Reversal of plasticity-like effects in the human motor cortex

Transcription

1 J Physiol (2010) pp Reversal of plasticity-like effects in the human motor cortex Ying-Zu Huang 1,JohnC.Rothwell 2,Chin-SongLu 1, Wen-Li Chuang 1, Wey-Yil Lin 1 and Rou-Shayn Chen 1 1 Department of Neurology, Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Taipei 10507, Taiwan 2 Sobell Department of Motor Neuroscience and Movement Disorders, Institute of Neurology, University College London, Queen Square, London, UK A number of experiments in animals have shown that successful induction of plasticity can be abolished if an individually ineffective intervention is given shortly afterwards. Such effects are termed depotentiation/de-depression. These effects contrast with metaplasticity/homeostatic plasticity in which pretreatment of the system with one protocol modulates the response to a second plasticity-inducing protocol. Homeostatic plasticity maintains the balance of plasticity in the nervous system at a stable level whereas depotentiation/de-depression abolishes synaptic plasticity that has just occurred in order to prevent ongoing learning. In the present study, we developed novel protocols to explore the reversal of LTP- and LTD-like effects in healthy conscious humans based on the recently developed theta burst form of repetitive transcranial magnetic stimulation (TBS). The potentiation effect induced by intermittent TBS (itbs) was completely erased by a short form of continuous TBS (ctbs150) given 1 min after itbs, whereas the depressive effect of continuous TBS (ctbs) was successfully abolished by a short form of itbs (itbs150). The reversal was specific to the nature of the second protocol and was time dependent since it was less effective when the intervention was given 10 min after induction of plasticity. All these features are compatible with those of depotentiation and de-depression demonstrated in animal studies. The development of the present protocols would be helpful to study the physiology of the reversal of plasticity and learning and to probe the abnormal depotentiation/de-depression shown in animal models of neurological diseases (e.g. Parkinson s disease with dyskinesia, dystonia and Huntingon s disease). (Resubmitted 12 April 2010; accepted after revision 20 July 2010; first published online 26 July 2010) Corresponding author R-S. Chen: Department of Neurology, Chang Gung Memorial Hospital, Taipei 10507, Taiwan. cerebrum@adm.cgmh.org.tw Abbreviations AMT, active motor threshold; ctbs, continuous theta burst stimulation; FDI, first dorsal interosseus muscle; ICF, intracortical facilitation; ISI, inter-stimulus interval; itbs, intermittent theta burst stimulation; LTD, long term depression; LTP, long term potentiation; MEP, motor evoked potential; PAS, paired associative stimulation; rtms, repetitive transcranial magnetic stimulation; SICI, short-interval intracortical inhibition; TBS, theta burst stimulation; tdcs, transcranial direct current stimulation; TMS, transcranial magnetic stimulation. Introduction The efficiency of synaptic transmission can be either potentiated or depressed by natural patterns of activity or by experimental intervention. Most investigations into these phenomena of synaptic long term potentiation (LTP) and long term depression (LTD) have been performed in reduced animal preparations. However, recent work using repetitive transcranial stimulation has shown that it is also possible to produce lasting effects on physiology and behaviour in humans that may involve changes in synaptic function within the brain (Chen et al. 1997; Nitsche & Paulus, 2000; Stefan et al. 2000; Peinemann et al. 2004). Although most of the effects induced in conscious humans last for a shorter time than those described in animals, several lines of evidence, e.g. the dependence on the N-methyl-D-aspartic acid (NMDA) receptor and its interaction with motor learning, suggest that the underlying mechanisms are similar to those of synaptic LTP/LTD (Nitscheet al. 2004; Ziemann et al. 2004; Huang et al. 2007; Teo et al. 2007; Gentner et al. 2008; Huang et al. 2008). Experiments in animals have shown that synaptic plasticity is controlled by several mechanisms. Thus, the ease with which it is possible to produce LTP/LTD depends on the previous history of activity in the system such that LTD is easier to produce after a history of high activity and LTP after a history of low activity (Bienenstock et al. 1982). This type of control is often termed DOI: /jphysiol

