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REPETITIVE TMS AND SYNAPTIC PLASTICITY : WHAT IS THE LINK ?

MASTERTHESIS Drs. J.M. Hoogendam

SUPERVISION V.Di Lazzaro, MD G.M.J. Ramakers, PhD

MASTERTHESIS J.M. H OOGENDAM Researchmaster & Cognition, Utrecht University February 2009 Studentnumber 3209008

SUPERVISION V. Di Lazzaro, MD Instituto di Neurol ogia, Università Cattolica, Rome, Italy G.M.J. Ramakers, PhD Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht, the Netherlands

2 Contents Repetitive TMS and synaptic plasticity – what is the link? 4

I. Synaptic plasticity 5 Short-term synaptic plasticity 5 Long-term synaptic plasticity 6 10 LTP, LTD and 10

II. Repetitive Transcranial Magnetic Stimulation 11 Principles of magnetic stimulation 11 Simple rTMS protocols 14 Patterned rTMS protocols 17

III. rTMS and the induction of synaptic plasticity 21 Results that correspond with key-concepts of 22 synaptic plasticity 1. rTMS has effects that outlast the period of 22 stimulation 2. The temporal pattern of pulses is important 22 3. Changes in excitability induced by rTMS 24 depend on the history of activation 4. rTMS interacts with learning 25 Results providing insight in the mechanism by 25 which rTMS induces effects 5. rTMS-induced effects in animal experiments 25 reflect more directly changes in synaptic plasticity 6. Pharmacological interventions shed light on the 28 mechanisms underlying rTMS-induced effects 7. The genetic background of subjects determines 28 the effects of rTMS

IV. Discussion 29 1. Uncertainty and variability 29 2. No direct evidence 30 3. Effects of rTMS on other forms of plasticity 31

V. rTMS in the future 32 Future research 32 The therapeutic potential of rTMS 34

VI. Conclusion 37

VII. References 38

3 Repetitive TMS and synaptic plasticity: what is the link?

During the last two decades, transcranial magnetic stimulation (TMS) has rapidly become a valuable method to investigate non-invasively the human . Depending on the stimulation parameters, TMS is able to excite or inhibit the brain, and offers opportunities to study various aspects of brain functioning. It can be used as a tool to explore the excitability of different regions, but it also offers the possibility to localize brain functions in space and time. Furthermore, repetitive TMS (rTMS) is able to induce changes in brain activity that last after stimulation. Therefore, rTMS has therapeutic potential in patients with neurological and psychiatric disorders like stroke, major depression disorder and schizophrenia. The question is, however, by which mechanisms rTMS influences the brain, resulting in these lasting effects. An appealing hypothesis is that the effects of rTMS on the brain are LTD- or LTP-like, as the lasting effects seem to implicate changes in synaptic plasticity. However, is this assumption supported by experiments and is it possible to be more specific about the way rTMS affects the brain? That is the topic of discussion in this thesis. In the first part, we will discuss several forms of synaptic plasticity and the mechanisms underlying these processes. Secondly, different rTMS protocols will be described. In part III, we present several lines of evidence that suggest a link between the effects of rTMS on the one hand and the processes involved in synaptic plasticity on the other. We then discuss these results (part IV), and state that, although these results are important and to a certain extent give insight into the mechanism by which rTMS modifies the brain, they do not provide direct evidence for the hypothesis that rTMS alters synaptic plasticity. In addition, focusing on the synaptic level seems to be too narrow because rTMS-effects can be widespread through the brain. In part V, we look forward to the future of rTMS. We strongly emphasize the need for innovating research in order to investigate the long-lasting effects of rTMS on the into more detail and give suggestions for such experiments. In addition, we point out the importance of such research for the improvement of rTMS as a treatment for neurological and psychiatric disorders. To conclude, we state there are strong indications for a link between rTMS and synaptic plasticity. However, the details of this link need to be further investigated. Future research needs to examine both the local effects of rTMS on the and the effects of rTMS on other, more global levels of brain organization. Only in that way can the after-effects of rTMS on the brain be completely understood.

Keywords rTMS; protocols; LTP; LTD; synaptic plasticity.

Abbreviations AMPA = alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; AMT = active motor threshold; BDNF = brain derived neurotrophic factor; CA1/CA3 = cornu ammonis 1/3; CaMKII = Ca 2+ calmodulin-dependent protein ; cAMP = cyclic adenosine monophosphate; CREB = cAMP response element-binding protein; cTBS = continuous theta burst stimulation; DLPFC = dorsolateral prefrontal cortex; D-wave = direct wave; EEG = electroencephalography; EPSP = excitatory ; FEF = frontal eye field; fMRI = functional magnetic resonance imaging; FST = forced swim test; HSF = high frequency stimulation; ISI = interstimulus interval; iTBS = intermittent theta burst stimulation; I-wave = indirect wave; IEG = immediate early gene; (E/L)-LTP = early/ late long-term potentiation; (E/L)-LTD = early/late long-term depression; M1 = primary motor cortex; MEG = magnetoencephalography; MEP = motor ; MT = motor threshold; NMDA = N-methyl-D-aspartate; PET = positron emission tomography; PKA = protein kinase A; PKC = protein kinase C; PPS = paired pulse stimulation; PTP = post-tetanic potentiation; QPS = quadripulse stimulation; rCBF = regional cerebral blood flow; RMT = resting motor threshold; (r)TMS = repetitive transcranial magnetic stimulation; SO = stimulator output; STDP = spike timing-dependent plasticity ; T = tesla; TBS = theta burst stimulation; TMS = transcranial magnetic stimulation; VBM = voxel based morphometry.

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I. Synaptic plasticity strengthened or weakened. These Plasticity is the ability of the brain to changes could occur in time scales reorganize itself, enabling short- and long- ranging from milliseconds to days or even term remodeling of neural communication longer ( figure 1 ) [18]. In the following, that outlasts an experimental manipulation both short-term and long-term changes in or period of training [12]. Goal of these synaptic strength will be discussed. In processes is the improvement of the addition, we will describe the concept of functioning of brain networks. Plasticity metaplasticity and the role of synaptic can occur at various levels of brain plasticity in learning. organization: from the ultra-structural to synaptic level [14]. Here we focus on Short-term synaptic plasticity synaptic plasticity, because the long- Short-term plasticity is a use-dependent lasting effects of rTMS are thought to change in synaptic strength that is originate from this type of plasticity [1]. observed in almost every synapse of the The synapse should not be considered to central [19]. There is, be static, but rather a dynamic connection however, no well-defined consensus of the between neurons that is constantly time-period in which these changes have changing in response to neural activity and to take place to be called short -term. other influences [17, 18]. By these Some researchers consider only changes changes, synaptic transmission can be lasting up to several seconds as belonging

Figure 1 Overview of changes in synaptic strength The x-axis indicates the time period in which changes take place and their durations, where t = 0 depicts stimulation of the neuron. The y-axis shows the direction of the change (increase or decrease in synaptic strength). E-LTD / L-LTD : early/late long-term depotentiation. E-LTP / L-LTP : early/late long-term potentiation. PTP : post-tetanic potentiation.

5 to short-term synaptic plasticity [19, 20], processes are present, which could while others use a broader time scale and interact with each other [22]. regard changes in synaptic strength Synaptic depression is considered to be lasting up to a few minutes as the result of the opposite of synaptic facilitation ( figure short-term plasticity [18, 21]. 1). In synaptic depression, periods of Typically, neurons communicate with each elevated activity could lead to a decrease other through fast chemical . A in synaptic strength [22]. This depression presynaptic propagates is usually attributed to a depletion of the down the axon and opens voltage-gated pool of presynaptic vesicles that are Ca 2+ channels. As a result, Ca 2+ enters the available for release into the synaptic cleft presynaptic nerve terminal and leads [18, 23]. However, it is thought that rapidly to the release of depression can also arise from feedback into the synaptic cleft. These activation of presynaptic receptors. The neurotransmitters lead, in turn, to a duration of depression ranges from postsynaptic response [22]. Due to hundreds of milliseconds to tens of synaptic plasticity both the presynaptic seconds and most likely reflects the time release of neurotransmitters as well as the required for the pool to be refilled [20]. postsynaptic response to the released Postsynaptic changes neurotransmitters could be altered, leading In addition to changes in presynaptic to an adapted synaptic connection [20]. neurons, also postsynaptic neurons could These changes will be described in the be involved in short-term synaptic following. plasticity. For instance, postsynaptic Presynaptic changes receptors could become less sensitive to Repeated activation of the presynaptic the released . This neuron by two or more action potentials in process is called desensitization and could close succession increases the occur at most ionotropic receptors [20]. concentration in the presynaptic terminal. The result of desensitization is smaller This is called facilitation (figure 1 ) and EPSPs. Another postsynaptic process results in more neurotransmitter being leading to a decrease in synaptic strength released into the synaptic cleft by a is saturation . In this case, fewer subsequent presynaptic action potential, postsynaptic receptors are available for and eventually in an increased excitatory neurotransmitter to bind on to. The exact postsynaptic potential (EPSP) [20, 22, 23]. effects of saturation depend on the type of Facilitation typically persists for several receptor that is saturated [23]. hundred milliseconds following presynaptic activity. Post-tetanic potentiation (PTP), Long-term synaptic plasticity however, could persist for several minutes Long-term changes in synaptic strength, (figure 1 ). PTP is a transient increase of like long-term potentiation (LTP) and long- glutamate release into the synaptic cleft term depression (LTD) have been the that results from brief periods of high- focus of an enormous amount of frequency stimulation. Like facilitation, investigation and are widely studied in a PTP is triggered by the elevation of variety of species [27]. However, LTP and intracellular Ca 2+ in presynaptic nerve LTD are not unitary phenomena, but broad terminals [24]. This results in increased terms that describe only the direction of a EPSPs. long-lasting change in synaptic efficacy Because the length of PTP outlasts the [28, 29]. LTP is an increase in the synaptic period of increased Ca 2+ levels, it is strength that could last for days or even hypothesized that the influx of Ca 2+ into the weeks or months, which could be induced neuron triggers other processes which in in experimental conditions as a result of turn leads to PTP, like the presynaptic brief high-frequency stimulation. LTD, on protein kinase C (PKC) pathway [25]. This the contrary, encompasses long-lasting pathway is critically involved in PTP [24, weakening of a neuronal synapse ( figure 26], but the exact molecular mechanism is 1) [14, 18]. However, the exact until now not completely understood. mechanisms underlying these plastic Hypothesized is that often multiple changes vary, depending on the synapses

6 and the circuits in which they operate. In (STDP), and later studies have defined the following, the induction, expression ‘critical windows’ for spike timing which are and maintenance of LTP and LTD will be on the order of tens of milliseconds [34]. described. Stimulating first the presynaptic neuron, Induction of LTP followed by stimulation of the postsynaptic LTP and LTD are most frequently studied neuron within a window of tens of in the CA1 region of the . In milliseconds induces LTP, while 1973, Bliss and Lømo described for the stimulation in the reverse order (post – first time that high-frequency stimuli trains pre) leads to LTD. No changes are (tetani) delivered to the axons of the observed if the presynaptic and pyramidal cells in the hippocampus lead to postsynaptic activities are separated by a long-lasting increase in EPSP-amplitude, more than 100 ms [35]. called LTP [18, 30]. Essential for the The cellular basis of this temporal window induction of LTP is 1 st the coincidence seems to be the NMDA receptor again, between pre- and postsynaptic activity and although other molecular mechanisms 2nd a specific temporal relationship cannot be excluded and different between these activities [27]. synapses seem to have cell-specific STDP Coincidence between pre- and windows [34, 36]. postsynaptic activity is at most synapses STDP has several functional implications. detected by the N-methyl-D-aspartate First of all, it probably reflects more (NMDA) receptor. This receptor is placed directly the physiological mechanism postsynaptically and binds glutamate that underlying changes in synaptic strength is released from the presynaptic neuron as than high-frequency stimulation does. In a result of an action potential. The NMDA- addition, STDP could serve as a receptor has an intrinsic cation channel, mechanism to code causality of external which is blocked by ions when events because of the specific relationship the postsynaptic cell is at its normal between pre- and postsynaptic activation resting [27]. Only [34]. Furthermore, the narrow time window when the postsynaptic neuron is allows the system to select inputs with a sufficiently depolarized by high-frequency millisecond precision. In this way, STDP stimulation, the Mg 2+ blockade will be can facilitate synchronization between expelled and the channel of the NMDA neurons. receptor will be opened. As a result, Ca 2+ This NMDA receptor-dependent mecha- enters the postsynaptic cell, initiating LTP nism explains several characteristics of induction [27]. So, coincidence between LTP, to begin with cooperativity. presynaptic (glutamate release) and Cooperativity refers to the fact that, when postsynaptic events (membrane high frequency stimulation is used to depolarization and release of Mg 2+ block) induce LTP, a crucial number of is necessary for LTP induction. This is in presynaptic fibers must be simultaneously accordance with Hebbs postulate, who activated to elicit LTP [37]. This implies the proposed as early as 1949 that correlated existence of a threshold for LTP induction, activity of pre- and postsynaptic neurons is depending on stimulus intensity, the important for the dynamics of synaptic number of pulses and the pattern of organization [31, 32]. stimulation. This property is explained by Secondly, the temporal relationship the above described mechanism: the between stimulation of pre- and postsynaptic cell must be sufficiently postsynaptic neurons has received depolarized to allow current (particularly considerable attention over the past Ca 2+ ) to flow through the NMDA receptor several years. In 1983, Levy and Steward channel, in order to induce LTP [37]. discovered that, when a weak and a However, this hypothesized mechanism is strong input were activated together, the recently questioned [38]. temporal order of the pre- and Closely related to cooperativity is postsynaptic spiking determined whether associativity. Although weak stimulation is LTP or LTD was induced [33]. This was not able to induce LTP in a certain called spike timing-dependent plasticity pathway, this pathway could undergo LTP

