Felipe A Tallabs G. Thesis submitted for the degree of Master of Science. Supervisor: Dr. Graeme Hammond-Tooke

Transcription

1 i Theta priming of 1 Hz repetitive transcranial magnetic stimulation in healthy volunteers. Felipe A Tallabs G Thesis submitted for the degree of Master of Science Supervisor: Dr. Graeme Hammond-Tooke University of Otago, Dunedin, New Zealand, 2011

2 ii ABSTRACT Objectives: Originally derived from animal experiments, the concept of priming in rtms experiments refers to a pre-treatment or pre-protocol stimulation that enhances the effect of the protocol. The purpose of this study was to investigate the use of theta priming with 1 Hz rtms to the motor cortex to induce corticospinal inhibition. The main question was whether there was any benefit in using theta priming instead of simply a longer period of 1Hz rtms. To address this, we compared 1 Hz rtms with and without theta priming, using a protocol in which the total period of stimulation was the same. Methods: 11 healthy volunteer participants aged between 18 and 45 (mean 27.6± 7.4) were given rtms to the right primary motor cortex in 3 separate sessions, in which the participant received one of three protocols: 1) 1 Hz rtms for 10 minutes, 2) 5 minutes of 5 Hz theta priming followed by 5 minutes of 1 Hz rtm or 3) 10 minutes of 1 Hz sham rtms using a placebo coil. In each session reaction times and pinchgrip and handgrip dynamometry were measured, followed by recording of active and resting motor evoked potentials from the left first dorsal interosseus muscle. One of three protocols was then administered, the order counterbalanced across participants, and this was followed by re-testing of motor evoked potentials, dynamometry and reaction times, in that order. Results: There was a significant effect of 1 Hz rtms alone on both RMEP and AMEP amplitude, p<0.05: both were significantly lower after 1Hz rtms compared to sham. RMEP amplitude was unchanged compared to sham rtms after the priming protocol, p= Hz rtms with and without theta priming significantly reduced right unimanual reaction time compared to sham. There was no significant effect of either protocol on cortical silent period or muscle strength, compared to sham rtms.

3 iii Conclusions: While theta priming abolished or interfered with the inhibitory effect of 1Hz rtms on corticospinal excitability, the effect on ipsilateral reaction times was the same. The negative effect of priming can be interpreted as a functional interference of the priming pre-protocol with the 1Hz protocol. The dissociation between the effects on MEP amplitude and ipsilateral reaction times suggests that MEP amplitude may not always predict behavioural changes mediated by transcallosal inhibitory mechanisms. Furthermore, the findings suggest that primed rtms may have no benefit over the use of longer durations of 1Hz stimulation.

4 iv ACKNOWLEDGEMENTS I would like to thank my supervisors, Dr Graeme Hammond-Tooke and Associate Professor Elizabeth Franz for their guidance and support. I would also like to thank the staff of the Neurology Department at Dunedin Hospital, Jill Lewis and Patsy Mason, as well as Jan Kettink and the staff of the Department of Medicine for their help and support.

5 v PREFACE Since the experiments of brain stimulation by Wilder Penfield in the 1940 s and 1950 s, neuroscientists have been interested in mapping the cerebral cortex and modifying behavior by cortical stimulation. Transcranial Magnetic Stimulation (TMS) and specifically repetitive TMS (rtms) has provided this option fairly recently. Research has been carried out since the 1980 s, and extensive use of TMS, legacy of the 1990 s, the so called decade of the brain has provided a plethora of stimulation protocols that are uncovering new ways to affect the brain and ultimately, behavior. As with any new tool there are controversies that have been raised, ranging from the inadequate control of the placebo effect to the controversy of improving someone s cognitive capacities as a result of stimulation. The mechanisms that are affected by this stimulation are still matter of investigation. It is generally acknowledged that long-term potentiation and depression (LTP and LTD) are the cellular mechanisms responsible for the effects of TMS in the human cortex. However, when talking about these cellular events, it is necessary to specify the particular type of synapse involved in the experiment, the time point in development and the triggering mechanisms (e.g., via N-Methyl-D-aspartate or metabotropic glutamate receptors). Different neurons express different arrangements of ion channels, which produce specific types of LTP and LTD mechanisms, which can be translated into specific firing patterns in every neuron type. This diversity of plasticity types is the major issue to be overcome by rtms protocols, since the neuron type of the target population in stimulation protocols is not controlled as with the animal experiments. This lack of control also produces interference and de-potentiation effects when two protocols are used, these protocols interfere with each other, and one protocol reverses the effect of the other protocol. These phenomena are still a matter of investigation but it appears that these interference effects are as varied as the different types of plasticity. Navigational TMS, a technique that uses brain maps taken from functional magnetic resonance studies to pin point TMS target areas in the brain. If used concomitantly with EEG this could provide a much more specific image of the anatomical and electrical characteristics of the target area, improving the specificity of rtms protocols and avoiding interference effects.