2 3684 Y.-Z. Huang and others J Physiol homeostatic plasticity. A second type of control determines how long the LTP/LTD may last. Depotentiation/de-depression describes a mechanism by which changes in plasticity that have been induced by successful protocols are abolished by a second protocol. Thus a high frequency conditioning protocol might induce LTP which would then be abolished by subsequent application of a short period of low frequency stimulation (Larson et al. 1993; Kulla & Manahan-Vaughan, 2000; Huang et al. 2001). Importantly, the latter would have no effects on LTP/LTD when applied alone. Data show that the ease of reversing LTP/LTD is greatest if the reversing protocols are applied immediately after the initial plasticity protocol and become gradually less successful with longer intervals (Larson et al. 1993; Staubli & Scafidi, 1999; Chen et al. 2001), suggesting that LTP/LTD consolidates over time. The phenomena of depotentiation/de-depression may account for retrograde interference with behavioural learning. In addition abnormal reversibility of LTP/LTD has been shown in animal models of Parkinson s disease with drug-induced dyskinesia (Picconi et al. 2003), DYT1 dystonia (Martella et al. 2009) and Huntington s disease (Murphy et al. 2000; Picconi et al. 2006). The present experiments sought to identify possible depotentiation/de-depression in the human brain using TMS of the motor cortex. We employed TBS of the motor cortex, which has been shown to produce LTP/LTD-like effects that outlast the period of stimulation for min (Huang et al. 2005; Huang et al. 2009). We then tried to abolish the after-effects by applying at different times afterwards a second very short period of TBS that when delivered alone had no effect on motor cortex excitability. Methods Subjects Healthy non-medicated subjects gave their informed consent prior to participation. The experiments were performed with the approval of the Institutional Review Board of the Chang Gung Memorial Hospital, Taiwan. All subjects were naive to the effects of TBS and unaware of the differences between itbs and ctbs protocols. In addition, control experiments were performed to avoid participant bias. It was not possible to study the same subjects in all experiments, but within each protocol all subjects were the same and hence we could directly compare their behaviour in test and baseline trials. One limitation of the designs we used was that the experimenter was not blinded to the type of stimulation. This is difficult to achieve in a TMS experiment since the protocols can be distinguished by the sound of the stimuli in the coil. A potential way around this would be to apply the depotentiating/de-depressing stimuli through a sham coil placed over (or under) a real coil that delivered the initial TBS patterns. Unfortunately we do not possess one of the new sham coils that is truly indistinguishable from a real device (see O Reardon et al. 2007) and were therefore unable to provide this gold standard control. However, it is an important point, which should be taken into account when considering the results to this and many other TMS studies. Recording and stimulation Subjects were seated in a comfortable chair. EMGs were recorded using Ag AgCl electrodes from the right (the dominant hand in all subjects) first dorsal interosseus muscle (FDI). EMG activity was recorded with a gain of 1000 and 5000 and filtered with a band-pass filter (3 Hz to 2 khz) through Digitimer D360 amplifiers (Digitimer Ltd, Welwyn Garden City, UK). Signals were recorded with a sampling rate of 5 khz and stored on a personal computer for later analysis by Signal software (Cambridge Electronic Design Ltd, Cambridge, UK) through a Power 1401 data acquisition interface (Cambridge Electronic Design). Trials in which the target muscle was not relaxed (as monitored by an oscilloscope) were rejected online, and that stimulus condition was repeated. Magnetic stimulation was given using a hand-held figure-of-eight coil with loop diameters of 70 mm (Magstim Co., Whitland, Dyfed, UK). Single pulse TMS was delivered by a Magstim machine, and TBS was delivered using a Magstim Rapid 2 stimulator. Stimulation was delivered over the motor hand area with the coil tangential to the scalp and the handle pointing in the posterior direction. The