7 if a neighboring pathway ending on the candidates are and arachidonic same postsynaptic neuron is strongly acid [40]. activated (associativity). Like cooperativity, LTP maintenance associativity is thought to play a role in Once induced and expressed, LTP is behavioral associative learning [38]. commonly divided in two forms: early- LTP Thinking back to the NMDA receptor, (E-LTP) and late-LTP (L- LTP) [41]. The weak stimulation reflects the situation in changes described above account for the which the postsynaptic neuron is not increase in synaptic strength that stays enough depolarized to expel Mg 2+ from the present for 30-60 minutes after induction NMDA receptor and the channel is not and thus reflect E-LTP. These changes opened. In contrast, strong activation in depend on post-translational modifications another pathway is able to depolarize this of existing proteins, such as protein postsynaptic neuron nevertheless, to open [42]. the NMDA receptor and induce LTP in Other mechanisms are responsible for the both pathways [37]. so called ‘late phase’ of LTP that could last Furthermore, LTP is input-specific . High hours, days or even weeks [28]. L-LTP frequency stimulation induces LTP only at depends on changes in gene expression active synapses, without affecting non- and the synthesis of proteins [43]. active synapses. This can be explained by Signaling molecules like protein kinase A the fact that the channel of the NMDA (PKA) are thought to activate immediate receptor will be opened only at synaptic early genes like zif268 [40, 44]. These inputs that are active. immediate early genes (IEGs) are rapidly LTP expression and transiently activated in response to For the expression of LTP, the Ca 2+ influx neuronal activation; they require no protein through the channel of the NMDA receptor synthesis to be activated and can in turn is essential. Several experiments have activate downstream targets [45]. Thereby, indicated that this Ca 2+ influx serves as a they play a role in the transition from E- second messenger signal [27]. In the LTP to L-LTP [44]. postsynaptic cell, Ca 2+ activates different For L-LTP, the transcription factor cAMP calcium sensitive signaling pathways, like response element-binding protein (CREB) Ca 2+ /calmodulin-dependent protein kinase needs to be activated. The importance of (CaMKII), cAMP and protein kinase C CREB for the maintenance of LTP is (PKC). These signaling mechanisms have demonstrated in mutant mice with a several downstream targets and induce targeted disruption of CREB. These mice both rapid as well as slow changes in the have shown deficits in sustainable LTP pre- and postsynaptic neuron that lead to [46]. CREB induces the transcription of increased synaptic strength [28]. How genes that code for several receptor- these intracellular cascades lead to LTP is associated and structural proteins. These until now not completely clear, but one of proteins are found in both presynaptic and the effects is the rapid insertion of postsynaptic neurons and are responsible additional alpha-amino-3-hydroxy-5- for the morphological restructuring and methyl-4-isoxazolepropionic acid (AMPA) growth of the synapse associated with L- receptors in the postsynaptic neuron. This LTP [40, 47]. Examples of such structural increases the cell’s sensitivity to changes are the so-called ‘slot proteins’ glutamate. Another component of LTP that are synthesized. These proteins are involves modification of the existing AMPA necessary for the preservation of the receptors by direct phosphorylation, supplementary AMPA receptors. The through which their permeability is protein PSD-95 is increased [39]. Furthermore, LTP can one of the synaptic proteins that has increase the ability of presynaptic neurons shown to control activity-dependent AMPA to release glutamate. To this aim, a receptor incorporation [48, 49]. retrograde messenger from the post- to It seems reasonable to assume that these the presynaptic neuron is required. Such a slot proteins are synthesized as a result of messenger is not yet identified [28], but CREB signaling. But how could these proteins, which are synthesized at some

8 distance from the sites at which they are presynaptic and postsynaptic activities are used to stabilize the strength of the separated by more than 100 ms. Like LTP, synapse, find their way to the appropriate LTD is a broad concept and encompasses synaptic terminals? The ‘synaptic tagging a rich diversity of mechanisms [28]. and capture’-hypothesis states that, during One of the mechanisms of LTD induction the induction of LTP, a synaptic tag is is NMDA receptor-dependent LTD, in generated. This tag is placed at the right which activation of the NMDA receptor position, in order to capture plasticity leads to a rise in the post-synaptic Ca 2+ related proteins traveling within the concentration, like it happens in LTP. The [41, 50]. Newly synthesized major determinant whether LTP or LTD proteins can bind to these tags and in that arises appears to be the nature of the Ca 2+ way, the synaptic ‘potentiality’ (i.e. E-LTP) signal. Large and fast rises in the calcium is converted into a longer lasting change ion concentration induce LTP by activating at those synapses at which the tags have specific pathways, small and slow rises been set (i.e. L-LTP) [51]. lead to LTD by turning on other pathways To conclude, several different and partly [18]. In case of LTD, Ca 2+ activates overlapping processes are induced by the phosphatases that cause internalization of influx of Ca 2+ in the postsynaptic neuron. postsynaptic AMPA receptors. Precisely Together, these changes lead to the how Ca 2+ -dependent phosphatases maintenance of LTP for a long period of reduce the surface expression of AMPA time. receptors is still uncertain, but a number of Other forms of LTP converging lines of evidence point to the Besides NMDA receptor-dependent LTP, AMPA receptor GluR2 subunit as a key other forms of LTP exist. For instance, regulator of the AMPA receptor LTP at the mossy fibers in the endocytosis [28]. Furthermore, LTD hippocampus does not require activation probably results from a net loss of slot of the NMDA receptor, but involves a proteins which act as placeholders for presynaptic increase in glutamate release AMPA receptors [28]. Altogether, the [52]. In addition, changes in synaptic decreased amount of AMPA receptors strength in the basal ganglia strongly reduces the sensitivity of the postsynaptic dependent on the activation of dopamine neuron to glutamate. receptors [53, 54]. There are until now a LTD maintenance number of controversies concerning the In analogy to LTP, LTD consists of an mechanism by which LTP is induced in early, short lasting phase (E-LTD) and a these areas [55], but these results indicate late, long-lasting phase (L-LTD). In the broadness of the concept of LTP and addition to the regulated internalization of the various mechanisms it comprises. AMPA-receptors which is important for E- LTD induction and expression LTD, protein synthesis is required to The opposite of LTP is long-term induce L-LTD [56]. A relative large and depression (LTD), in which synaptic heterogeneous set of neural proteins is transmission is depressed for a long involved in maintaining both LTP and LTD, period of time. LTD can reverse the effects and the distinction between LTP and LTD of LTP (‘depotentiation’) or be induced ‘de must lie either in the synthesis of a limited novo’. In vitro, LTD is induced by low- number of critical proteins, or in the frequency stimulation, and in contrast to creation of distinct synaptic tags that allow the short trains of stimulation used for recruitment of distinct subsets of proteins LTP-induction, long periods of stimulation [42, 57]. Recently, it has been shown that (600-900 pulses) are required to induce there are indeed process-specific synaptic LTD [39]. Furthermore, LTD can be tags that regulate the effects of plasticity induced when stimulation of a post- related proteins in either LTP or LTD [58]. synaptic neuron is followed by stimulation This seems to extend the ‘synaptic tagging of the presynaptic neuron within a specific and capture’- hypothesis of Frey and window of tens of milliseconds, relating to Morris [41, 50, 51, 59], in this way that the the concept of STDP [35]. No changes in tags are not only important to determine synaptic strength are observed if the the exact place of protein binding but also

9 the nature of the proteins that are able to order of events is not important in this form bind to the tags. of metaplasticity [62]. Other forms of LTD One important implication of metaplasticity Besides the above-described NMDA is that the effect of a conditioning receptor-dependent LTD, also other forms stimulation cannot be predicted unless the of LTD exist. For example, an aberrant previous stimulation history of the tissue is form of LTD is observed in the , known [61]. Second, it is important to keep in which activation of a metabotropic in mind that plasticity and metaplasticity is required [18, 28]. For share some mechanisms and therefore this so-called mGluR-dependent-LTD, could be operative at the same time. This activation of the NMDA-receptor is not makes it difficult to distinguish which necessary. Instead, glutamate binds to changes in synaptic efficacy are due to mGluR1 and the AMPA receptor, and as a plasticity and which to metaplasticity [62]. result several second messengers are activated. This ultimately leads to the LTP, LTD and learning internalization of AMPA receptors, like in Because the mechanisms underlying LTP NMDA receptor-dependent LTD, and to and LTD are able to modify the strength of the loss of synapses [60]. synapses for a long period of time, LTP and LTD are the most widely held Metaplasticity candidate mechanisms for learning [2, 28]. Activity-dependent modifications of However, because of the diversity of synaptic strength as described above are mechanisms underlying LTP and LTD on important for the functioning of the brain. the one side and the different forms of They are, however, not the only way in learning on the other side, the claim that which synaptic strength can be altered. ‘LTP equals learning’ is too general to be Prior synaptic or cellular activity (or: true [59]. Despite these differences inactivity) could also lead to a persistent between forms of LTP and forms of change in the direction or degree of learning, the underlying general 'synaptic synaptic plasticity that is elicited by a given plasticity and ’ hypothesis states pattern of synaptic activation [61]. This the following: ‘plasticity of synaptic plasticity’ is called Activity-dependent synaptic plasticity is metaplasticity , and most probably reflects induced at appropriate synapses during a form of homeostasis of excitability. memory formation, and is both Metaplasticity entails an extensive range necessary and sufficient for the of mechanisms, many of which overlap information storage underlying the type with the mechanisms of conventional of memory mediated by the brain area plasticity [62]. Both NMDA receptors and in which that plasticity is observed [59]. metabolic glutamate receptors seem to Space is lacking here to discuss this play a role in metaplasticity. hypothesis and the criteria that need to be The ‘synaptic tagging and capture’- fulfilled to confirm this hypothesis in an hypothesis of Frey and Morris [41, 50] extensive way, but some important results provides a possible mechanism by which supporting a link between LTP and metaplasticity could take place. Normally, learning will be summarized in short. weak high frequency stimulation (HFS) is Rioult-Pedotti and colleagues showed that not able to induce L-LTP, but it does set a the strengthening of synapses that takes synaptic tag. When this weak HFS is place during learning was indeed the followed by strong HFS in a second result of LTP [2]. Rats were trained on a pathway, the first pathway is also able to motor task, in which they had to reach with establish L-LTP because the synaptic tag one forelimb into a box to receive food. in this pathway is able to capture the After five days of training, less LTP could proteins that have been synthesized in be induced in the trained primary motor response to the strong HFS in the second cortex (M1) compared to the opposite, pathway. This synaptic tagging is also untrained M1. In contrast, the trained M1 effective when strong HFS precedes weak showed more LTD than the untrained M1. HFS [51]. This indicates that the temporal The authors take these results to indicate

10 that learning induced LTP and that because by this method the pain fibers are because of a certain ceiling (a maximum not stimulated [66]. synaptic strength), less LTP could be TMS follows Faraday’s fundamental induced additionally in the trained principles of electromagnetic induction hemisphere. In the untrained hemisphere, (figure 2 ). An electrical current in a however, LTP could be easily induced. So, stimulation coil produces a magnetic field. learning shifts baseline synaptic strength This magnetic field could readily penetrate away from its floor and toward the upper the scalp and skull and because the modification range limits [2]. magnetic field changes rapidly, a flow of Although this experiment is a strong electric current is induced in the brain [67]. indication for the role of LTP in learning, it Induced currents in the brain flow parallel does not directly demonstrate that learning to the coil, but in the opposite direction. can induce LTP. Using an inhibitory Dependent on the stimulator used, the avoidance paradigm, Whitlock and current lasts for 100-500 µs. Note that, colleagues were able to provide evidence despite its name, the magnetic part of the for this [63]. One-trial inhibitory avoidance transcranial magnetic stimulus does not learning in rats produced the same play a role in stimulating the brain: the changes in hippocampal glutamate magnetic field simply carries an electrical receptors as induction of LTP through high stimulus across the scalp and skull [65]. frequency stimulation did. Furthermore, in The waveform of the magnetic pulse could accordance to the findings of Rioult be monophasic, which induces current Pedotti and colleagues, Whitlock et al. flow in one direction in the brain, or showed that this learning-induced synaptic biphasic, in which current is first induced in potentiation blocked the subsequent one direction and then in the other [68]. It induction of LTP [63]. is reported that monophasic TMS is more To summarize: these results show that the effective than biphasic TMS in altering ability to induce LTP or LTD is changed by neuronal function [68, 69]. This is probably learning, because of an underlying because monophasic pulses preferentially ‘homeostatic’ rule: learning reduces LTP activate a relatively uniform population of and increases LTD [64]. Finally, LTP neurons, which are oriented in the same seems to be the mechanism by which direction. Therefore, monophasic rTMS newly learned information is encoded. leads easily to summed effects on

II. Repetitive Transcranial Magnetic Stimulation

Principles of magnetic stimulation Transcranial magnetic stimulation (TMS) is a method of delivering electrical stimuli to the brain through the intact scalp. Thereby, TMS interacts with normal brain functioning. Conventional electrical stimulation could also be used to stimulate the brain but has some disadvantages, which make it less suitable for human experiments. First, the scalp and skull have a high resistance to electrical current Figure 2 which makes it hard for electrical Principles of transcranial magnetic stimulation to reach and stimulate the stimulation brain [65]. Furthermore, electrical A brief , high-current pulse in produced in the magnetic coil ( C), which is placed above the stimulation is painful because pain fibers scalp [1] . This current generates a magnetic in the scalp are activated [1]. Magnetic field ( MF ) that induces an electric field ( EF ) in stimulation, on the contrary, enables the brain. Arrows indicat es direction of current stimulation of the brain with little or no pain flow.