6 vi We will probably see, in the future, a more active role of non-invasive brain stimulation techniques in clinical settings from neurology to clinical psychology, and possibly even in enhancing the capabilities of normal subjects.

7 vii TABLE OF CONTENTS CHAPTER Chapter I PAGE Introduction and Literature Review Introduction Repetitive TMS rtms Motor Inhibition...15 Chapter II Methods and Results Participants Tests Experimental Procedures Results..26 Chapter III Discussion Introduction Limitations of the study Effects of protocols Overcoming Interference Behavioural effects Conclusions...42 References. 43 Appendix...50

8 viii LIST OF TABLES Chapter 1 Table 1.1 inhibition. Table 1.2 inhibition. Chapter 2 Table 2.1 Table 2.2 Table 2.3 Table 2.4 Chapter 3 Introduction and Literature Review Summary of studies using unpatterned rtms to produce corticospinal Studies using pattern rtms or priming to produce corticospinal Methods and Results Participants. Neurophysiological measures. Reaction times. Strength tests. Discussion No Tables

9 ix LIST OF FIGURES Chapter 1 Figure 1. Figure 1.2 Figure 1.3 Figure 1.4 Figure 1.4 Figure 1.5 Figure 1.6 Chapter 2 Figure 2.1 Chapter 3 Figure 3.1 Figure 3.2 Introduction and Literature Review Direction of Current Flows Induced in the human cortex. patterns of electric fields are determined by coil shape. BCM Threshold. Metaplasticity timeline. Facilitation of plasticity type dependant of receptor pre-activity. Facilitation of plasticity type dependant of receptor pre-activity. Similarities between LTP/LTD in vitro and rtms. Methods and Results Structure of a typical experimental session. Discussion Stimulation paradigms. Co-stimulation protocol with negative correlation.

10 x LIST OF ABBREVIATIONS Adj.MEP ALS AMEP AMT AP C CSP ctbs DF DLPFC EMG FDI GABA Hz IFCN ISI itbs KG LICI LTD LTP M1 MEP mglur msec mv Intensity of Stimulation Adjusted to a MEP Amyotrophic Lateral Sclerosis Active Motor Evoked Potential Active Motor Threshold Action Potential Path- Conditioned Pathway Cortical Silent Period Continuous Theta Burst Stimulation Degrees of Freedom Dorsolateral Prefrontal Cortex Electromyography First Dorsal Interosseus Muscle Gamma aminobutyric acid Hertz International Federation of Clinical Neurophysiology Interstimulus Interval Intermittent Theta Burst Stimulation Kilograms Long Intracortical Inhibition Long term Depression Long-term Potentiation Primary motor cortex Motor Evoked Potential Metabotropic glutamate receptors Milliseconds Microvolts

11 xi NA NMDAR NS PAS PAS PPS QPS RMEP RMT RSA RT rtms SD STDP SICI TBS tdcs TBS TES TMS T-Path Not Available N-Methyl-D-aspartate receptors Not Significant Paired Associative Stimulation Paired Associative Stimulation Paired Pulse Stimulation Quadripulse Stimulation Resting Motor Evoked Potential Resting Motor Threshold Rhythmical Slow-wave Activity Reaction Time Repetitive Transcranial Magnetic Stimulation Standard Deviation Spike-Timing-Dependant Plasticity Short Intracortical Inhibition and Facilitation Theta Burst Stimulation Transcranial Direct Current Stimulation Theta burst stimulation Transcranial Electrical Stimulation Transcranial Magnetic Stimulation Test Pathway