motor hand area was defined as the location on the scalp where magnetic stimulation produced the largest MEP from the contralateral FDI when the subject was relaxed (the motor hot-spot ). The stimulation intensity of TBS was defined in relation to the active motor threshold (AMT) of the subject. The AMT was defined for each Magstim machine separately as the minimum intensity of single pulse stimulation required to produce an MEP of greater than 200 μvonmorethan 5 out of 10 trials from the contralateral FDI while the subject maintained a voluntary contraction of the FDI to about 20% maximum. Visual feedback of EMG level wasprovidedtohelpsubjectsmaintainaconstantlevelof contraction. TBS protocols The protocols used for TBS are based on those that we previously reported (Huang et al. 2005). They comprised bursts of three pulses at 50 Hz at an intensity of 80% AMT repeated at 200 ms intervals (i.e. at 5 Hz) and were all given over the motor hot-spot. Four TBS protocols were used in this study: (1) intermittent TBS (itbs): a

3 J Physiol Depotentiation and de-depression in humans s train of TBS repeated every 10 s for 20 repetitions to have 600 pulses in total; (2) continuous TBS (ctbs): a 20s train of uninterrupted TBS containing 300 pulses; (3) a shorter form of ctbs containing 150 pulses (ctbs150): a 10 s train of uninterrupted TBS; and (4) a shorter form of itbs containing 150 pulses (itbs150): a 2 s train of TBS repeated every 10 s for five repetitions. itbs was used to potentiate motor cortical excitability, while ctbs was used to depress excitability. ctbs150 and itbs150 applied alone had no effect on cortical excitability and were designed to test the reversibility of potentiation and depression respectively. Each experiment was performed at least 1 week apart in a pseudo randomised order. In all the following experiments, the intensity of stimulation for MEP assessment was set to that required to produce an MEP of approximately 1 mv in the baseline condition. In animal studies, depotentiation or de-depression is usually induced by a protocol milder than those used toinduceltdorltp,andwhichonitsownhasno effect on naive cortex (for review, see Zhou & Poo, 2004). Thus, depotentiation is usually produced by applying an LTD-like protocol (e.g. low frequency stimulation ranging from 1 to 5 Hz) (O Dell & Kandel, 1994; Staubli & Scafidi, 1999; Huang et al. 2001) at lower stimulus intensity or for a shorter time than usual. We decided to use a shorter form of ctbs (i.e. ctbs150) to test the reversibility of the effects induced by itbs for three reasons. (1) We did not employ a lower intensity protocol since we were unsure whether stimulation at a reduced intensity would activate the same circuits as those involved in the LTP-like effects of itbs. This was also the reason for not using protocols that are believed to act via different mechanisms, e.g. transcranial direct current stimulation (tdcs) or paired associative stimulation (PAS), as the reversal protocol. (2) We chose to use 150 pulses as the stimulus since a previous study showed that trains of TBS shorter than 75 pulses tend to be excitatory (Huang et al. 2005). A ctbs train of 150 pulses was therefore a compromise between too short a train causing facilitation and too long a train causing inhibition. (3) A final possibility would have been to use regular rtms at a fixed rate. However, this usually requires a lengthy period of stimulation which would have blurred the time course of the interaction we observed. As regards the protocol for de-depression, although less well studied in animal experiments, intermittent high frequency stimulation has been commonly used (Chen et al. 2004; Kumar et al. 2007). Two shorter forms of TBS, i.e. itbs150 and ctbs150, were tested as reversal stimulation for de-depression. Experimental design (Fig. 1) Experiment 1: itbs150 or ctbs150 interventions at 1 min after the end of itbs. Eight subjects (1 man, 7 women; mean age, 33.3 ± 10.3 years) were recruited for this experiment. We first tested the effect of ctbs150 on the size of MEPs. Baseline MEP was determined as the average of 30 MEPs evoked by a standard TMS pulse delivered every s. ctbs150 was then given, and the effect on motor cortex excitability was assessed using single pulses of TMS delivered in trains of 12 pulses given every s every 1 min for 6 min then every 2 min until 21 min after the end of TBS. In separate