11 synaptic plasticity. Biphasic TMS, in evoked potentials (MEPs; see box 1 ). contrast, activates differently oriented Furthermore, single pulse TMS is able to neurons, imaginably resulting in both disrupt normal cognitive functioning, if the inhibitory and facilitatory effects that could magnetic pulse is applied at a crucial cancel each other out [69]. moment during task performance to a part Single pulse TMS and rTMS of the brain necessary for carrying out the TMS can be delivered in single pulses or task. The depolarization of the neuron in trains of several TMS pulses. Single caused by the magnetic pulse interferes pulse TMS is mostly used to investigate with the ability of the subject to perform the excitability of the brain by measuring the task and creates a transient ‘virtual the motor threshold or by evoking motor lesion’ [70, 71].

Box 1 Methods to investigate the effects of TMS

Motor threshold The m otor threshold (MT) is an indicator of the excitability of the motor cortex and is defined as the minimal intensity of stimulation ap plied to the primary motor cortex that produces a visible movement in the contralateral hand in at least 5 out of 10 stimulations. Resting motor threshold (RMT) is measured during complete relaxation, while the active motor threshold (AMT) is determined in a voluntary contracted muscle. Muscle contraction lowers the MT [3] , probably because an increasing number of spinal motor neurons is brought to fire by the stimulation [4] . As a result, the AMT is lower than the RMT. Motor evoked potentials The muscle responses induced by TMS could also be recorded by electrodes. These so - called motor evoked potentials (MEPs) are thoug ht to reflect the excitability of the motor cortex, the sub-cortex and the spinal tract [7] . MEPs are widely used to study the physiology of corticospinal conduction. Several parameters of the MEP could be studied, of which the size (or amplitude) of the MEP is most used. Theoretically, the s ize of the MEP relates to the number of activated corticospinal neurons and thereby reflects the excitability of the cortex [4]. Invasive epidural recordings Another method to investigate more directly the effects of TMS on the brai n are invasive epidural recordings. In conscious human subjects with no abnormalities in the central nervous system, electrodes are implanted in the spinal cord for the relief of otherwise intractable pain [10]. In this way, descending volleys in response to TMS could be registered directly. Invasive epidural recordings are the analog of experiments in animals, in which an electrode placed in the spinal cord records a series of high-frequency waves in response to a single electrical pulse to the motor cortex [11] . In these experiments, anodal stimulation recruited a single descending volley in the pyramidal tract. This wave is hypothesized to reflect direct stimulation of the pyramidal tract axons and is therefore called the D (direct)-wave. If the intensity of stimulation is increased, this D-wave is followed by other volleys with a periodicity of approximately 1.5 ms. These waves are thought to be produced by indirect, transsynaptic activati on of the pyramidal tract neurons and are therefore called I (indirect)-waves [13] . These waves could also be measured with invasive epidural recordings in humans. Thereby, invasive epidural recordings provide a useful insight into the after-effects of TMS: the descending volleys reflect the effectiveness of synaptic input to fast-conducting corticospinal neurons [15] . That way, descending volleys are a more direct reflection of cortical changes than MEPs, because the size of MEPs is not only related to the number of activated corticospinal neur ons, but is also influenced by spinal excitability [16] . However, MEPs could be recorded without restrictions in all human subjects, while invasive epidural recordings could be measured in just a small, specific group of patients.

12 When TMS is applied repetitively, it is able of the effect [5]. Furthermore, it appeared to change and modulate activity beyond that high-frequency stimulation (> 5 Hz) the stimulation period. There are several produces facilitatory after-effects, while protocols for this so-called repetitive TMS low-frequency stimulation (< 1 Hz) induces (rTMS), with different long-lasting effects. inhibitory effects in the brain [73]. Variables influencing the effects are, Recently, studies have identified the among others, stimulus frequency, structure of the pulse train as an important stimulus intensity, duration of the parameter in determining the effects of a application period and the total number of certain protocol [72]. Therefore it seems stimuli [72]. Earlier research investigating most useful to describe rTMS protocols as the effects of rTMS has shown that the either simple , in which individual stimuli duration of the after-effect is proportional are spaced apart by identical inter stimulus to the length of stimulation: the longer the intervals (ISIs) or patterned , in which stimulation period, the longer the duration different ISIs are employed (figure 3 ).

Figure 3 Overview of rTMS protocols Simple rTMS protocols have an identical interstimulus interval (ISI) between the differen t pulses. Shown are a low-frequency protocol ( 1 Hz stimulation ) and a high-frequency protocol ( 5 Hz stimulation ). Patterned protocols are characterized by different ISIs. Thetaburst stimulation uses a pattern in which 3 pulses of stimulation are given at 50 Hz (ISI 20 ms), repeated every 200 ms ( 5 Hz). In c ontinuous thetaburst stimulation ( cTBS ), a 40 s train of uninterrupted TBS is given (i.e. 600 pulses). In intermittent thetaburst stimulation ( iTBS ), a 2 s train of TBS is repeated every 10 seconds for a total of 600 pulses (190 seconds). In paired pulse stimulation ( PPS ), 2 pulses are delivered with a variable ISI, but an ISI of 1.5 ms is mostly used. Pairs are delivered every 5 s (0.2 Hz). Quadripulse stimulation ( QPS ) uses short trains of 4 pulses th at are also repeated every 5 s (0.2 Hz). Like in PPS, the ISI is variable in QPS. * Length of ISI can be varied

13 Box 2 Intensity of stimulation

In order to standardize the intensity of stimulation used across subjects and studies, several methods could be applied. The most used method is to express the intensity of stimulation as a percentage of the motor threshold (MT) . Another approach is to use the size of the MEP (expressed in mV) as a reference point to determine the intensity of stimulation. Both methods take into account the inter-individual variance in excitability of the motor cortex. This is not the case in studies in which a fixed intensity, as a percentage of the maximum stimulator output (SO) is used for all subjects. This method is time-efficient and most used for stimulating cortical regions other than the motor cortex . The reason for this is that it could be questioned if the MT is an appropriate reference for determining the correct intensity of stimulation above these cortical areas [5, 6] . When one would use the MT to determine the intensity of stimulation over, for example, the pre frontal cortex, one should at least correct for the differences in scalp to brain distances between the motor cortex and the prefrontal cortex [8] . Furthermore, the differential susceptibility of brain areas to stimulation should be taken into consideration, because the assumption that the effects of TMS are similar across cortical areas seems to be wrong [9] . This indicates that the MT is not self-evidently an appropriate measure to determine the intensity of stimulation, and that a fixed intensity could be a reasonable alternative.

In the following, the most important and However, the studies investigating the widely used rTMS protocols will be effects of rTMS on motor excitability vary described. Protocols using electrical in the intensity of stimulation used and the stimulation, like transcranial electrical number of pulses applied (see table 1 A). stimulation and paired associative The effects of 1 Hz rTMS are in most stimulation, in which magnetic and cases inhibitory, although stimulation at electrical stimulation are combined, will not relatively low intensities (90% RMT, 90% be discussed. Because of the link to AMT) sometimes fails to induce a processes of synaptic plasticity, measurable effect on motor excitability. exclusively protocols that have shown to However, one has to keep in mind that induce effects lasting for at least several there is a large inter-individual variability minutes are considered. Results of studies when applying 1-Hz rTMS, with some using these protocols are summarized in subjects even showing facilitatory effects table 1 (simple protocols) and table 2 as shown by Daskalakis et al. [75]. (patterned protocols). Although the effects Furthermore, the duration of the effects of rTMS could be investigated on various shows a lot of variation. This could be levels, only papers investigating rTMS- attributed to technical reasons (the wave- induced changes in the size of the motor form used, the number of pulses applied) evoked potential are, for reasons of clarity, [68], but also to inter-individual variability. included in these tables (for some Few studies applied stimulation at very low important concepts: see box 1 and 2 ). frequency (0.2 or 0.6 Hz) [76, 77]. Focus will be on recent papers, published Therefore, these paradigms will not be in the last five years. For an overview of further discussed. earlier papers, we refer to Fitzgerald and Primed 1 Hz rTMS colleagues [74]. In 2003, Iyer and colleagues introduced an adaptation of the 1 Hz protocol [78]. In this Simple rTMS protocols (table 1) so-called primed 1-Hz protocol , 1 Hz One- hertz rTMS stimulation was preconditioned (‘primed’) By now, 1 Hz rTMS is the most frequently by sub-threshold stimulation at high used protocol to investigate structure- frequency (6 Hz). The priming consisted of function relationships in the brain, both in 20 trains that lasted 5 s each with an inter- the motor cortex as in other brain areas. train interval of 25 s [78]. The rationale

14 Table 1 A Simple rTMS protocols (low-frequency): effects and effect-durations on Motor Evoked Potential (MEP)-size

Protocol Study n Intensity # pulses MEP Duration Note 0.2 Hz Ikeguchi et al. (2005) [76] 38 90 % RMT 180 ↓ 20 min 0.6 Hz vd Werf et al. (2006)[77] 12 90 % RMT 560 ↔ NA High inter-individual variability 1 Hz Bagnato et al. (2005) [79] 9 90 % RMT 900 ↔ NA Chouinard et al. (2003) [80] 7 90 % RMT 900 ↓ 10 min Daskalakis et al. (2006) [75] 12 90 % RMT 900 ↔ NA High inter-individual variability Heide et al. (2006) [81] 11 115 % RMT 900 ↓ 2 min MEP-size of non-stimulated motor cortex increased (up to 30 min) Lang et al. (2008) [82] 9 90 % RMT 1200 ↓ 4 min Decrease in MEP more pronounced after pre-treatment with D1/D2 receptor agonist Modugno et al (2003) [83] 14 90 % RMT 900 ↔ NA O'Shea et al. (2007) [84] 5 90 % AMT 900 ↓ > 15 min Pal et al. (2005) [85] 13 115% RMT 900 ↔ NA Rizzo et al (2004) [86] 8 90 % AMT 1500 ↓ NI Suppa et al. (2008) [87] 15 90 % AMT 1500 ↓ 30 min Taylor et al. (2007) [68] 12 90 % RMT 900 ↓ 5 min Monophasic waveform: sign.effect at 5 min after rTMS; biphasic at 20 min after rTMS Zafar et al. (2008) [88] 9 90 % RMT 900 ↓ NI Primed 1 Hz Daskalakis et al. (2006) [75] 10 90 + 115 % RMT 600 + 600 ↔ NA Iyer et al. (2003) [78] 11 90 + 115 % RMT 600 + 600 ↓ > 60 min 2 Hz Todd et al. (2006) [89] 31 80 % AMT 600 ↓ 30 min Stimulation at 70 % AMT: ns . Stimulation at 90% AMT: high inter-individual variability AMT : active motor threshold. NA : not applicable. NI : not investigated. NS : not significant. RMT : resting motor threshold. ↓: decrease. ↑: increase. ↔: no significant change.