12 Chapter I- Introduction and literature review 1

13 2 1.1-INTRODUCTION Transcranial Magnetic Stimulation (TMS), is a neurophysiological technique used to investigate neural connectivity and brain mapping as well as a method to assess the integrity of the corticospinal pathways in disease. It has been used to induce a virtual lesion to study cognitive function and more recently it has been investigated as a therapy for neurological conditions. The basic idea is that by inducing a magnetic flux in the cerebral cortex, an electric field is induced which can excite or inhibit the stimulated region. The evolution of TMS began with a previous technique called Transcranial Electrical Stimulation (TES) which was developed with the idea of stimulating the human brain relatively non-invasively (Merton & Morton, 1980). However, non-invasive stimulation of the brain dates back from before the 19 th century. Luigi Galvani and Alessandro Volta used direct current stimulation, looking for the source of electricity in animals. Electrical stimulation was originally used to stimulate muscles directly, but it was soon applied to the motor cortex. Although it was successful in evoking a motor evoked potential (MEP), it proved to be painful. It was in 1804 that non-invasive brain stimulation was first used for therapeutic reasons; Aldini used transcranial direct current stimulation (tdcs) to treat patients with depression (melancholics). Despite the apparent success of this technique (according to Utz et al., 2010), it was soon replaced by electroconvulsive therapy. In 1985 Anthony Baker and collaborators at the University of Sheffield presented the first clinically useful device capable of producing painless brain magnetic stimulation of the motor cortex (Barker et al., 1985). TMS uses a coil of wire that produces a magnetic field perpendicular to the plane of the coil, when a high electrical current is passed through it (see Figure 1.1). Such a magnetic field can reach an intensity of 2 Tesla, with a duration of 100µs. The topography of the intensity of the stimulating field depends mostly on the shape of the coil, and although there are coil shapes for specialized use, two main designs of coils are available: circular coils and figure-of-eight coils. The first type is quite useful for stimulating larger topographical areas, whereas the second type has a much more restricted field, being able to stimulate a smaller target area. The spatial resolution of a typical figure-of-eight coil is 1 cm 2, with a depth of 2cm (Merabet & Pascuale-Leone, 2009). A TMS discharge also produces an electric field that runs perpendicular to the magnetic field, and can stimulate neurons. However the most important stimulation

14 3 effect is produced by the induced currents in the brain (Hallett et al., 2007). In the case of a circular coil, the magnetic field will be strongest along the circumference of the coil, the induced currents in the brain decrease in strength as they flow to the centre of the coil. In a figure-of-eight coil the field strength is highest at the intersection of the coils (Cohen et al., 1990) (See Figure 1.2). Figure 1.1 Direction of Current Flows Induced in the human cortex. The direction of current flow in the cortex is in the opposite direction to that in the coil.

15 4 Figure 1.2 patterns of electric fields as determined by coil shape. The electric field induced by a circular coil (A) is maximal around the perimeter of the coil (C); in the case of a figure of eight coil (B) the field is maximal where the coils intersect (D). (Cohen et al., 1990). Therapeutic applications have emerged from the use of a TMS variant known as repetitive (rtms), where repetitive pulses are applied at various frequencies. Modern TMS devices can produce magnetic pulses from low frequencies like 0.1Hz up to 50Hz. In general terms it is thought that low frequencies, around 1Hz induce inhibition in the cortex while higher frequencies, 5Hz and above, induce excitation or facilitation in neural tissue. rtms can induce seizures, and safety guidelines prescribing limits on frequency, train length and intensity must be followed to avoid potential dangers (Wasserman et al., 1998). TMS has been widely used for brain mapping, that is to say, to find the exact cortical areas involved in specific tasks. Several paradigms of stimulation have been developed to achieve this. One of the most common is mapping the motor cortex, which means utilizing the capacity of TMS to evoke MEP. These are also used in the measurement of

16 5 central motor conduction times, the time that an action potential (AP) takes to travel from the motor cortex to the spinal cord. The conduction times can be used to assess the motor pathways in neurological conditions like multiple sclerosis and amyotrophic lateral sclerosis (ALS) TMS parameters By mapping the motor cortex we have come to understand better the functional divisions of cortical areas such as the motor areas for the hands and legs and their relative overlapping. Also, the function of different pathways like weak ipsilateral and strong contralateral motor pathways and their interrelations in complex movements have been better described (Wassermann et al., 1992). The assessment of cortical excitability can be evaluated by a number of different measures that describe specific changes in neural excitability, including the stimulation intensity threshold (the minimum stimulus required to produce a MEP). The level of neural population excitability as reflected in the size of the MEP, also indicates membrane excitability since is directly related to sodium (Na) and calcium (Ca) channel changes. Another basic concept is the recruitment curve. This plots the MEP size as a function of stimulus intensity) and represents the topographical shape of the spreading of neural tissue activation as result of stimulation caused by any given TMS pulse. Normally this curve is related to the shape of the coil (see Figure 2.2). Besides these two basic concepts, there are more complex measures, which are discussed below. Cortical Silent Period (CSP): this is a period of inhibition of the motor cortex that is reflected in a cessation of motor activity in the the target muscle for a period of time after a TMS pulse, and is thought to represent intracortical inhibition mediated by gamma-aminobutyric acid-b (GABA-B) receptors (Werhahn et al., 1999). The first part is thought to be related partly to spinal cord mechanisms. It is recorded by applying a TMS pulse during voluntary contraction of the target muscle. Various methods have been used for its measurement, including measuring the latency of the onset of 25% of pre-stimulus muscle activity. Short intracortical inhibition and facilitation (SICI): this may be demonstrated by applying two TMS stimuli in close succession (called paired pulse stimulation). The first stimulus is a conditioning stimulus of low intensity, so that no MEP is perceptible (below threshold). However it does exert a cellular change in the stimulated neurons, so