experiments we examined how the effect of itbs was modulated by ctbs150 given 1 min after the end of itbs. Subjects came for two sessions in a random order. (1) The itbs session: baseline MEPs were evoked using 30 pulses delivered every s. itbs was then applied to the subject. MEP size was assessed using single pulses of TMS delivered in trains of 12 pulses given every s every 1 min for 6 min, then every 2 min until 21 min after the end of itbs. (2) The depotentiation (DePo) session: the procedure was similar to that of the itbs session, except that ctbs150 was given over the motor hot-spot 1 min after the initial assessment of MEP after the end of itbs. In addition, a control study (DePo-i) was tested in eight subjects (2 men, 6 women; mean age, 32.1 ± 3.0 years) using a protocol similar to that of the DePo session, in which ctbs150 was replaced by itbs150. This was a control experiment to test whether any short period of stimulation could reverse the plasticity induced by itbs. It also provides useful information that differentiates reversal of plasticity from homeostatic plasticity. Experiment 2: itbs150 or ctbs150 interventions at 1 min after the end of ctbs. Seven subjects (4 men, 3 women; mean age, 28.7 ± 3.6 years) were recruited for this experiment. We first tested the effect of itbs150 on the size of MEP as described above for ctbs150. Then subjects were asked to come for a further two sessions in a random order to examine the effect of ctbs on MEP size and how this was modulated by itbs150 given 1 min after the end of ctbs. (1) The ctbs session: the effect of ctbs was assessed as described above for the itbs session. (2) The de-depression (DeDe) session: the procedure was similar to that of the ctbs session except that itbs150 was given over the motor hot-spot 1 min after initial assessment of MEPs after the end of ctbs. In addition, a third session (DeDe-c) was tested using a protocol similar to that of the DeDe session, in which itbs150 was replaced by ctbs150. Experiment 3: intervention at 10 min after TBS. Eight subjects(3men,5women;meanage,32.1± 3.8 years), who did not participate Experiment 1 and 2, participated in this experiment. Subjects came for two sessions: (1) DePoX session: ctbs150 was given 10 min after the end of itbs; and (2) DeDeX session: itbs150 was given 10 min after the end of ctbs. Thirty baseline MEPs were recorded

4 3686 Y.-Z. Huang and others J Physiol as in previous experiments. After itbs or ctbs was given, MEP size was assessed in trains of 12 pulses given every s every 1 min for 6 min, then every 2 min until 21 min. In between, ctbs150 or itbs150 was given 10 min after the end of itbs or ctbs, respectively. Experiment 4: effects on intracortical inhibition and facilitation. Given that short-interval intracortical inhibition (SICI) and intracortical facilitation (ICF) are modified by TBS (Huang et al. 2005, 2009), we tested if the reversal of the effects on MEP size could also be observed on SICI and ICF. SICI and ICF were tested before and at 5 min after the end of the TBS protocols in this part of the experiment. The conditioning stimulus was given at 80% AMT. The test stimulus was set to produce an MEP of 1 mv and was not adjusted after TBS conditioning since we expected no change in MEP size after the TBS protocols in this experiment. Subjects received in a random order either the test stimulus alone or conditioning test stimuli atinter-stimulusinterval(isi)of2,3,4,7,10and12ms for a total of 10 trials per condition. Six subjects (2 men, 4 women; mean age, 30.3 ± 3.1 years) came for four sessions with different TBS protocols: itbs150, ctbs150, DePo (ctbs150 given at 1 min after itbs) and DeDe (itbs150 givenat1minafterctbs). Data analysis Data were analysed using SPSS. One-way repeated measures ANOVA on the absolute amplitude of MEPs or two-way repeated measures ANOVA on MEP amplitudes Figure 1. Experimental design In experiment 1, depotentiation was studied in two separate sessions: itbs and DePo sessions. In experiment 2, de-depression was evaluated in three separate sessions: ctbs, DeDe and DeDe-c sessions. In experiment 3, the time dependency of depotentiation and de-depression was evaluated in two separate sessions: DePoX and DeDeX sessions. In experiment 4, SICI and ICF before and after ctbs150, itbs150, depotentiation protocol (itbs+ctbs150) and de-depression protocol (ctbs+itbs150) were tested in 4 separate sessions.