15 Table 1 B Simple rTMS protocols (high-frequency): effects and effect-durations on Motor Evoked Potential (MEP)-size

Protocol Study n Intensity # pulses MEP Duration Note 5 Hz Esser et al. (2006) [90] 7 90 % RMT 1500 ↑ NI Fitzgerald et al. (2007) [91] 10 90 % RMT 750 ↔ NA Gilio et al. (2007) [92] 7 120 % RMT variable ↑ < 15 s Number of pulses: 5-60 Peinemann et al. (2004) [93] 8 90 % RMT 150 ↔ NA Peinemann et al. (2004) [93] 5 90 % RMT 900 ↑ 20 min Peinemann et al. (2004) [93] 8 90 % RMT 1800 ↑ > 40 min Perez et al. (2005) [94] 6 Adj.1.2 x MEP size 300 ↑ 200 ms MEP measured at tibialis anterior and soleus muscle Quartarone et al. (2005) [95] 10 90 % AMT 1500 ↔ NA Quartarone et al. (2005) [95] 10 90 % RMT 1500 ↑ NI Rizzo et al (2003) [86] 11 90 % AMT 1500 ↑ > 60 min Stimulation at 70 % and 80% AMT: no effect on MEP-size Suppa et al. (2008) [87] 15 90 % AMT 1500 ↔ NA Takano et al. (2004) [96] 10 90 % AMT 150 ↑ 12 min Zafar et al. (2008) [88] 9 90 % RMT 1200 ↑ NI 6 Hz Todd et al. (2006) [89] 31 80 % AMT 600 ↓ 30 min Stimulation at 70 % AMT : ↔. Stimulation at 90% AMT: high inter-individual variability 10 Hz Arai et al. (2007) [69] 11 90 % AMT 1000 ↔ NA Only monophasic waveform induces significant effect Arai et al. (2007) [69] 11 90 % RMT 1000 ↑ 10 min Daskalakis et al. (2006) [75] 12 90 % RMT 900 ↔ NA Jung et al. (2008) [97] 20 80% RMT 300 ↑ up to 120 min Trains of 1.5 sec Jung et al. (2008) [97] 20 80% RMT 1000 ↓ up to 90 min Trains of 5.0 sec 20 Hz Daskalakis et al. (2006) [75] 12 90 % RMT 900 ↔ NA 25 Hz Khedr et al. (2007) [98] 11 130 % RMT 1500 ↑ NI Adj : intensity of stimulation adjusted to a MEP of a certain magnitude. AMT : active motor threshold. NA : not applicable. NI : not investigated. NS : not significant. RMT : resting motor threshold. ↓: decrease. ↑: increase. ↔: no significant change.

16 behind the priming relates to the fact that intensity and effect is also indicated by the brief pretreatment with stimulation in the 5- results of Quartarone et al. [95] and Arai et 6 Hz-range greatly increases the ability of al. [69] ( table 1 B). Both studies reported subsequent 1 Hz stimulation to induce in no effect of high-frequency rTMS (5 or 10 vitro LTD [61]. In fact, Iyer et al. also found Hz respectively) at 90% of the active that primed 1 Hz rTMS significantly motor threshold (AMT), but did find an increased the amount of cortical effect of rTMS at 90% of the resting motor depression, relative to 1 Hz stimulation threshold (RMT), which is generally higher without precedent priming rTMS. The than the AMT, as explained in box 1 . depression after primed 1 Hz rTMS lasted Thus, these studies indicate that the for at least 60 minutes ( table 1 A) [78]. intensity of HFS is of great influence on This indicates that priming could increase the effect that is induced. the inhibitory effects of 1 Hz rTMS. Most likely this relates to processes involved in Patterned rTMS protocols (table 2) metaplasticity, in which the activation Theta burst stimulation history of the neurons is important for the Theta burst stimulation (TBS) is an rTMS after-effects that are induced. protocol based on the naturally occurring Nonetheless, Daskalakis and colleagues theta rhythm (5 Hz) of the hippocampus. did not find a significant change in MEP This protocol is a modification of the theta after application of primed 1 Hz stimulation burst protocol used in animal experiments, [75]. They explained this contrast by in which it has proven to alter synaptic stating that their study did not aim to plasticity [101]. investigate changes in MEPs and that, The basic element of the TBS rTMS with their experimental design, small protocol is a burst of 3 stimuli at 50 Hz, changes in corticospinal excitability could which is repeated every 200 ms, i.e. at the have been missed [75]. However, frequency of the theta rhythm ( figure 3 ) Langguth et al. recently also reported that [102, 103]. It is important to note that primed 1 Hz rTMS did not result in a better different patterns of delivery of TBS have clinical outcome compared to a standard 1 different, opposite effects on excitability Hz protocol in patients with tinnitus. In (table 2 A). Forty seconds of continuous addition, priming did not reduce the inter- theta burst stimulation (cTBS) reduces the individual variability of treatment response amplitude of MEPs for ~ 60 minutes, while in this study [99]. So, although it seems intermittent TBS (iTBS), in which a 2 s reasonable, based on animal research and train of TBS is repeated every 10 s for a the results of Iyer et al. [78] to assume that total of 190 s, increases MEPs for at least priming has the potential to decrease 15 minutes [103]. Note that in both motor excitability additionally, this effect is protocols the total number of stimuli is not consistent across studies. equal (600 pulses). The authors suggest rTMS at frequencies above 1 Hz that TBS in humans produces a mixture of Just one study used stimulation with a facilitatory and inhibitory effects, while frequency of 2 Hz [89]. Because of the facilitation builds up faster than inhibition depressing effect on motor excitability, it [103]. They further assume that the effects seems that this protocol has similar effects on both inhibition and facilitation saturate as 1-Hz stimulation and therefore this at some level. In that case, the short trains study is included in table 1 A. In contrast, in iTBS would favor rapid build-up of high rTMS frequencies (5-25 Hz) tend to facilitatory effects, while in the longer increase cortical excitability ( table 1 B). lasting train of cTBS the initial facilitatory However, the after-effects of such effects are eventually replaced by protocols depend highly on the intensity of dominating inhibitory effects [103]. This stimulation as shown by Modugno and could also explain the results of Gentner et colleagues [100]. They reported a al., who found that 300 pulses of cTBS decrease in excitability after HFS at low increased MEP amplitude, while 600 intensities and an increase in excitability pulses of the same protocol decreased the after HFS at higher intensities (not shown size of the MEP [104]. However, this in table) [100]. This relationship between model is speculative yet.

17 Table 2 A Patterned rTMS protocols: effects and effect-durations on Motor Evoked Potential (MEP)-size

Protocol Study n Intensity # MEP Duration Note pulses

cTBS Cheeran et al. (2008) [105] 18 80 % AMT 300 ↓ > 30 min Influence of genotype: only in individuals with Val/Val group significant change of MEP Di Lazzaro et al. (2005) [102] 4* 80 % AMT 300 ↓ 8 min Gentner et al. (2007) [104] 36 70 % RMT 300 ↑ 25 min Decrease in MEP-size ( up to 21 min) if isometric contraction (5 min) preceded iTBS Gentner et al. (2007) [104] 36 70 % RMT 600 ↓ 17 min Huang et al. (2005) [103] 9 80 % AMT 600 ↓ 60 min Huang et al. (2007) [106] 6 80 % AMT 600 ↓ NI No change in MEP-size after pretreatment with NMDA receptor-antagonist Huang et al. (2008) [107] 9 80 % AMT 300 ↓ 20 min No change in MEP-size if stimulation was given during contraction FDI Iezzi et al. (2008) [108] 10 80 % AMT 300 ↓ > 30 min Increase in MEP-size if finger-movements preceded cTBS Martin et al. (2006) [109] 8 80 % AMT 600 ↓ 35 min Murakami et al. (2008) [110] 6 80 % AMT 600 ↓ 35 min Stefan et al. (2008) [111] 18 70 % RMT 300 ↓ NI Stefan et al. (2008) [111] 18 70 % RMT 600 ↓ 35 min Zafar et al.(2008) [88] 9 80 % AMT 600 ↓ 30 min Effects independent of current direction or pulse configuration iTBS Cheeran et al. (2008) [105] 18 80 % AMT 600 ↑ > 30 min Influence of genotype: only in individuals with Val/Val group significant change of MEP Di Lazzaro et al. (2008) [112] 3* 80 % AMT 600 ↑ > 6 min In one subject increase ns . Just one measurement after rTMS Di Lazzaro et al. (2008) [112] 18 80 % AMT 600 ↑ > 6 min High inter-individual variability; Just one measurement after rTMS Huang et al. (2005) [103] 9 80 % AMT 600 ↑ 15 min Huang et al. (2007) [106] 6 80 % AMT 600 ↑ NI No change in MEP-size after pretreatment with NMDAR-antagonist Huang et al. (2008) [107] 7 80 % AMT 600 ↑ 20 min No change in MEP-size if stimulation was given during contraction FDI Iezzi et al.(2008) [108] 10 80 % AMT 600 ↑ > 30 min Decrease in MEP-size if finger-movements preceded iTBS Murakami et al. (2008) [110] 6 80 % AMT 600 ↑ 15 min Teo et al. (2007) [113] 6 80 % AMT 600 ↑ 20 min Zafar et al. (2008) [88] 9 80 % AMT 600 ↑ 30 min Effects independent of current direction or pulse configuration Adj : intensity of stimulation adjusted to a MEP of a certain magnitude. AMT : active motor threshold. cTBS : continous theta burst stimulation. FDI : first dorsal interosseus muscle. iTBS : intermittent theta burst stimulation. NA : not applicable. NI : not investigated. RMT : resting motor threshold. ↓: decrease. ↑: increase. ↔: no significant change. * : patient with epidural electrode.

18 Table 2 B Patterned rTMS protocols: effects and effect-durations on Motor Evoked Potential (MEP)-size

Protocol Study n Intensity # MEP Duration Note pulses PPS-1.5 ms Benwell et al. (2006) [114] 8 100 % AMT 360 ↑ 11 min Di Lazzaro et al. (2007) [115] 1* Adj.MEP-size 0.5 mV 312 ↑ > 3 min Just one measurement after rTMS Di Lazzaro et al. (2007) [115] 5 Adj.MEP-size 0.2 mV 312 ↑ > 3 min Just one measurement after rTMS Fitzgerald et al. (2007) [116] 10 Adj. MEP-size 0.5-1 mV 360 ↔ NA Hamada et al. (2007) [117] 5 90 % AMT 1440 ↑ 5 min Hamada et al. (2007)[117] 16 Adj.MEP-size 0.4 mV 720 ↑ 10 min Hamada et al. (2007) [117] 10 Adj.MEP-size 0.3-0.5 mV 720 ↑ 10 min Thickbroom et al. (2006) [118] 8 Adj. MEP-size 0.5 - 1 mV 720 ↑ 10 min PPS-3 ms Khedr et al. (2004) [119] 12 80% AMT 1000 ↓ NI PPS-100 ms Daskalakis et al. (2008) [120] 15 Adj.MEP-size 1 mV 100 ↓ NI QPS-1.5 ms Hamada et al. (2007) [121] 7 130 % AMT 720 ↑ 10 min Hamada et al. (2007) [121] 7 90 % AMT 1440 ↑ > 75 min Hamada et al. (2007) [121] 5 90 % AMT 720 ↔ NA Hamada et al. (2007) [121] 16 Adj.MEP-size 0.4 mV 1440 ↑ 30 min Hamada et al. (2008) [122] 10 90 % AMT 1440 ↑ > 75 min After priming (10 min QPS with ISI 5 ms): no significant MEP-change QPS-5 ms Hamada et al. (2008) [122] 10 90 % AMT 1440 ↑ > 75 min After priming (10 min QPS with ISI 5 ms): no significant MEP-change QPS-10 ms Hamada et al. (2008) [122] 10 90 % AMT 1440 ↑ > 45 min After priming (10 min QPS with ISI 5 ms): depression ( > 30 min) instead of facilitation QPS-30 ms Hamada et al. (2008) [122] 10 90 % AMT 1440 ↓ 20 min After priming (10 min QPS with ISI 5 ms): prolonged depression ( > 30 min) QPS-50 ms Hamada et al. (2008) [122] 10 90 % AMT 1440 ↓ 90 min After priming (10 min QPS with ISI 5 ms): enhanced depression QPS-100 ms Hamada et al. (2008) [122] 7 90 % AMT 1440 ↓ 45 min After priming (10 min QPS with ISI 5 ms): enhanced depression QPS-1.25 s Hamada et al. (2008) [122] 8 90 % AMT 1440 ↔ NA

Adj : intensity of stimulation adjusted to a MEP of a certain magnitude. AMT : active motor threshold. NA : not applicable. NI : not investigated. PPS-x: Paired pulse stimulation with inter-stimulus interval of x. QPS-x: Quadripulse stimulation with inter-stimulus interval of x. RMT : resting motor threshold. ↓: decrease. ↑: increase. ↔: no significant change. * : patient with epidural electrode.