17 6 that the amplitude of a second, supra-threshold stimulus varies depending on the interstimulus interval (ISI). If the second stimuli follows after 5 msec, the effect will be inhibitory; if it follows after an interval of 8-30 msec, the effect will be excitatory. The effect is mediated by GABA-A receptors (Di Lazzaro et al., 2000), and reflects the effect of modulatory effect of interneurons synapsing on motor neurones (Ziemann et al., 1996). Long intracortical inhibition (LICI): This refers again to paired pulse stimulation similar to SICI, but in this case both stimuli are strong enough to produce MEP (suprathreshold stimulation). The second test pulse follows at around msec later, and only achieves inhibition (Sanger et al., 2001) and like CSP is possibly mediated by GABA-B receptors. Connectivity: TMS can also be used to demonstrate connectivity between different cortical areas by paired pulse methods where one area is stimulated followed by second stimulus to another, producing facilitation or inhibition of the MEP where the second stimulated area is the motor cortex. Two examples: 1-Premotor cortex inhibition: this type of inhibition is achieved by stimulating the premotor cortex, generally with low frequency pulses; however other paradigms can be used. This can be achieved stimulating the PMC ipsilaterally or contralaterally (Civardi et al., 2004). 2-Transcallosal inhibition: If the contralateral motor cortex is stimulated briefly before MEP are elicited, an inhibitory effect can be demonstrated (Ferbert et al., 1992) Plasticity. The effects of repetitive TMS (rtms) in the human brain are thought to involve the neural mechanisms of plasticity, the ability of the nervous system to change as a result of experience. One form of neural plasticity is due to the decrease or increase of synaptic strength as a result of previous neural firing or external stimuli. This dynamic ability to change is fundamental for the process of learning. The cellular mechanisms of plasticity include long-term potentiation (LTP) and longterm depression (LTD). Long-term potentiation is a facilitation or enhancement of a synapse or group of synapses as a result of a previous activity in those synapses. Long-

18 7 term depression is the dynamic opposite to potentiation, that is, an induced depression or inhibition of a certain synapse or group of synapses. These two mechanisms are the result of receptor changes, mostly postsynaptically. It is considered that LTP/LTD mechanisms are due to the activation of metabotropic glutamate receptors (mglurs) and N-Methyl-D-aspartate receptors (NMDAR). Most stimulation protocols provoke a synergistic interaction of these two types of receptors in order to obtain an outcome that is inhibitory or excitatory. In general terms it has been observed that high frequency stimulation or tetanic stimulation tends to be excitatory whereas low frequency is inhibitory. If LTP is the fundamental mechanism to strengthen synaptic connections, LTD is a selective system to weaken synapses in order to have continuous ability and availability to encode new information. With just LTP, the nervous system would rapidly become synaptically inflexible, and at a certain point it would be impossible to learn anything new. In 1982, a classic paper introduced the Bienenstock-Cooper-Munro (BCM) model. which described the interaction between LTP and LTD as a result of postsynaptic activity (Figure 1.3). This interaction can be understood as the postsynaptic response to presynaptic dynamics; such response is the basis of synaptic plasticity (Bienenstock et al., 1982). Figure 1.3 We see a threshold designated Θ m and two non-linear functions, the blue curve is a synapse before activity and the red curve is after, we can see that the red