5 J Physiol Depotentiation and de-depression in humans 3687 (ISI of 10 and 12 ms) and compared with Student s t test. A P < 0.05 was considered statistically significant. Results The effect of ctbs150 and itbs150 A one-way ANOVA showed that ctbs150 and itbs150 given alone did not change the size of MEPs (F 14,98 = 0.601, P = 0.859; F 14,84 = 0.660, P = 0.806, respectively) (Fig. 2). Figure 2. Effect of ctbs150 and itbs150 on MEPs There was no after-effect on the size of MEPs when ctbs150 or itbs150 was given alone. normalised to the pre-tbs baseline amplitude was used to examine the time course of changes in MEP amplitude and to test the effects of ctbs or itbs and the influence of the ctbs150 or itbs150 interventions. SICI and ICF before and after conditioning were compared with two-way repeated measures ANOVA. The data were also separated on apriorigrounds into SICI (ISI of 2 and 3 ms) and ICF itbs150 or ctbs150 interventions at 1 min after the end of itbs Figure 3 compares the after-effect of applying itbs alone with itbs followed by ctbs150 at 1 min (DePo) and itbs followed by itbs150 at 1 min (DePo-i). When it was applied alone, itbs facilitated MEPs (one-way ANOVA of all time points for itbs, F 14,98 = 1.799, P = 0.049). Addition of ctbs150 abolished the facilitation. This was confirmed in the two-way ANOVA of the time points after application of ctbs150, which showed a significant main effect of PATTERN (itbs and DePo) (F 1,7 = , Figure 3. Results of depotentiation experiments A, averaged EMG traces from one subject are shown at each measured time epoch. B, ctbs150 successfully abolished the potentiation following itbs by bringing it back to the baseline level when it was given at 1 min after itbs. C, however, itbs150 given at 1 min after itbs failed to change the effect of itbs. Black line and asterisk: statistically significant; grey line and ns: not significant.

6 3688 Y.-Z. Huang and others J Physiol P = 0.011). Paired t tests demonstrated that there was no difference between the initial amount of facilitation in the first minute after itbs (t = 0.689, P = 0.513). In contrast to the effect of ctbs150, application of itbs150 did not have any effect on the response to itbs (two-way ANOVA: no significant main effect of PATTERN (F 1,14 = 0.025, P = ) or PATTERN TIME interaction (F 12,168 = 0.689, P = 0.761). itbs150 or ctbs150 interventions at 1 min after the end of ctbs Figure 4 compares the result of ctbs alone with that of ctbs followed by itbs150 (DeDe) and ctbs followed by ctbs150 (DeDe-c). When it was applied alone, ctbs suppressed MEPs (one-way ANOVA of all time points for ctbs, F 14,84 = 2.666, P = 0.003). Addition of itbs150 abolished the suppression. This was confirmed in the two-way ANOVA of the time points after application of itbs150 which showed a significant main effect of PATTERN (ctbs vs. DeDe) (F 1,6 = 8.284, P = 0.028). In contrast to the effect of itbs150, application of ctbs150 did not have any effect on the response to ctbs (two-way ANOVA: no significant main effect of PATTERN (F 1,6 = 0.263, P = ) or PATTERN TIME interaction (F 11,66 = 1.142, P = 0.344)). One-way ANOVA demonstrated that there was no difference between the initial amount of suppression in the first minute after ctbs in all three sessions (F 2,12 = 0.697, P = 0.517). Intervention at 10 min after TBS Figure 5A compares the result of DePoX session with that of itbs session. There was neither a PATTERN (itbs and DePoX) effect (F 1,14 = 0.024, P = 0.891) nor a PATTERN TIME interaction (F 13,182 = 0.856, P = 0.601) using two-way ANOVA. Further comparisons on the time points before and after ctbs150 were performed separately and confirmed that there was no significant difference between the two sessions. (F 1,14 = 0.096, P = 0.761; F 1,14 = 0.220, P = 0.646, respectively). Figure 5B compares the result of DeDeX session with that of ctbs session. There was neither a PATTERN (ctbs and DeDeX) effect (F 1,13 = 2.740, P = 0.132) nor a PATTERN TIME interaction (F 13,169 = 1.154, P = 0.318) using two-way ANOVA. However, a further comparison on the time points after itbs150 revealed a significant effect of PATTERN (F 1,13 = 6.298, P = 0.026) such that MEPs returned more quickly towards baseline Figure 4. Results of de-depression experiments A, averaged EMG traces from one subject are shown at each measured time epoch. B, itbs150 successfully abolished the depression that follows ctbs by returning it to the baseline level when it was given at 1 min after ctbs. C, however, ctbs150 given at 1 min after ctbs failed to change the effect of ctbs. Black line and asterisk: statistically significant; grey line and ns: not significant.