19 Epidural recordings in patients with an probably relates to spike timing-dependent electrode implanted in the spinal cord models of synaptic plasticity [39, 124]. showed that iTBS and cTBS had different Quadripulse stimulation (QPS) effects on corticospinal activity [102, 112]. Recently, Hamada and colleagues This further supports the hypothesis that adapted the PPS protocols described the pattern of TBS has great influence on above [121]. Based on animal experiments the circuits in the cortex that are in which the number of pulses per train is modulated and therefore on the after- a very potent factor to influence the level effects that are induced [112]. of hippocampal synaptic plasticity, they When comparing the studies applying TBS hypothesized that, by increasing the listed in table 2 A, it is noteworthy that the number of pulses per train, the facilitatory results show more consistency than the effect of stimulation could be increased. results of the simple rTMS protocols This new protocol consisted of trains of discussed above ( table 1 A and 1 B). This four magnetic pulses at 1.5 ms interval, is probably caused by the fact that across repeated every 5 s, and was called the studies the intensity of stimulation and quadripulse stimulation (QPS) ( figure 3 ) the number of applied pulses are roughly [121]. Results showed that 30 minutes of equal, in contrast to the variation in QPS significantly increased MEP-sizes for intensity and number of pulses in simple at least 75 minutes after stimulation ( table rTMS protocols. However, it could also 2B) and the authors indicated that QPS reflect that TBS elicits more consistent was more effective than PPS with the results than simple rTMS protocols. same inter-stimulus interval [121]. In a Furthermore, it is important to notice that later experiment, the effect of ISI interval the effects of TBS last for a considerable on the after-effects was investigated [122]. period of time after the end of stimulation. It was shown that QPS with short inter- Repetitive Paired-Pulse TMS (PPS) stimulus intervals (ISI 1.5 ms, 5 ms or 10 Thickbroom et al. [118] were the first to ms) increased the size of the MEP, while describe repetitive paired-pulse TMS QPS at long ISIs (30 ms, 50 ms, 100 ms) (PPS) at I-wave periodicity. In this induced MEP suppression ( table 2 B and protocol, paired TMS pulses of equal 3). ISI- length also influenced the duration intensity are delivered with an inter- of the effect. The longest depression was stimulus interval (ISI) of 1.5 ms. The pairs induced by QPS with an ISI of 50 ms (up are repeated every 5 s ( figure 3 ). The ISI to 75 minutes), while facilitation lasted of 1.5 ms reflects the periodicity of indirect longest after QPS with an ISI of 1.5 and 5 (I)-waves (see box 1 ) and refers to the ms (at least 75 minutes) [122]. high-frequency (~ 600 Hz) repetitive These results indicate several things. First, discharges of corticospinal fibers that are the long-lasting facilitatory effect of QPS- 5 elicited by single-pulse TMS of the motor ms (at least 75 minutes) raises the cortex [15, 118, 123]. Thickbroom and question whether the I-wave periodicity of colleagues reported that 30 minutes of 1.5 ms is the critical factor in PPS and PPS with an ISI of 1.5 ms (PPS-1.5) QPS. At least, these results suggest it is increased MEP amplitudes four-fold up to not. Second, there seems to be a non- 10 minutes after stimulation [118]. In an linear relationship between the sign and epidural study, Di Lazarro and colleagues the duration of plastic changes induced by also found an increase in MEP amplitude QPS with different ISIs. (table 2 B) [115]. Table 3 – Influence of ISI - length on the Khedr et al [119] and Daskalakis et al effect of PPS and QPS

[120] also applied PPS, but with an ISI of 3 Increase in Decrease in ms and 100 ms respectively. Surprisingly, MEP-size MEP-size this longer ISI reversed the sign of the effect: while PPS-1.5 increased the MEP, Paired pulse PPS- 1.5 PPS- 3 PPS-3 and PPS-100 decreased MEPs stimulation PPS- 100 Quadripulse QPS- 1.5 QPS- 30 (see table 2 B and, more specific, table 3 ). stimulation QPS- 5 This specific temporal relationship QPS- 50 QPS- 10 QPS- 100

20

III. rTMS and the induction of discussed in two sections. First, findings of rTMS-studies that correspond with some synaptic plasticity key-concepts of synaptic plasticity will be

considered. Second, we will present In the following, findings of rTMS-studies several results that prove more directly will be discussed in light of synaptic that the mechanism by which rTMS plasticity. We will present several lines of induces changes in the brain resembles evidence that make it reasonable to those of synaptic plasticity. assume that rTMS is indeed able to alter synaptic strength through processes like LTP and LTD. These findings are summarized in figure 4 , and will be

Figure 4 Similarities between the induction of synaptic plasticity in brain slices and the induction of rTMS effects in the normal human brain The two bars indicate the time period in which the stimulation (electrical stimulation in vitro or rTMS) induces lasting changes. The boxes show different processes that play a role in both methods of stimulation, and the time point at which they are important. All processes are described in detail in section III of this thesis.

21

RESULTS THAT CORRESPOND WITH and second, these effects must be able to influence subsequent task performance. KEY -CONCEPTS OF SYNAPTIC Studies applying this approach are PLASTICITY summarized in table 5 . Although not all studies explicitly reported the duration of 1. rTMS has effects that outlast the the rTMS-induced effect, the fact that period of stimulation there is an effect on task performance Changes in excitability evidently indicates that the effect of rTMS One of the most important features of outlasts the period of stimulation. synaptic plasticity, as indicated by an Number of pulses determines duration enormous amount of research, is that Several studies indicated that the duration changes in synaptic efficacy last after of the effect is dependent of the number of induction. Therefore, the most obvious pulses applied. Peinemann and reason to suggest that rTMS has effects colleagues [93] showed that 150 pulses of on synaptic plasticity is the fact that rTMS 5 Hz rTMS had no significant effect on is also able to induce changes in MEPs, while 900 and 1800 pulses of 5 Hz excitability that outlast the period of stimulation increased MEPs for stimulation. respectively 20 and more than 40 minutes These lasting changes could be easily (table 1 ). A similar relationship between measured in the motor system and are the number of pulses and the duration of usually expressed as an increase or the effect was demonstrated by Nyffeler decrease in the size of the motor evoked and colleagues [125], see table 5 . These potential (see box 1 ). Studies reporting findings are in accordance with results of effects of rTMS on the size of the MEP in animal studies, in which the persistence of healthy subjects are summarized in table LTP in vivo may be prolonged up to days 1 and 2, and already discussed in section depending on the number of stimulation II. Note that not all studies examined the trains applied [126]. duration of the effect. Although the studies In sum, studies of different aspects of investigating the duration of the rTMS- brain functioning indicate that rTMS effect on excitability fail to yield consistent induces long-lasting effects. These effects results, in most studies an effect outlasting most likely reflect changes in synaptic the period of stimulation is reported. efficacy. Neuroimaging In addition to these studies on motor 2. The temporal pattern of pulses is excitability, neuroimaging studies have important been performed to investigate the effects Classical studies of LTP and LTD have of rTMS on the brain. Most of these shown that several temporal aspects of studies reported long-lasting effects of the induction protocols are of great rTMS. Various methods are used to importance for the sign and duration of the investigate the duration and pattern of effect on synaptic efficacy. these effects, as indicated by the studies First, LTP in vitro can be typically induced listed in table 4 . The majority of studies by a so-called high frequency tetanus, concern functional imaging using PET, while low frequency stimulation leads to fMRI or EEG, and their results show LTD [127, 128]. It is hypothesized that this significant lasting effects of rTMS on brain rate dependence is related to the finding activity. that the velocity of increase of the Task performance postsynaptic calcium-levels determines The effects of rTMS could also be the direction of the induced effect, as investigated at the level of task described in part I of this thesis. performance. In light of this thesis the so- This frequency-dependency also holds for called off-line approach, in which rTMS rTMS. Low-frequency rTMS (~1 Hz) precedes task performance, is most induces in most cases a decrease in interesting [5]. This approach depends on excitability, while high-frequency rTMS (> two assumptions. First, the effects of rTMS 5 Hz) generally increases excitability have to outlast the period of stimulation (table 1 ).

22

Table 4 Neuroimaging studies investigating the effect duration of rTMS on the brain Protocol Study n Intensity # pulses Method Stimulated area Effect Effect duration 1 Hz Eisenegger et al. (2008) [129] 12 54 % SO 900 PET right DLPFC Increase in rCBF 9 min Lee et al. (2003) [130] 8 90 % RMT 1800 PET left M1 Widespread changes in rCBF > 60 min Rounis et al. (2005) [131] 16 90 % RMT 1800 PET left M1 Widespread changes in rCBF > 60 min 5 Hz Esser et al. (2006) [90] 7 90 % RMT 1500 EEG M1 Potentiation of EEG responses NI Huber et al. (2007) [132] 19 90 % RMT 1500 EEG left M1 Localized potentiation of cortical EEG responses up to 60 min Rounis et al. (2005) [131] 16 90 % RMT 1800 PET left M1 Widespread changes in rCBF > 60 min Takano et al. (2004) [96] 6 90 % AMT 150 PET left M1 Increased rCBF 8 min Tegenthoff et al. (2005) [133] 12 90 % RMT 2500 fMRI left M1 Enlargement of index finger representation 120 min cTBS Hubl et al. (2008) [134] 7 80 % RMT 600 fMRI right FEF Decreased BOLD signal at stimulated area up to 35 min Schindler et al. (2008) [135] 4 80 % RMT 600 EEG right FEF Increased neuronal synchronization up to 20 min AMT : active motor threshold. cTBS : continuous theta burst stimulation. DLPFC : dorsolateral prefrontal cortex. EEG : electroencephalography. FEF : frontal eye field. fMRI : functional magnetic resonance imaging. M1 : primary motor cortex. NI : not investigated. PET : positron emission tomography. rCBF : regional cerebral blood flow. RMT : resting motor threshold. SO : maximum stimulator output.

Table 5 Studies investigating the effect of rTMS protocols on task performance (‘offline paradigms’) Protocol Study n Intensity # pulses Task Stimulated area Effect Effect duration 1 Hz Chambers et al. (2006) [136] 16 92 % RMT 900 Stop-signal task right IFG Impaired ability to stop initiated action at least 7.5 min Johnson et al. (2007) [137] 7 105 % RMT 600 Divided attention task DLPFC Impaired performance on task up to 21 min Knoch et al. (2006) [138] 27 100 % RMT 900 Decision making task right DLPFC More disadvantageous decision making at least 7 min Terao et al. (2007) [139] 13 105 % RMT 1200-2400 Motor preparation task Various Delayed reaction times after rTMS up to 90 min Primed 1 Hz Aleman et al. (2008) [140] 7 45% SO 600+ 600 Digit span task right DLPFC Disruption of task performance NI 5 Hz Tegenthoff et al. (2005) [133] 33 90 % RMT 2500 Two point discrimination left M1 Improvement of tactile perception 120 min 20 Hz Wagner et al. (2006) [141] 17 100 % RMT 1600 Divided attention task left DLPFC Impaired performance on task 30-60 min cTBS Nyffeler et al. (2006) [125] 5 80 % RMT 600 Measurements of eye right FEF Inhibitory effect on saccades 30 min movements Nyffeler et al. (2006) [125] 4 80 % RMT 1200 Measurements of eye right FEF Inhibitory effect on saccades > 170 min movements Nyffeler et al. (2006) [125] 1 80 % RMT 2400 Measurements of eye right FEF Inhibitory effect on saccades > 650 min movements iTBS Ragert et al. (2008) [142] 23 80 % AMT 600 Two point discrimination left M1 Improvement of tactile perception > 30 min

Modified TBS Silvanto et al. (2007) [143] 6 60 % SO 200 Motion detection task Visual areas Impaired detection of motion NI AMT : active motor threshold. cTBS : continous thetaburst stimulation. DLPFC : dorsolateral prefrontal cortex. FEF : frontal eye field. IFG : inferior frontal gyrus. iTBS : intermittent thetaburst stimulation. M1 : primary motor cortex. NI : not investigated. rCBF : regional cerebral blood flow. RMT : resting motor threshold. SO : maximum stimulator output.