19 8 curve has moved to the right, indicating that LTD is enhanced, and thus becomes harder to obtain LTP from these changed conditions (from Bienenstock et al., 1982). We can see in Figure 1.3 that a response below the threshold represents LTD, and above LTP. According to the BCM theory this threshold is the result of the average amount of postsynaptic activity (ibid). According to this model, LTD or LTP is dependant on on the state of the postsynaptic neurone and there is a sliding threshold which separates the two (Toyoizumi et al., 2005). As mentioned before, low frequency stimulation induces LTD whilst higher frequency stimulation induces LTP. This stimulation response is due to a similarity between stimulation protocols and specific levels of postsynaptic activity. Quoting Ireland et al., 2009: The modification threshold is itself a function of the degree of cell firing recently generated by the postsynaptic neuron, if the neuron has been recently very active, presumably driven by synaptic inputs, then the modification threshold shifts to the right, making it harder to generate LTP and easier to induce LTD. The neuron firing rate then is the key element for switching from one mechanism to another, and the fundamental element for firing rate is calcium levels. LTP requires a fast and high rise of calcium levels and LTD requires a steady but slow rise of calcium levels (Purves et al., 2008). These two types of calcium dynamic are mediated mostly by postsynaptic receptors. As we mentioned before there is a synergy among these receptors, namely NMDAR and mglur. Their pre-activity in the synapse dictates if LTP or LTD will proceed. In general, when presynaptic NMDAR activity is preponderant, plasticity will be shifted towards LTD, but when mglur activity is preponderant, plasticity will be shifted towards LTP (Abraham & Tate, 1997) (Figure 1.4). Complex molecular explanations of LTP/LTD processes are beyond the scope of this thesis and due to fact that the experiments on this thesis are inductive to LTD, I will extend my explanation of metaplasticity to that specific mechanism only.

20 9 Figure 1.4 The effects of mglur and NMDA receptor (NMDAR) activation on subsequent LTD and LTP. Pre-activation of NMDA receptors shifts synaptic plasticity away from LTP and towards LTD Metaplasticity According to the concept of metaplasticity, a synaptic change will remain until a strong enough bout of activity erases or modifies such change (Abraham and Bear., 1996). This activity can be the result of previous cell firing, endocrine or paracrine signals, but also external stimuli that can be chemical, electrical or magnetic in nature (See Figure 1.5).

21 10 Figure 1.4 Metaplasticity timeline. Reproduced from Ireland et al., According to Ireland et al., 2009: Metaplasticity refers to an activity-dependent and persistent regulation of subsequently induced synaptic plasticity, such as long-term potentiation (LTP) or long-term depression (LTD). Essentially, metaplasticity entails a change in the physiological or biochemical state of neurons such that the ability to generate synaptic plasticity is altered. A key feature of metaplasticity is that this change, once established, outlasts the triggering ( priming ) bout of activity and persists until a second bout of activity occurs to induce LTP or LTD. Metaplasticity can be of two different kinds, homosynaptic: which means that the facilitation only affects those same synapses that were stimulated in the first instance, and heterosynaptic, in which the original stimuli is applied to neighboring synapses to the one that presents the metaplasticity (Abraham & Tate, 1997). LTD by artificial means is normally achieved by low frequency (1-5Hz ) stimulation, and this has been translated into the rtms field (Chen et al., 1997). Further research has found that the most efficient way to depress a synapse is by of the use of priming. This means that previous stimulation or bout of activity predisposes the synapse to produce an enhanced depression with a second bout of stimulation. If, for example, a high frequency train is presented at below threshold, it will not elicit LTP; however it may alter synaptic function, so that a second bout of activity at low frequency and above the threshold enduces enhanced LTD (Abraham & Tate, 1997). There is a series of phenomena that one should take into consideration before designing a priming/depotentiation protocol. There is a certain inductive period that has been found in priming protocols. For example Fuji et al. (1991) found that if stimulation for

22 11 depotentiation is given 100 minutes after LTP, there will be a 50% reduction in the LTD induced compared to 20 minutes after. Other researchers found that it was impossible to elicit LTD after an hour (O Dell and Kandel., 1994). This means that there is a limited time frame for the induction of LTD after priming. There is a difference between priming and depotentiation: the latter term is used when LTP has already been produced and is then reduced by a stimulation protocol. The priming effect is the result of the activity of synapses that were engaged in the induction protocol, that is to say, is homosynaptic, whilst depotentiation is the result of neighboring activated neuromodulatory interneurons that received an LTP and this in turn enhances the induction of LTD with a subsequent train of stimuli. In other words it is heterosynaptic (Muller et al., 1995). 1.2-REPETITIVE TMS Repetitive TMS is a modality where trains of magnetic pulses are applied to the brain, a technique used mostly for stimulating or inhibiting the human cortex. The mechanisms are thought to be very similar to LTP and LTD, following the same general rules: low frequencies (~1Hz) for inhibition and higher frequencies (>5Hz) for excitation. The effects of rtms outlast the period of stimulation, usually by an equivalent period of time. It is interesting to note that there is a high level of inter-individual variability in results of the trials in the literature; this is thought to be the result of differences in the individual state of cortical excitability (Hoogendam et al., 2010). This, as can be deduced from the results of metaplasticity research, is most likely due to the fact that each individual s previous state of postsynaptic activity will shape the outcome of the stimulation protocol. (See Figure 1.6 for a comparison between synaptic plasticity in brain slices and rtms.) Although the lasting effects of rtms are thought to involve an LTP/LTD like mechanism, it is speculated that longer lasting modulatory effects might be the result of gene induction. The specific mechanisms are still unclear and require further investigation (Merabet & Pascual-Leone., 2009) Applications Despite the variability of experimental results, therapeutic applications of rtms have been used in a variety of neurology and psychiatric conditions including depression,