7 J Physiol Depotentiation and de-depression in humans 3689 after itbs150. There was no significant PATTERN effect (F 1,13 = 0.269, P = 0.613) or PATTERN TIME interaction (F 7,91 = 0.870, P = 0.533) before the itbs150 intervention. SICI/ICF The amplitude of control MEPs was not changed by ctbs150 (t = 1.077, P = 0.331), itbs150 (t = 0.925, P = 0.397), DePo (t = 0.800, P = 0.460) and DeDe (t = 1.354, P = 0.234). Figure 6 compares SICI/ICF before and after ctbs150 (Fig. 6A), itbs150 (Fig. 6B), DePo (Fig. 6C) and DeDe (Fig. 6D). In each of the sessions, there was neither a main effect of CONDITION (before and after) (ctbs150: F 1,5 = 0.000, P = 0.989; itbs150: F 1,5 = 0.339, P = 0.586; DePo: F 1,5 = 0.287, P = 0.615; DeDe: F 1,5 = 0.044, P = 0.842) nor a CONDITION ISI (2, 3, 4, 7, 10 and 12 ms) interaction (ctbs150: F 4,20 = 0.284, P = 0.885; itbs150: F 4,20 = 0.411, P = 0.799; DePo: F 4,20 = 1.231, P = 0.329; DeDe: F 4,20 = 1.208, P = 0.338). Since the underlying mechanism is known to be different, we performed separate comparisons on SICI (at ISI of 2 and 3 ms), ICF (10 and 12 ms) and the effect at an ISI of 7 ms. None of SICI, ICF or the effect at an ISI of 7 ms was changed by ctbs150 (P = 0.807, 0.651, 0.962, respectively), itbs150 (P = 0.161, 0.766, 0.782, respectively), DePo (P = 0.482, 0.958, 0.319, respectively) or DeDe (P = 0.541, 0.451, 0.383, respectively). Note that in Fig. 6C and D, SICI/ICF was first evaluated at baseline prior to any TBS. It was tested again after itbs+ctbs150 or ctbs+itbs150. At this time SICI/ICF was not different from baseline. ICF. Importantly, the effect of itbs was not changed by applying a short session of itbs150 rather than ctbs150 and the effect of ctbs was not changed by ctbs150, indicating that depotentiation and de-depression were specific to the nature of the second protocol. Finally, these effects depended on the time interval between protocols. Thus ctbs150 had no effect on facilitation if it was applied 10 min after itbs. The reversal of depression was still present if itbs150 was applied at 10 min after ctbs, but in contrast to the very quick reversal when itbs150 was given at 1 min, it now took 5 min to build up. As noted in Methods, although subjects were blinded to the experimental design, the experimenters were not. This is a potential limitation that is difficult to overcome with present TMS technology. However, advances in coil designs that have been piloted in clinical studies of depression (O Reardon et al. 2007) may make this feasible in future studies. Our hypothesis is that these effects are compatible with the phenomena of depotentiation and de-depression that have been described in experiments on brain slices in areas Discussion As reported previously (Huang et al. 2005, 2007, 2008), itbs potentiated the amplitude of MEPs for approximately 20 min after the end of stimulation whereas ctbs depressed the amplitude of MEPs for 20 min or so. The shorter protocols of ctbs150 and itbs150 had no after-effects on the size of MEPs nor on the amount of SICI and ICF. Despite this, both of them could interact with the longer lasting after effects of conventional ctbs/itbs. Thus, ctbs150 abolished potentiation produced by itbs if it was given 1 min after the end of itbs; similarly, depression produced by ctbs was abolished when itbs150 was delivered 1 min after the end of ctbs. Importantly there was no change in SICI/ICF measured at the time that the MEP amplitude had returned to the baseline level after depotentiation/de-depression. This suggests that all the effects of itbs and ctbs reported previously (Huang et al. 2005) were abolished by the second stimulation and that the reversal of MEP size was not due to a shift in balance between SICI and Figure 5. Reversal of plasticity is time dependent A, ctbs150 failed to abolish the potentiation induced by itbs when it was given at 10 min after itbs. B, similarly, itbs150 given at 10 min after ctbs abolished the depression induced by ctbs, although in this case, the effect took about 5 min before it was complete. Black line and asterisk: statistically significant; grey line and ns: not significant.

