cortex that share a number of physiological properties with LTP as tested in brain slices [45]. LTP studies in brain slices preparations from several HD mouse models have shown the presence of synaptic abnormalities at striatal synapses and alterations in long-term plasticity [28,29,13,14]. In particular, a well-studied HD mouse model, R6/1, exhibits defective neocortical plasticity [16,17,33] that is associated with deficits in a cortical-specific associative discriminatory task [17]. These observations, together with findings in lesional models of HD [43,39], strongly suggest that in HD there are deficits in the plastic properties of glutamatergic synapses. A variety of pre- and post-synaptic changes has been proposed to explain LTP deficits in HD animal models and such alterations are relevant to understand our findings. There is accumulating evidence that postsynaptic signaling pathways, including those associated with NMDA receptor activation, are impaired in HD. It has been hypothesized that increased release of glutamate from the cortex or hypersensitive postsynaptic glutamate receptors, would result in neuronal dysfunction and cell death [12,13]. These alterations of glutamatergic transmission, disruption of intraneuronal signaling [53] and dysregulation of activity-dependent gene expression [6] could be responsible of the reduction of the PAS LTP-like plasticity. PAS-induced plasticity follows the Hebbian rules and is NMDA receptor dependent [45,55]. These two properties are consistent with LTP as the underlying mechanism of PAS-

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induced plasticity. Our data assume clinical relevance when considering that learning and memory processes are likely mediated by changes in the strength of synapses in neural circuits. Neural activity during learning gives rise to long-term changes in synaptic strength, which allows memories to be stored and later retrieved [32]. Interestingly, motor sequence learning is abnormal in both symptomatic and presymptomatic HD patients [10,27,20]. The abnormalities in PAS-induced potentiation might represent the neurophysiological correlate of these behavioral abnormalities. Future studies are needed to test specifically this hypothesis. The deficits we described might also result from abnormalities in sensory processing often reported in HD [1]. In fact, basal ganglia dysfunction leads to abnormal thalamocortical drive that induces changes in the excitability of cortical sensorimotor areas. However, we included in the study only patients without N30 alteration. It’s thus likely that our findings result from impaired M1 plasticity. Previous TMS studies addressing motor cortical excitability in HD yielded controversial results [2,21,40], probably due to the wide variability of the TMS protocols used and the heterogeneity of the patients’ population. A recent paper [30] reported deficit of motor cortex plasticity in HD by using 5 Hz repetitive TMS (rTMS). The present findings confirm and extend their observations. The MEP facilitation induced by short-trains of rTMS probably involves mechanisms of short-term synaptic plasticity [23,24]. Thus, the lack of progressive increase in MEP size Lorenzano and colleagues found with their protocol during rTMS might be interpreted as a lack of post-tetanic or short-term potentiation [23]. In fact, according to the widely accepted “multiple phase” model of LTP, changes in synaptic efficacy which lasts only seconds to a few minutes are defined post-tetanic potentiation. Changes in synaptic efficacy of less than 30 min duration are considered short-term potentiation and early LTP refers to the time between 30 and 60 min after tetanus and is caused by post-translational modifications of existing proteins. PAS has been used to test plasticity in neuropsychiatric disorders with significant motor disturbances [41,49,35,8]. Because of the physiological properties of PAS [45], the present study provides a strong evidence for a deficit of early LTP-like plasticity in HD patients.

Short-term plasticity of the blink reflex, studied with R2 recovery curves [26], has been reported to be abnormal in HD patients [11,19,18,4,51]. With the protocol used in this study, we addressed, for the first time, impairment of long-term plasticity in the blink reflex circuit. The input–output relations of the area of the R2 response was similar in patients and controls, demonstrating that basal synaptic transmission within the blink reflex circuit was not impaired in HD patients. This finding suggests that the lack of potentiation in HD is not related to a problem in basal synaptic transmission but it is likely due to a synaptic failure to sustain activity-dependent changes. As recently suggested, the LTP-like plasticity of the R2 represents a long-term plasticity of the WDR neurons [31] that is impaired in basal ganglia diseases [7,42]. Thus, the lack of potentiation we reported could be due to abnormal WDR neuronal activity either at the presynaptic level (primary afferent terminals) or at the postsynaptic terminal in HD. Furthermore, altered activity of local interneurons modulating the WDR neurons function might significantly contribute to the HD plasticity deficit. The lack of potentiation we found could be due to functional abnormalities of neural structures other than the brainstem. In fact, central regulation of blinking involves cortex, extrapyramidal motor systems and rostral brainstem structures [5,15]. Thus, abnormal activity in these structures [30,34] might affect brainstem excitability and its capacity to undergo to LTP-like, activity-dependent changes. 6. Conclusion We used PAS and HFS to induce long-lasting changes in MEP amplitude and R2 area and showed that M1 and brainstem LTP-like plasticity is impaired in HD patients. Thus, in addition to short-term plasticity deficit previously described, symptomatic HD patients also display abnormal motor and brainstem long-term plasticity. Our study takes advantage of experimental approaches designed to investigate neuroplasticity in basic research and applies these paradigms to the clinical domain. Future application of this model might well yield significant insights on the neurophysiological bases of associative learning deficit in HD.

5.2. Brainstem plasticity Disclosure statement In addition to abnormal M1 LTP-like plasticity, our symptomatic HD patients exhibited abnormal LTP-like plasticity of the R2 response, a late bilateral blink reflex component. The relationship between R2 latency and the plastic properties of the blink reflex circuits are not known. Increased R2 latency has been reported in several studies in both presymptomatic and symptomatic HD patient [18,36,51]. Increased synaptic delay, decreased release of neurotransmitters within the reflex pathway and dysfunctions of the basal ganglia output to the brainstem might account for this abnormality [51]. We selected patients with normal R2 latency; thus our results point towards remarkable plasticity deficits even in HD patient with normal R2 latency.

The protocol was approved by the IRB of the University of Messina, Italy. Conflict of interest The Authors do not have actual or potential conflicts of interest. Acknowledgements Supported by MIUR, Italy and PCS-CUNY grants.

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