23 Not only the frequency of stimulation but Prior stimulation also the temporal pattern of the electrical Various rTMS studies have indicated that pulses is important in the experimental priming (i.e. pretreatment with stimulation) induction of synaptic plasticity in brain influences the result of the subsequent slices. In addition to the standard high stimulation. Iyer et al [78] showed that 6 frequency stimulation, other stimulation Hz priming enhanced the depressant protocols exist to elicit LTP in vitro . An effect of the subsequent 1 Hz rTMS. In example of such a protocol is theta burst addition, Hamada and colleagues [122] stimulation (TBS), that resembles the reported that priming (with QPS with an ISI naturally occurring firing pattern of of 5 ms for 10 minutes) blocked the effect neurons in the hippocampus [101, 127]. It of subsequent QPS-1.5ms and QPS-5 ms was shown that, in rat hippocampal slices, (see table 2 B). Priming furthermore TBS (4 pulses at 100 Hz, repeated with reversed the facilitatory effect of QPS-10 200 ms interburst intervals) produced a ms: after priming, QPS-10 ms depressed greater change in the field excitatory post- MEP-sizes for at least 30 minutes [122]. in the early phase of Physiological activity LTP than 100 Hz stimulation with the Three studies recently demonstrated that same total number of pulses did [144]. physiological activity is also able to This result demonstrates that the temporal influence the effects of rTMS over the pattern of the stimulation protocol motor cortex. First, Gentner and influences the LTP induction in vitro . colleagues [104] reported that application This temporal relationship between pulses of cTBS for a duration of 20s (300 pulses) is also important when rTMS is applied to without prior voluntary motor activation human subjects. When comparing table 1 facilitated subsequently evoked MEPs. In (simple rTMS protocols, in which the contrast, the size of the MEPs was interstimulus intervals are identical) and depressed when this stimulation was table 2 (patterned rTMS protocols, with preceded by voluntary activation of at least different interstimulus intervals), it is 5 minutes ( table 2 A) [104]. A similar result noteworthy that the most consistent and was found by Iezzi and colleagues [108], prominent effect durations are reported for who revealed that a brief sequence of patterned rTMS protocols like cTBS finger movements prior to stimulation (decrease for approximately 30 min), iTBS reversed the after-effects of both iTBS and (increase for 15 min) and QPS (in general cTBS [108]. Huang et al [107] investigated effects of at least 30 min). This indicates the effect of physiological activity during that the pattern of stimulation is of great and after stimulation. They showed first importance for the duration of rTMS- that voluntary contraction of the first dorsal induced effects, like it is in LTP induction interosseus muscle during stimulation protocols. abolished the effect of both iTBS as cTBS. In contrast, contraction of the same 3. Changes in excitability induced muscle after stimulation resulted in a by rTMS depend on the history of stronger facilitatory after-effect of iTBS activation compared to iTBS without contraction. Metaplasticity (‘the plasticity of plasticity’) Surprisingly, this was not the case with is an important concept concerning cTBS: contraction directly after stimulation synaptic plasticity. It holds the ability of the reversed the effect of cTBS from inhibition synapse to induce long-term potentiation to facilitation [107]. or depression dependent on the history of Taken together, these studies provide activation of a synapse [61]. Hereby strong evidence for the hypothesis that the metaplasticity is a form of homeostasis to after-effects of stimulation are sensitive to maintain synaptic strength in balance. prior activation of the synapse, either by Several rTMS studies have shown results priming stimulation or by physiological resembling these ideas about activity. Several studies suggested that changes in the membrane potential or in metaplasticity. Both prior stimulation as 2+ well as physiological activity have the Ca concentration of postsynaptic influence on the rTMS effect. neurons cause this altered response to the

24 stimulation [104, 107, 108], but more LTP, and given the fact that iTBS is a research is needed to clarify this point. potentiating protocol [103], one would expect that iTBS given before learning 4. rTMS interacts with learning decreases the speed of the subsequent As outlined before, LTP and LTD are learning. However, this is not the case and prime candidates for mediating memory therefore one should think of another and learning [28, 59]. This hypothesis is mechanism by which iTBS influences supported by the results of several animal learning. Possibly iTBS increases the studies, indicating that LTP indeed excitability of the stimulated motor area, underlies learning [2, 63]. When which improves subsequent learning [64]. connecting processes like LTP and LTD to To conclude, these studies demonstrate the effects of rTMS, it is therefore that rTMS is able to influence learning, important to examine if rTMS also although the exact mechanism is not clear influences learning. This is, in fact, done in [64, 145]. Because learning and LTP are two experiments. closely linked, these findings strongly Muellbacher and colleagues investigated suggest that rTMS is able to alter synaptic the effects of rTMS on motor learning strength. [145]. Subjects learned a simple thumb abduction movement, in which they had to RESULTS PROVIDING INSIGHT IN THE maximize the acceleration of the MECHANISM BY WHICH R TMS movement with relation to a visual display. In the first practice session, acceleration INDUCES EFFECTS increased with 25-50%. If the subjects performed the task again the next day, 5. rTMS-induced effects in animal they started with this higher performance, experiments reflect more directly indicating that they learned to perform the changes in synaptic plasticity task at a higher speed. Furthermore, they Animal experiments provide more direct showed additional improvement of their insight in the effects of rTMS, because performance in this second session [145]. changes in synaptic plasticity could be A 15-min train of 1 Hz rTMS (intensity investigated at the cellular level using 115% RMT), applied directly after the first brain slices. Different kinds of experiments practice session, impaired learning: the have been performed (see table 6 ) and significant increase in acceleration gained are reviewed in the following section. It will during practice session 1 was lost at the become clear that, although they differ in start of session 2. However, the ability to the method used, all studies provide some improve performance during session 2 kind of evidence for the hypothesis that was unaffected [145]. These results rTMS affects synaptic strength through indicate that rTMS interacts with the LTP or LTD. consolidation of learning and suggests that rTMS directly impairs or facilitates LTP rTMS applied directly after learning is able in rat to disturb expression and maintenance of In two experiments, Ogiue-Ikeda and LTP. colleagues investigated the effect of high- rTMS could also improve learning. frequency stimulation at different Swayne and Teo reported that iTBS to the intensities on long-term potentiation in the motor cortex hand area, given 10 minutes rat hippocampus [146, 147]. They reported before testing, increased the speed of that 25 Hz rTMS at 0.50 and 1.00 Tesla learning in a subsequent thumb abduction (T) did not affect LTP, while stimulation at task [64]. In light of the homeostatic rule of 1.25T significantly depressed LTP and learning and plasticity, in which there is a stimulation at 0.75T enhanced LTP in the balance between learning and the maintenance phase (10-60 minutes after possibility to induce LTP or LTD, this result tetanus stimulation). The authors have no is quite surprising. Based on the study of explanation for this particular pattern of Rioult-Pedotti and colleagues [2], who effects, but stated that the effects of rTMS showed that learning reduced subsequent

25 Table 6 Animal experiments investigating the effect of rTMS protocols

rTMS protocol Study Animal (n) Intensity # pulses Method Effects 1 Hz Aydin-Abidin et al. (2008) [148] rat (6) 30-33 % SO 3600 pulses Immunohistochemistry Increased c-Fos expression in various cortical regions 1 Hz, 4 Hz Allen et al. (2007) [149] cat (8) 100 % SO variable Electrophysiology Increase in spontaneous spike rate up to 60 s, decrease in evoked 8 Hz (short trains of 1-4 s) firing rate for more than 5 min. Perturbed phase relationship among neural responses 5 Hz Hayashi et al. (2004) [150] monkey (10) 35% SO 2000 pulses PET Changes in uptake of 18 F-fluorodeoxyglucose that lasted for at least 8 days 10 Hz Aydin-Abidin et al. (2008) [148] rat (6) 29-32% SO 2400 pulses Immunohistochemistry Increased c-Fos expression in various cortical regions Kim et al. (2006) [151] rat (9) 1.4 T 1000 pulses/ day; Behaviour (FST) rTMS had effects on behavioral (decreased immobility time) and stimulation for 7 days and electrophysiology cellular level (increased EPSPs after LTP induction). 25 Hz Ogiue-Ikeda et al. (2003a) [146] rat (32) 0.50 T and 1000 pulses/ day; Electrophysiology Stimulation at 1.25T inhibited LTP; no effect of stimulation at 0.50T. 1.25 T stimulation for 7 days

Ogiue-Ikeda et al. (2003b) [147] rat (40) 0.75 T and 1000 pulses/ day; Electrophysiology Stimulation at 0.75T enhanced maintenance phase of LTP (10 to 60 1.00 T stimulation for 7 days min after tetanus stimulation), no long effect of stimulation at 1.00T. iTBS Aydin-Abidin et al. (2008) [148] rat (6) 25-30% SO 3000 pulses Immunohistochemistry Increased zif268 protein expression in limbic cortices. FST : forced swim test. MT : motor threshold. PET : positron emission tomography. SO : maximum stimulator output. T: Tesla.

26 depend on the stimulus intensity and that experiment was performed in monkeys, rTMS administered at the appropriate because the high dose of radiation is not stimulus intensity may potentially activate applicable to studies in human. They hippocampal function [147]. They further found rTMS-induced changes in the suggest that the facilitatory effect of 0.75T uptake of FDG that lasted for at least 8 stimulation depends on the expression of days. Because cerebral glucose genes involved in LTP induction like c-Fos. metabolism correlates with excitatory Another interesting research method is the glutamatergic synaptic activity, it is combination of electrophysiological reasonable to assume that the long-lasting research with the investigation of behavior. changes in the glucose metabolism can be Kim et al. [151] compared the effect of a attributed to changes in neuronal seven-day treatment with 10 Hz rTMS with excitability analogous to LTP or LTD [150]. the effect of a seven-day treatment with Thereby, this study indicates that rTMS the antidepressant drug fluoxetine on the has long-lasting effects on the brain, forced swim test (FST). The FST is the corresponding to changes in synaptic most popular animal model of depression. plasticity. In this test, immobility time represents rTMS is able to increase the expression behavioral despair in the animal. Earlier of genes involved in synaptic plasticity experiments have shown that stress (and Recently, Aydin-Abidin and colleagues so the FST procedure) disrupts LTP in the investigated the effect of three rTMS rat hippocampus [152]. Therefore, Kim et protocols on the expression of c-Fos and al. induced LTP in different groups of rats zif268, two immediate early gene (IEG) (group 1: rTMS treatment + FST; group 2: proteins [148]. 1 Hz stimulation and 10 Hz sham-stimulation + FST; group 3: stimulation increased the expression of c- fluoxetine treatment + FST; group 4: naive Fos in various cortical areas like the frontal group which received no treatment and cortex, the motor cortex and the parietal was not exposed to the FST). Ultimately, cortex compared to both sham stimulation Kim and colleagues showed two important and control rats that did not receive things. First, treatment with rTMS stimulation at all. iTBS specifically decreased the immobility time in the FST increased expression of zif268 in limbic compared to sham-stimulation (acoustic cortices. The increased expression of stimulation only), while the animals treated zif268 is most interesting, because with fluoxetine only showed marginal experiments on synaptic plasticity improvement in their immobility time after indicated that expression of this gene is FST. Second, as shown in earlier studies, essential for the induction and persistence LTP induction was impaired in the sham- of LTP [44]. Thereby, this result provides stimulation and fluoxetine group due to the evidence for the influence of rTMS on LTP stress evoked by the FST. On the maintenance. contrary, the group of rats that received rTMS desynchronizes the phase rTMS showed a significant increase in field relationship among neural responses excitatory postsynaptic potentials after With a methodological innovating method, LTP induction, equal to the naive group Allen and colleagues [149] investigated who was not exposed to the FST. This the effects of short rTMS trains (1-4s) at indicates that a seven-day treatment with various frequencies (1-8 Hz) on neural rTMS can reverse the impaired synaptic processing in cat visual cortex. They efficacy caused by the FST, most likely by measured local field potentials and found acting at the cellular level [151]. that the short rTMS trains initially Altogether, these studies provide evidence increased neural firing (~ 1 minute), for the hypothesis that rTMS has direct followed by a decrease in firing for several effects on LTP and LTD. minutes. In addition, they investigated the rTMS induces effects that last for more temporal distribution of spikes. Compared than a week to pre-TMS, there appeared to be a strong Hayashi and colleagues combined rTMS desynchronization between spontaneous with repeated neuroimaging, namely 18 F- spikes and theta oscillations after rTMS fluorodeoxyglucose (FDG) PET [150]. This [149].