23 12 where high frequency (20Hz) protocols have been applied to the left dorsolateral prefrontal cortex (DLPFC) or low frequency (1Hz) protocols to the right DLPFC, achieving antidepressant effects in some subjects (Simons and Dierick., 2005). Other therapeutic use has also been investigated with motor conditions like Parkinson s disease and dystonia (Wassermann & Lisanby., 2001). In Parkinson s disease, rtms has been applied to motor areas to enhance reaction times and motor coordination, resulting in improved motor abilities. Also, inhibitory 1Hz stimulation has been used to improve writer s cramp in subjects with focal hand dystonia. (ibid) Figure 1.6. Similarities between LTP/LTD in vitro and rtms. From: Hoogendam et al., 2010.

24 Experiments One of the most important aspects of rtms is the time length of stimulation. The duration of the effects are related to the number of pulses or duration of protocol (Nyffeler et al., 2006). For example, one study found that 150 pulses at 5Hz did not affect the measured MEPs, but 900 pulses did (Peinemann et al., 2004). Several studies have assessed the metabolic response to rtms protocols, for example; they have observed changes in EEG spectrum (Esser et al., 2006; Huber et al., 2007; Litvak et al., 2007; Schindler et al., 2008) or changes in regional cerebral blood flow (Lee et al., 2003; Rounis et al., 2005). These studies have helped to clarify if in fact there is a change in cortex activity Protocols Many rtms protocols have been used for experimental trials; I will describe only those relevant to inhibition of motor cortex. Depending on variables like pulse waveform (monophasic or biphasic), frequency, duration of the application period and intensity, the following protocols are configured to achieve inhibition of the motor cortex. Two of the most important things to notice in these protocols is the inter-stimulus interval (ISI); this is the time that lapses from the first stimuli train to the second. This time relationship is of fundamental importance in patterned protocols (protocols with more than one type of stimuli or more than one train), for it will shape the outcome of TMS-induced plasticity. The second is the intensity of the stimulus, which also modifies the outcome of rtms. Some studies for example, indicate that even high frequencies induced at lower intensities can cause inhibition instead of excitation (Modugno et al., 2001). The most common protocol used with rtms to achieve inhibition is the 1Hz protocol, applied mostly at intensities of 90% of resting motor threshold (RMT; the stimulation intensity threshold for producing a motor evoked potential, with the target muscle at rest) or above, although some studies have related the stimulation intensity to the active motor threshold (AMT; the stimulation intensity threshold for producing a motor evoked potential, with the target muscle contracting). Used at various durations, from 60 pulses (1 minute) to 1600 pulses (26 minutes), this protocol has been the most consistent in achieving inhibition across different intensities and durations (See Table 1).

25 14 Most protocols achieving inhibition are among those designated as special or patterned protocols; this means that they follow more complex patterns of induction than a regular protocol where only one intensity, one frequency and one timing are used. Paired Associative Stimulation (PAS): In this protocol, a peripheral nerve, usually the median nerve, is stimulated briefly before stimulation to the contralateral motor cortex. This type of stimulation is based on the concept of Spike-Timing-Dependant Plasticity (STDP). In this paradigm, the order of presynaptic and postsynaptic spike will determined the outcome (LTP or LTD) when a strong and a weak input is received in the same nerve pathway (Levy & Steward., 1983; Dan & Poo., 2004). With peripheral stimulation there is a significant delay before the action potential reaches the cortex; for it to arrive at a similar time to the TMS pulse, the peripheral nerve is stimulated first. If the TMS pulse is applied 25 milliseconds after a stimulus to the median nerve in the antecubital fossa, the result will be excitatory (Stefan et al., 2000; Stefan et al 2002; Stefan et al., 2004; Stefan et al., 2006; Tecchio et al., 2008). If the TMS pulse is induced 10 milliseconds after, the result will be inhibitory (Stefan et al., 2007; Murakami et al., 2008; Di Lazzaro et al., 2009). We can infer that PAS is a heterosynaptic method that can replicate heterosynaptic LTD if applied 10 milliseconds after, which is just a little time shorter than the time needed for the afferent input to reach the cortex. This provokes a inhibitory on the potential, whereas if applied 25 milliseconds after, it induces an increase in the MEP amplitude. Paired Pulse Stimulation (PPS) : This type of protocol is also base on the STDP concept (Huang et al., 2009; Thickbroom, 2007). PPS works by applying pairs of pulses with a brief ISI to the same cortical area. A range of 1.5ms between one stimulus and the next is used for excitation, whereas 3 msec or higher can be used for inhibition (Khedr et al., 2004; Daskalakis et al., 2004). Again, the principle of disruptive or summatory effect is the same as in PAS. Quadripulse stimulation (QPS): Developed from PPS, this induces double the quantity of pulses than PPS every 5 seconds, ISI is usually 1.5ms for excitation, again; if ISI is msec the effect is inhibitory (Hamada et al., 2007). It has been shown to be more efficient than PPS using the same ISI. Theta burst stimulation (TBS): This type of stimulation uses trains of very high frequency >50Hz, usually 3 every 200msec (Di Lazzaro et al., 2005; Huang et al., 2005).