8 3690 Y.-Z. Huang and others J Physiol including hippocampus, cerebellum and corticostriatal pathways (Huang et al. 2001; Picconi et al. 2003; Coesmans et al. 2004; Kumar et al. 2007) as well as in the brains of freely moving animals (Staubli & Scafidi, 1999; Kulla & Manahan-Vaughan, 2000). It is important to distinguish depotentiation and de-depression from the similar but unrelated phenomenon of synaptic metaplasticity, which describes how synaptic plasticity can be modulated by prior synaptic activity (Abraham & Bear, 1996). The latter involves giving a priming protocol that changes the plasticity induced by a subsequent protocol whereas depotentiation/de-depression require that plasticity is first induced by a standard protocol but is then abolished by a second protocol. In metaplasticity, the priming protocol may or may not cause a long term effect on the system under test (Abraham, 2008). On the contrary, the abolition protocol in depotentiation/de-depression on its own usually has no long term effect (Zhou & Poo, 2004). Metaplasticity prevents LTP/LTD from driving a system into instability whereas depotentiation/de-depression is thought to abolish previously acquired learning. For example, Xu et al. (1998) demonstrated that LTP induced in the hippocampus of freely moving rats was completely erased when the rat entered a new environment. There have been many studies in humans of the interaction between various types of rtms and tdcs (transcranial direct current stimulation) protocols, but all of these have been designed to test for metaplasticity rather than depotentiation/de-depression. In some cases, a weak priming protocol was followed by a reliable test protocol (Iyer et al. 2003; Hamada et al. 2008; Todd et al. 2009), but in many cases, each protocol alone was capable of producing long term effects on cortical excitability (Siebner et al. 2004; Quartarone et al. 2005; Muller et al. 2007; Potter-Nerger et al. 2009). As far as we are aware, there is only one previous study in humans in which an effective priming protocol was followed by an ineffective test protocol. Siebner et al. (2004) studied the interaction between an effective tdcs protocol and a 15 min train of 1 Hz rtms at 85% rest motor threshold which had no significant effect on MEPs when given alone. In this case, the 1 Hz rtms did not simply abolish the effect of preceding tdcs, as expected from depotentiation/de-depression; instead it reversed the effect. Thus Siebner et al. (2004) interpreted their results in terms of a homeostatic interaction between a potentiating or depressing pretreatment with tdcs and a potentially suppressive effect of 1 Hz rtms. In fact, as Siebner et al. (2004) pointed out, although 1 Hz rtms did not produce a significant effect on MEPs in their study, several other studies had found that very similar 1 Hz rtms protocols, sometimes at a slightly higher intensity (e.g. 90% rest motor threshold), produced long term effects on MEP size (Romero et al. 2002; Lang et al. 2006) and SICI/ICF (Romero et al. 2002; Modugno et al. 2003). Indeed, this form of 1 Hz rtms can have clinical benefits in patients with laevodopa induced dyskinesia (Brusa et al. 2006). It should be noted that in the present study, the protocols for abolishing previously induced plasticity (ctbs150 and itbs150) are much milder and shorter than those used by Siebner et al. (2004) and produced Figure 6. SICI and ICF before and after depotentiation/de-depression SICI/ICF was not changed by ctbs150 (A), itbs150 (B), depotentiation protocol (C) or de-depression protocol (D).