27 This is an important finding, because this cycloserine and found that this agonist ability of TMS to disrupt the precise timing reversed the after-effect of iTBS from of signals between neurons is considered facilitation to inhibition. This further to be a direct indication of the way by indicates that the after-effects of TBS are, which rTMS alters brain plasticity [153]. at least in part, dependent on NMDA To summarize this section, the animal receptor activity. However, it is unclear experiments discussed above support the why activation of the NMDA receptor hypothesis that rTMS is able to influence changes the excitatory effects of iTBS into processes in synaptic plasticity. rTMS can inhibitory effects. facilitate or inhibit LTP induction, probably Dopaminergic receptors by altering the expression of genes The role of dopaminergic receptors in the involved in plasticity and/or by acting on induction of the after-effects of 1 Hz rTMS the synchrony between neural responses. was topic of investigation in a study This results in long-lasting changes in the performed by Lang and colleagues [82]. brain, which could persist for more than a There is evidence that dopaminergic week, as indicated by neuroimaging. mechanisms are involved in plasticity because D1 and D2 receptor activation 6. Pharmacological interventions modifies LTP and LTD in animals [82, shed light on the mechanisms 154]. Lang et al. used the dopamine underlying rTMS-induced effects receptor (D1 and D2) agonist pergolide, By using drugs that act on receptors and found that the suppression of involved in , one could corticospinal excitability by 1 Hz rTMS was provide pharmacological evidence that the more pronounced after pergolide intake after-effects of rTMS involve plasticity-like compared to the suppression after changes. Recently, some studies placebo. In addition they showed that this investigated the role of the NMDA receptor suppression was a real effect of the and dopaminergic receptors in the interaction with rTMS: sham stimulation induction of rTMS effects on motor after pergolide intake did not change excitability. corticospinal excitability [82]. NMDA receptor Comparing the results of these three The role of the NMDA receptor in synaptic studies, it is important to notice that the plasticity has been described extensively study of Huang et al [106] provides the above. In short, activation of the NMDA strongest evidence for the hypothesis that receptor by stimulation induces the influx rTMS induces its effects by acting on of Ca 2+ postsynaptically, which in turn processes critical for synaptic plasticity. triggers a series of reactions that lead to The results of the other two studies are long-term changes in synaptic efficacy more difficult to interpret, because they [27]. used a receptor agonist. In particular the In a double-blind placebo-control study reversing effect of the NMDA receptor Huang and colleagues used the NMDA agonist D-cycloserine on the effect of iTBS receptor antagonist memantine and on excitability is remarkable [113]. It investigated the effect of this blockade on suggests that the relationship between the the induction of after-effects by iTBS and NMDA receptor and rTMS is complex. cTBS [106]. They found that memantine Nonetheless, overall it is clear that the blocked the suppressive effect of cTBS NMDA receptor and dopaminergic and the facilitatory effect of iTBS on the receptors play a role in the induction of size of MEPs. This result provides direct rTMS effects. evidence for the hypothesis that the NMDA receptor is important for the 7. The genetic background of induction of long-lasting after-effects of subjects determines the effects iTBS and cTBS [106]. of rTMS Teo and colleagues provided further If rTMS indeed induces effects that reflect evidence for the NMDA-dependence of the changes in synaptic plasticity, then it is after-effects of TBS in humans [113]. They reasonable to investigate the influences of used the NMDA receptor agonist D- genes involved in plasticity on rTMS

28 effects [64]. Recently, Cheeran and IV. Discussion colleagues investigated whether the effects induced by rTMS are significantly Several lines of evidence, as described in influenced by a polymorphism of the the previous section, support the BDNF gene [105]. Brain derived hypothesis that rTMS affects the brain by neurotrophic factor (BDNF) has been mechanisms like LTP and LTD. In the first identified as one of the key neural signals place, there are various aspects of rTMS- orchestrating synaptic plasticity. In induced effects that correlate closely with humans, the gene coding for BDNF shows conceptual key features of synaptic a common single nucleotide polymorphism plasticity, like 1 st the duration of the effect which produces a Valine to Methionine that exceeds the period of stimulation, 2 nd substitution at codon 66 [155]. Subjects the dependency of the effect on with this Val66Met genotype appear to stimulation frequency, 3 rd the importance have smaller hippocampal volumes, of the history of activation and 4 th the role reduced grey matter in several areas of in learning and memory. Furthermore, the frontal cortex, poorer episodic memory animal studies as well as pharmacological and abnormal hippocampal activation and genetic studies indicate that the compared to Val/Val subjects [156]. mechanism by which rTMS induces effects Furthermore, it was demonstrated that on the brain resembles mechanisms that subjects with the Val66Met genotype could change synaptic strength. showed decreased experience-dependent However, several critical remarks plasticity in the motor cortex, as indicated concerning these results and their by a smaller cortical map representation of implications for the link between rTMS and the motor cortex after a repetitive key synaptic plasticity should be made. To press task, and a smaller increase in start, both rTMS and synaptic plasticity are MEPs compared to Val/Val subjects [155]. broad phenomena which makes it difficult, Based on these findings, Cheeran and maybe even impossible, to find one link colleagues hypothesized that the Val/Met between them. Secondly, it should be polymorphism in the BDNF gene would noted that the results described in section influence the response to rTMS protocols. III are not able to link rTMS effects directly This appeared to be true: after cTBS and to mechanisms underlying synaptic iTBS, MEPs were significantly changed in plasticity. Third, the effects of rTMS on the the Val/Val individuals but not in the Non- brain appear to be not restricted to Val/Val individuals with one or two Met- changes at the synaptic level. Also other alleles [105] (see also table 2 A). This forms of brain plasticity could be result is important in different respects. influenced by stimulation. Therefore, First, it points to the important role of focussing on the link with synaptic BDNF in the induction of rTMS effects, plasticity in order to understand the effects and, because BDNF is important for of rTMS on the brain may be too narrow. synaptic plasticity, this finding thereby These aspects will be discussed in the further supports the hypothesis that rTMS following. indeed affects synaptic plasticity. Furthermore, it implicates that genetic 1. Uncertainty and variability variation in the normal population can Bringing together the concepts underlying produce significant differences in the after- synaptic plasticity and the mechanism by effects of rTMS protocols. Thereby, it which rTMS influences the brain is difficult, could explain the inter-individual variability because both are not unitary phenomena. in studies and the different outcomes of Synaptic plasticity, on the one hand, is a studies discussed above. Future studies broad concept comprising various short- could benefit from this knowledge and and long-term changes as described in include genotype as a potential covariate part I of this thesis. Despite an enormous in the analysis of data. amount of research, the molecular mechanisms underlying these processes are until now not exactly known. More

29 research is needed to get more insight into In vitro studies these mechanisms. The knowledge about synaptic plasticity rTMS, on the other hand, could have mainly comes from research done in different effects depending on stimulation hippocampal slices. In these slices, LTP parameters like frequency, intensity and and LTD could be easily induced and duration of stimulation. So, when measured. When comparing these results considering the results described above, with the effects of rTMS on the human one should keep in mind that there are brain (as investigated for example by many known (and maybe even unknown) measuring MEPs), the following things factors influencing them. This variability should be remembered. and these uncertainties complicate the In the first place, it is important to notice search for a clear link between the two that the induction of LTP or LTD in vitro by concepts. means of stimulation reflects an artificial mechanism, and therefore probably differs 2. No direct evidence from the physiological mechanism by Although the results presented above which changes in synaptic plasticity occur support the hypothesis that rTMS induces in the normal functioning human brain. its effects on the brain by mechanisms Secondly, rTMS-effects in humans are in similar to LTP and LTD, they do not most cases investigated over the motor provide direct evidence for this hypothesis. cortex and not on the hippocampus, which More specific: that rTMS seems to have makes it difficult to compare these results the same effects on the excitability of the with those of in vitro studies in which brain (measured as the amplitude of hippocampal tissue is used. There are two MEPs) as seen in ‘ in vitro’ experiments major reasons to perform rTMS over the after the induction of LTP or LTD is no human motor cortex instead of over the causal proof that the underlying hippocampus. First, stimulation of the mechanisms are identical. The same holds hippocampus is impossible: this structure for the influence of genetic background is located too deep into the brain to be and drugs: the fact that they influence both reached with magnetic stimulation. the effects of rTMS and the induction of Furthermore, stimulation of the LTP/ LTD does not automatically ascertain hippocampus has no well-defined that the underlying processes are the behavioral output which can be used as a same. control of stimulation [27]. Therefore, most The only way to investigate in a direct way studies deliver rTMS over the motor cortex whether the effects of rTMS on the human where limb movements or MEPs could be brain are really due to changes in the easily measured. However, it could be efficacy of synaptic transmission is by questioned to what extent the motor invasive recording of synaptic responses neurons, which are stimulated by rTMS, in conscious humans [27]. However, it is are comparable to the CA1 and CA3 unnecessary to say that such experiments pyramidal neurons in the hippocampus in are not applicable. The second best option which LTP and LTD are induced. is to perform such experiments in A third point of consideration when monkeys. Unfortunately, these comparing the results of in vitro studies experiments have, to our knowledge, until with those of rTMS studies is the fact that now not been performed. the intensity and frequency of stimulation Therefore, the studies described in this that is applied in vitro is in general much thesis used other methods to get more higher than those used for stimulating the insight into synaptic plasticity and the human motor cortex. Most important effects of rTMS, like in vitro studies and reason for the relatively low intensity and animal experiments. Advantages and frequency in human rTMS studies is the disadvantages of both methods will be risk of inducing an epileptic insult [157]. discussed in the following. Moreover, stimulation at high intensity over the motor cortex could lead to undesirable movements by stimulating hand-, face- or leg muscles.

30 The fact that the intensity and frequency of brain regions in small animals. In addition, rTMS is much lower than those used in it has been argued that stimulation with a LTP induction protocols could explain the small coil is less effective because of the difference in effect duration that is found diminished induced electric field [70, 158]. between these two types of stimulation. Second, when performing experiments in While rTMS induces effects that last anesthetized animals like cats, monkeys, maximal up to several hours, LTP could one should take into account the effects of last for months. anesthesia. In general, anesthetics tend to Finally, it is important to realize that the depress neuronal firing and excitatory stimulation used for LTP-induction in vitro synaptic transmission, and increase is very focal, in contrast to rTMS over the synaptic inhibition through interactions human brain that has a low spatial with multiple proteins [159, 160]. It thus resolution. In brain slices , small electrodes remains to be seen whether the effects of are used to stimulate specific fibers rTMS on are similar in projecting to just one neuron. rTMS, on the non-anesthetized animals on the one hand other hand, has a spatial resolution of at and conscious human subjects on the least mm 2 or maybe even cm 2 [70]. In other hand [153]. addition, the brain’s irregular surface To conclude this point, the results of in raises the possibility that rTMS vitro studies and animal experiments could differentially affects neurons in the sulci not be simply translated to human rTMS and gyri [70]. experiments, although they provide Altogether, these arguments indicate that valuable knowledge about synaptic it is not realistic to expect the same results plasticity and the effects of rTMS. A critical after rTMS in vivo as after LTP or LTD consideration of these findings is essential induction in in vitro studies. Therefore, one to determine what they mean in the could state that the comparison between context of rTMS in the human brain. these different kinds of studies is not useful. However, the same above- 3. Effects of rTMS on other mentioned arguments could also be used forms of plasticity to defend another position: although rTMS Plasticity in the brain is broader than just is applied at a low intensity and with a synaptic plasticity. Not only at the synaptic relatively low frequency and low spatial level, also at other levels of brain resolution, this stimulation is nevertheless organization remodeling could take place able to induce measurable changes in in order to improve the functioning of the motor excitability as indicated by changes brain. Examples of such plastic changes in the size of MEPs. We therefore would are structural modifications and altered argue that this fact could be taken as a connections between functional networks strong indicator for the unique [14]. So, when thinking about the opportunities rTMS offers to affect non- physiological basis of the effects of rTMS, invasively the human brain. it may be too limited to look only at the Animal studies effects of rTMS on synaptic plasticity. Animal studies as discussed in this thesis Indeed, several studies indicated that provide a lot of information about the rTMS is also able to modify plasticity at effects of rTMS on the brain and on other levels of brain organization. We will synaptic plasticity. However, several shortly discuss these studies in the things should be kept in mind when following. translating these findings to the effects of rTMS induces structural changes stimulation in human. In a voxel-based morphometry (VBM) First of all, it is important to realize that, study, May and colleagues investigated because of the difference in brain size the effects of a 5-day treatment with 1 Hz between humans and e.g. rats, stimulation rTMS at an intensity of 110% MT [161]. in the latter until now could not be They were able to show that rTMS administered to a specific brain area. delivered to the superior temporal cortex There are technical limits on building coils caused macroscopic cortical changes in of the size required to stimulate distinct gray matter as early as within 5 days of