26 15 It is called theta burst stimulation because in one second you can have 5 bursts, that is to say, 5 oscillations, which correspond to theta rhythm (4-7 Hz). This protocol was developed due to successful animal experiments using electrical stimulation to induce synaptic plasticity (Larson et al., 1986). Theta burst stimulation has two variants; continuous TBS (ctms) and intermittent TBS (itbs). In ctbs, a continuous pulse of theta bursts normally achieves inhibition if applied for 40 seconds. In itbs, a two second train is repeated every 10 seconds for duration of 190 seconds, achieving an increment in the length of MEP. It is been speculated by Huang et al (2005) that this technique causes a complex relationship of inhibition and excitation. itbs caused reduction in both short interval cortical inhibition (SICI) and facilitation (SICF). The authors suggest that ctbs initally produces facilitation, but eventually LTD with the build up of inhibitory effects. Primed 1Hz rtms: This is a variation of the traditional 1 Hz protocol, which in the majority of cases is inhibitory at intensities of 90% of resting motor threshold (RMT). Because the efficacy of this protocol is very variable (Hoogendam et al., 2010), Iyer et al (2003) proposed a new version of this protocol called primed 1 Hz stimulation, where stimulation of 6 Hz is applied with a subthreshold intensity and in bursts of 5 seconds each, after which the subject receives conventional 1Hz rtms. This pre-treatment to the 1 Hz protocol is thought to induce excitation in homosynaptic pathways that will in turn enhance the heterosynaptic inhibition of 1Hz TMS rtms MOTOR INHIBITION Several studies have compared methodologies for achieving facilitation and inhibition. However due to the nature of this thesis, I will only address corticospinal inhibition. It is interesting to observe the different types of protocols that have caused inhibition in the past, As we can in see in Table 1, there are a variety of protocols in terms of frequency, intensity and duration (number of pulses) that can induce inhibition, but, which protocols are more efficient is still a matter of discussion,. It is speculated that priming protocols like the theta priming protocol introduced by Iyer et al (2003), might induce inhibition through a complex interaction between homosynaptic and heterosynaptic pathways, enhancing the efficiency for plasticity. The inhibitory effects achieved by the PPS, QPS and PAS protocols are thought to result from STDP or spike-timing dependent plasticity (in this paradigm, the order of presynaptic and postsynaptic spike will determine the

27 16 outcome (LTP or LTD) when a strong and a weak input is received in the same nerve pathway. In ctbs protocols, 50Hz stimulation is applied in bursts at theta frequency (5-6 Hz). Since, this does not involve applying different stimulation patterns in a before and after fashion, this protocol presumably does not rely on a priming effect related to metaplasticity. However the effects may alter depending on the duration of treatment (Huang et al., 2005). In Tables 1.1 and 1.2 studies using simple or patterned rtms protocols that induced decrement in MEP are presented. Protocols Study N Pulses Intensity 0.2Hz Ikeguchi et al., %RMT 1Hz Wassermann et al., NA 125%RMT Fitzgerald et al., %RMT Stinear and Byblow, %RMT Sommer M, Lang N et al., %RMT Romero et al., %RMT Siebner HR et al., 1999a %RMT Siebner HR et al., 1999b %RMT Maeda et al., 2000a %RMT Maeda F et al., 2000b %RMT Plewnia et al., %RMT Chouinard et al., %RMT Heide et al., %RMT Lang et al., %RMT O Shea et al., %AMT Rizzo V et al., %AMT Taylor et al., %RMT Zafar et al., %RMT Suppa et al., %AMT 2Hz Todd et al., %AMT 5Hz Modugno et al., %RMT 6Hz Todd et al., %AM T 10Hz Jung et al., %RMT 20Hz Pascual-Leone et al., NA 150%RMT