9 J Physiol Depotentiation and de-depression in humans 3691 no visible effect on either MEPs or SICI/ICF. Thus we think that the present protocol is a close parallel to the depotentiation/de-depression protocols used in animal literature. We cannot exclude the possibility that the results are an example of partial homeostatic interactions in which a slightly more effective protocol would have led to a reversal rather than abolition of the priming effect. However, this is less likely since itbs did not prime itbs150 to produce inhibition and ctbs did not prime ctbs150 to produce facilitation or at least to block the ongoing potentiation from itbs and depression from ctbs. Experiments in reduced preparations have shown that depotentiation/de-depression have specific underlying cellular mechanisms. Depotentiation for example is not simply the summation of LTP and LTD. Studies have shown that some forms of depotentiation may activate protein phosphatase-1 and -2A and reverse the phosphorylation of Ser831 on the GluR1 subunit of the AMPA receptor caused by LTP induction and thereby stop ongoing LTP (Huang et al. 2001). In contrast, the Ser845 on GluR1, which is dephosphorylated to produce LTD, is unaffected during depotentiation (for review, see Zhou & Poo, 2004). Recent studies suggest that metaplasticity occurs through different mechanisms in which the priming stimuli modify NMDA receptors (NMDARs) and NMDAR-mediated calcium influx (Abraham, 2008) and may also change the activity of some types of potassium channel (Surmeier & Foehring, 2004). As in animal studies, our experiments showed that reversibility of potentiation caused by itbs is time dependent. When ctbs150 was given at 10 min, instead of 1 min after itbs, it was no longer able to abolish potentiation: the facilitated MEP remained unchanged for another 10 min and was not different from that observed in itbs session alone. In contrast, depression induced by ctbs was still reversible by itbs150 at 10 min after the end of ctbs, although there was a few minutes delay before MEPs returned to the baseline. This difference in the time course of the effects of ctbs150 and itbs150 may relate to experiments in animals which have shown that the time window during which depotentiation may occur depends on a number of factors including the protocol used to induce LTP, the protocol for induction of depotentiation and the pathway under study: it may range from a few minutes (Larson et al. 1993; Huang et al. 1999) to tens of minutes or even more (Burette et al. 1997). The effect of the depotentiation decreases as the interval between LTP induction and delivery of depotentation stimuli is increased (Chen et al. 2001). A weaker depotentiation protocol leads to a narrower time window for depotentiation (Staubli & Scafidi, 1999). Less is known about the time dependency of de-depression although it is usually assumed that it shares common features with depotentiation. Given these data, our current results could be explained by postulating that the baseline LTP/LTD-like effects of itbs/ctbs are equally powerful but that the reversing effect of ctbs150 is slightly stronger than itbs150. Thus, itbs150 cannot reverse ctbs at 10 min whereas ctbs could reverse itbs effects at a similar timing. It has been suggested that reversal of LTP and LTD in the adult might be important in preventing acquisition of inappropriate learning and might contribute to the initial instability of memory before final consolidation (Huang & Hsu, 2001); it may also play a role in developmental refinement of neural circuits (Zhou & Poo, 2004). In the motor system it is well known that motor memories are unstable after a period of learning and vulnerable to interference by subsequent practice of a different task (Walker et al. 2003; Robertson et al. 2004; Krakauer & Shadmehr, 2006). This is compatible with the properties of the reversibility of LTP and LTD. However, the direct connection between synaptic depotentiation and de-depression and interference learning has not been studied. It may be that the protocols developed in the present study would be helpful in understanding such linkage and probing the underlying physiological implications of the reversal of synaptic plasticity. Inconclusion,wehaveshownforthefirsttimethatitis possible to reverse LTP/LTD-like effects in the conscious human brain with short bursts of stimulation that on their own have no apparent effect on cortical excitability. We suggest that this is an example of the phenomenon of depotentiation/de-depression described in the animal literature. This will be helpful to probe natural processes of memory consolidation and forgetting. Moreover, animal models of several pathological conditions are characterised by an abnormality of mechanisms that reverse plasticity such as Parkinson s disease with laevodopa-induced dyskinesia, DYT1 dystonia and Huntington s disease (Murphy et al. 2000; Picconi et al. 2003; Picconi et al. 2006; Martella et al. 2009). 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