31 intervention. Similar macroscopic changes be of great importance in the future. That have been induced in another experiment is the topic of the following section. after 3 months training of juggling [162]. The result of this VBM-study therefore V. rTMS in the future indicates that a relatively short treatment What will be the future of rTMS? At the with rTMS could have a considerable end of this thesis, we provide recommen- effect on brain structure. The time-course dations for future research. In our opinion, of the changes in the study of May et al. this research is necessary to unravel in suggests that the changes in gray matter more detail the mechanism by which rTMS reflect fast adjustment of neuronal affects the brain. The results of this systems at the cellular level, such as spine research are not only of fundamental and synapse turnover rather than slower importance for the use of this method in evolving mechanisms as neuronal or glial modern neuroscience, they also offer the cell genesis [161, 163]. possibility to improve the application of rTMS has effects on functional rTMS as a treatment for various brain connectivity disorders [27]. To clarify this, we will Several neuroimaging studies have discuss the therapeutic potential of rTMS indicated that the neural consequences of in depression and stroke. rTMS are not restricted to the site of stimulation [164]. Even a short train of Future research high-frequency rTMS at sub-threshold To achieve a better understanding of the intensity modulates activity of remote physiological basis of rTMS and the areas in the brain, which are connected mechanism by which it induces long- with the site of stimulation through cortico- lasting changes in the brain, several cortical connections [165]. In addition, hypotheses need to be investigated. rTMS is able to alter resting-state activity Therefore, various study methods have to in the brain. Recently, Zandbelt and be used. colleagues showed that primed 1-Hz rTMS The most important and fundamental over the prefrontal cortex increased hypothesis that has to be tested is whether resting-state activity locally for at least 45 rTMS truly acts on the synapse and, in line minutes, as detected by fMRI [166]. with this point, if rTMS indeed induces Together, these results indicate that long-lasting changes in the efficacy of one focusing at the synaptic level could lead to or more synapses. Microelectrode a too narrow view when considering the recordings in experimental animals, effects of rTMS on the brain. Changes at preferable monkeys, should be used to the macroscopic level and alterations in measure single-spike responses and the connectivity of the brain also need to changes in field potentials during and after be studied in order to get a complete rTMS, like Logothetis and colleagues did overview of the way in which rTMS affects with fMRI [167]. In our opinion, the brain. simultaneous intra-cortical electro- Thus, to sum up this discussion, we have physiology and rTMS in awake monkeys stated that it is difficult to link two broad could provide valuable and important concepts like synaptic plasticity and rTMS. knowledge about the effects of rTMS, for Second, we indicated that a direct link example concerning the direction of the between synaptic plasticity and rTMS rTMS-effect (excitatory or inhibitory) and effects on the brain could, until now, not the duration of this effect as measured by be made, and pointed out the strengths increases or decreases in EPSPs. and weaknesses of in vitro studies and Furthermore, the differences between animal research. In addition, we showed rTMS-protocols could be investigated in that rTMS has also effect on other levels this way. Electrophysiology should also be of brain plasticity. However, despite these used to determine which cells in the cortex critical remarks and the remaining are especially activated by rTMS. uncertainties about the exact mechanism Secondly, it would be a great step forward underlying rTMS-induced changes in the if TMS-coils could be technically improved, brain, we have confidence that rTMS could

32 Figure 5 Suggestions for further research In imitation of the experiment of Rioult-Pedotti and colleagues [2], the effect of rTMS on the induction of LTP and LTD could be investigated (experiment 1). In addition, the effect of rTMS on learning could be examined (experiment 2). For more details, see text. * = compared to untrained M1 and paired control rats ** = compared to control rats who received sham stimulation

enabling local stimulation in e.g. small 5). Rioult-Pedotti and colleagues showed rodents. Such miniaturized coils could that learning a motor task decreased the open new perspectives to study further the amount of LTP and enhanced the amount neurochemical and neurophysiological of LTD in the trained motor cortex of rats. effects of rTMS on the (rodent) brain. In It would be interesting to investigate addition, local stimulation in rodents will whether rTMS over the rat motor cortex is make it possible to investigate the also able to influence subsequent LTP and relationship between rTMS-induced LTD induction ( figure 5 , experiment 1). effects, LTP- and LTD induction, and We hypothesize that after excitatory rTMS learning. To this goal, we suggest (for example high-frequency rTMS, iTBS experiments extending the findings of or QPS with an ISI of 1.5, 5 or 10 ms) the Rioult-Pedotti and colleagues [2] ( figure amount of LTP would be reduced, like

33 Rioult-Pedotti and colleagues found after complete overview of the effects of rTMS learning [2]. On the contrary, we expect on the brain. that inhibitory rTMS (for instance low- frequency rTMS and cTBS) would The therapeutic potential of rTMS enhance the amount of LTP that could be Despite the fact that the physiological induced after stimulation. In a second mechanism underlying rTMS is not experiment, the effect of rTMS on learning completely known, its therapeutic potential should be investigated by stimulating rats in psychiatric and neurological disorders before or after they learn the motor task has already been investigated. The (figure 5 , experiment 2). In this way, one rationale behind the use of rTMS as a could investigate 1 st whether rTMS before treatment is based on the finding that it subsequent learning affects performance induces long-lasting functional effects in on the motor task, as suggested by the the brain [27]. We will shortly discuss data of Swayne and Teo [64]; 2 nd if rTMS some promising results of rTMS as a after learning influences performance on treatment for depression (as an example the motor task, as described in humans by of a psychiatric disorder) and stroke Muellbacher et al. [145]; 3 rd what the (neurological disorder), and point out differences are between excitatory and which questions still remain to be inhibitory rTMS; and 4 rd how rTMS and addressed to improve these treatments in learning together affect subsequent LTP/ the future. LTD induction. Such experiments would Depression provide more insight into the relationship Major depressive disorder (MDD) is a between rTMS-induced effects and severe illness and a significant percentage learning. of patients fails to respond to standard Furthermore, the molecular basis of the therapies [168]. In these patients, rTMS-induced changes needs to be electroconvulsive therapy (ECT) has long investigated in more detail. Several been used as last treatment option. ECT is pharmacological studies showed that an extreme measure that has the risk of NMDA- and dopamine receptors play an inducing memory loss and other cognitive important role in the induction of rTMS deficits [27]. rTMS could be a good effects [82, 106, 113]. However, their alternative for ECT, because it has less precise role needs to be further clarified. It adverse effects and does not require is important that future pharmacological anesthesia. studies replicate the findings of these Therefore, several clinical trials experiments by investigating both the investigated the effectiveness of rTMS in effects of receptor-agonists and treatment-resistant depressive illness. antagonists. In addition, the involvement of Based on earlier studies suggesting a left other receptors in the induction of rTMS- prefrontal lobe dysfunction in MDD, most effects needs to be analyzed, for example trials used high-frequency rTMS (between the AMPA-receptor. This receptor plays a 5 and 20 Hz) over the left dorsolateral role in the induction of LTP and we prefrontal cortex (DLPFC) in order to therefore hypothesize that it is also reverse the dysfunction of this area [168, involved in the induction of rTMS-effects. 169]. An alternative paradigm, involving Finally, several kinds of neuroimaging low-frequency stimulation of the right studies have to be carried out in order to prefrontal cortex, has also been applied. get a complete view of the effects rTMS Although the first clinical studies had induces in the brain. Structural MRI rather mixed results, more recent studies experiments should be performed in both have presented better outcomes and humans and animals to investigate in higher effect sizes [169, 170]. For more detail what effects different rTMS- example, Fitzgerald and colleagues protocols have on gray and white matter showed that bilateral stimulation of both properties. Functional MRI, EEG and MEG the left and right dorsolateral prefrontal should be used to study the spread of cortex had substantial treatment efficacy rTMS effects throughout the brain. Such compared to sham stimulation [168]. In studies are indispensable to get a this trial, a significant therapeutic response

34 was achieved in about a half (44%) of the other stimulation protocols are used. treatment-resistant MDD patients. However, we expect that combining rTMS Furthermore, 36% of patients showed with pharmacological interventions will clinical remission [168]. Other clinical improve its therapeutic efficacy in MDD. studies, summarized in a recent meta- Stroke analysis [170], achieved about the same Stroke is the most important cause of results. Another trial showed that high- disability in the adult population of western frequency rTMS over the left DLPFC and industrialized countries [173]. In reaction low-frequency stimulation over the right to the vascular event, the brain undergoes DLPFC were equally efficacious in the plastic changes in an attempt to recover treatment of MDD [171]. Since low- the function of the targeted area. Also frequency stimulation has a considerable changes in remote brain areas are lower risk of inducing a seizure (compared present, and these changes are probably to high-frequency rTMS), and because maladaptive [174]. This leads to an patients appear to tolerate low-frequency imbalanced activity between the two rTMS better than high-frequency rTMS, hemispheres and may be an important low-frequency stimulation over the right causal factor for motor function impairment DLPFC may be preferred to high- [174]. As a result, many patients have frequency stimulation over the left DLPFC. remaining symptoms of the motor system However, although these results suggest when they survive the first year after the that rTMS, in particular low-frequency occurrence of the stroke. These symptoms stimulation over the right DLPFC, is a make it for two- third of the patients promising antidepressant strategy for impossible to continue their profession treatment-resistant MDD, several after stroke [175]. Therefore, the reduction important aspects still need to be of such symptoms by rTMS would be of investigated in order to improve the results great value for these patients. in the future. In fact, rTMS has already been used to First, the maintenance of the treatment modulate the imbalance between motor effects is of major interest. Most studies cortices. Two different approaches can be only evaluated the effects of rTMS up to applied to achieve this [173, 176]. The first only a few months. Therefore, it still has to approach is to stimulate the hemisphere in be examined whether the beneficial effects which the stroke occurred using of rTMS can be extended for a longer stimulation protocols that increase cortical period of time [170]. excitability [173, 176, 177]. Goal is to Secondly, until now all trials used simple enhance the output of the damaged motor rTMS protocols. However, as discussed cortex and the response to physiotherapy before, recent studies in healthy subjects [176]. In an epidural study, Di Lazzaro and showed that new, patterned protocols like colleagues showed that iTBS indeed TBS, PPS and QPS are more efficient enhanced corticospinal activity in the than simple rTMS protocols [103, 118, affected hemisphere [177]. Furthermore, it 121, 124]. We therefore suggest that these was shown that 10 days of 3 Hz patterned protocols would have longer stimulation (employed as an add-on effect durations and consequently could intervention to normal physical and drug be more effective in the treatment of MDD therapies) improved immediate clinical [172]. This needs to be investigated. outcome in early stroke patients [178]. Thirdly, to further improve the efficacy of a However, an important theoretical treatment with rTMS, the combination of disadvantage of this approach is the rTMS with neuropharmacology seems to increased risk of inducing a seizure: be useful. Recently, a pharmacological remember that a damaged (and so study showed that the combination of 1 Hz vulnerable) brain area is stimulated with rTMS with the agonist an excitability-enhancing stimulation pergolide induced a more pronounced protocol [173, 174]. suppression of the MEP than 1 Hz rTMS Therefore, the second approach in the alone did [82]. It still has to be investigated treatment of stroke patients targets the whether this effect is also present when intact hemisphere. Stimulation protocols

35 are used that suppress cortical excitability, practice [182]. This suggests that it is based on the hypothesis that the worthwhile to investigate whether the unaffected hemisphere inhibits the combination of rTMS with these drugs affected hemisphere [174]. Several studies indeed leads to an ameliorated treatment. investigated the efficacy of this approach Finally, because stroke is a hetero- with in general positive results. It was geneous condition, stimulation in these shown that 1 Hz stimulation over the patients will be most optimal when its unaffected hemisphere resulted in a parameters are individually determined, significant improvement of the motor based on the type and extent of the clinical function performance in chronic stroke symptoms, the location and size of the patients [174, 175]. Furthermore, this lesion and comorbidities [173]. Future approach appeared to be safe and research should therefore be performed in feasible for children with arterial ischaemic large groups of patients, in which these stroke, and seemed to improve hand covariates and their effects on treatment function in these patients [179]. with rTMS could be extensively studied. However, many questions still need to be Despite these remaining questions, we are answered. Until now it is not known which convinced that rTMS could be very useful approach (stimulation of the affected or in the treatment of both neurological and the unaffected hemisphere) is the most psychiatric disorders. The ability of rTMS optimal. This needs to be investigated in to induce improvements in patients in the near future [173]. In addition, the which other therapies failed (treatment- possibility of stimulating other areas than resistant MDD patients and chronic stroke the primary motor cortex should be patients) implies that it is a powerful examined. It has been suggested that method that could be useful for a variety of secondary sensorimotor areas, like the brain disorders. supplementary motor, the posterior parietal cortex and the dorsal and ventral premotor cortex also play a role in the functional reorganization after stroke. Because these areas are highly interconnected with the primary motor cortex, they could be promising targets for future rTMS studies in patients with motor deficits [173]. Another question that needs to be answered is when stimulation should be performed to achieve the best results. Most studies are executed in chronic (> 1 year) stroke patients, because the deficits of the patients are stable in that phase and the risk of inducing seizures is low [173]. However, an earlier intervention is possibly more beneficial, considering the fact that there is enhanced metabolic activity in diverse brain areas shortly after the occurrence of the stroke [173, 180]. Other points of attention are, as described above for the treatment of MDD, the duration of the therapeutic effect and the question whether rTMS-treatment could be improved by combining it with pharmacological interventions or by motor practice [181]. Earlier research indicated beneficial effects for drugs such as and l-dopa on motor

36

VI. Conclusion

Goal of this thesis was to investigate whether the long-lasting effects of rTMS on the brain could be linked to changes in synaptic plasticity. Although we could not find direct evidence for the hypothesis that rTMS acts on the brain by the same mechanisms that underlie synaptic plasticity, we presented seven lines of evidence that do support this assumption. We think these are strong indicators that rTMS affects the brain by mechanisms like LTP and LTD. However, future innovating research needs to investigate in a more direct way whether rTMS indeed has effects on the synapse. Furthermore, we stress that focusing on the synapse may be too narrow when considering the effects of rTMS on the brain. Future research should therefore also take into account the after-effects of rTMS on other levels of brain organization. Only in that way can the effects of rTMS on the brain be completely understood, and this is of fundamental importance to improve the application of rTMS, in neuroscience and as a treatment for severe neurological and psychiatric disorders.

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