28 17 Table 1.1 Summary of studies using unpatterned rtms to produce corticospinal inhibition. AMT= active motor threshold; ctbs= continuous theta burst stimulation; Hz= hertz; MEP= motor evoked potential; PAS= paired associative stimulation; PPS= paired pulse stimulation; QPS= quadripulse stimulation NA=not available; RMT= resting motor threshold. Protocol Study N Pulses Intensity ctbs Di Lazzaro et al., %AMT Huang et al., %AMT Gentner et al., %RMT Iezzi et al., %AMT Huang et al., %AMT Cheeran et al., %AMT Zafar et al., %AMT Murakami et al., %AMT PPS-3ms Khedr et al., %AMT PPS-100ms Daskalakis et al., MEP size 1mV QPS-30ms Hamada et al., %AMT QPS-50ms Hamada et al., %AMT QPS-100ms Hamada et al., %AMT PAS 10ms Murakami et al., pairs MEP size 1mV Quartarone et al., pairs 90%AMT Rosenkranz et al., pairs MEP size 1mV Stefan et al., pairs 130%RMT Di Lazzaro et al., pairs MEP size 1mV PAS N20-5 Muller et al., pairs MEP size 1mV Jung and Ziemann, pairs MEP size 1mV Ziemann et al., pairs MEP size 1mV PAS various Wolters et al., pairs 130%RMT 6Hz primed 1 Hz Iyer et al., % RMT Table 1.2. Studies using patterned rtms or priming to produce corticospinal inhibition. AMT= active motor threshold; ctbs= continuous theta burst stimulation; Hz= hertz; MEP= motor evoked potential; PAS= paired associative stimulation; PPS= paired pulse stimulation; QPS= quadripulse stimulation NA=not available; RMT= resting motor threshold.

29 Contralateral inhibition Another important issue for rtms is transcallosal contralateral inhibition of the opposite motor cortex during rtms protocols. It is possible to inhibit the primary motor cortex (M1) by exciting the opposite M1, or release it from inhibition by inhibiting the opposite M1. These effects are mediated by transcallosal connections. A transcallosal inhibitory effect may be important in patients with stroke. It is thought that excessive inhibition from the unaffected hemisphere results in impairment of function of the side affected by stroke. In this situation, the contralateral motor cortex is over-exerting inhibition on the damaged motor area (Strens et al., 2005). The two motor areas apparently have a reciprocal inhibitory balance between them (Ward and Cohen., 2004), but when one area is damaged by stroke or other cause, this equilibrium is lost, and the non-lesioned side is 1) released from the contralateral inhibition, and 2) keeps exerting its inhibitory influence towards the damaged counterpart. Because the non-lesioned side is released from contralateral inhibition, it can increase its function to a level of overactivity. This overactivity represents vicariation (where one part of the brain takes over the function of a damaged area) in some instances (Butefisch et al., 2003; Rau et al., 2003Luft et al., 2004; Lotze et al., 2006), but it can also play a maladaptive role by inducing an excess of inhibition on the lesioned area, impairing its expeditious recovery (Shimizu et al., 2002; Martin et al., 2004). It is thought that rtms might become a means for improving stroke recovery either by upregulating the lesioned motor area (Kehdr et al., 2005), or by downregulating the contralesional motor area (Mansur et al., 2005). One study used priming theta burst stimulation followed by 1Hz for downregulating the contralateral non-lesioned area with apparent positive results as measured by fmri activity of the lesioned M1 area (James et al., 2010). One way to assess the effects of transcallosal contralateral inhibition is by using reaction times (RT) tests. Transcallosal inhibition can modify reaction times, particularly bimanual reaction times. Bimanual RTs are slower due to the effect of transcallosal inhibition, a phenomenon which can be measured as bimanual cost (bimanual cost = bimanual RT-unimanual RT) (Franz & Fahey, 2007; Hammond-Tooke et al., 2010). Preliminary data suggest that bimanual cost can be modified by rtms to the ipsilateral motor cortex (Hammond-Tooke et al., 2011) but simple motor reaction times have also been shown to been modifiable by rtms guided by a navigational system to ensure

30 19 accurate application of the TMS pulses in respect to the patient s neuroimaging (Bashir et al., 2010) Priming the motor cortex To prime means to prepare, or to predispose for something. In rtms experiments it refers to a pre-treatment or pre-protocol stimulation. The concept of priming stimulation was proposed in animal experiments by Christie and Abraham in a now classic paper (Christie and Abraham, 1992). Long-Term depression or LTD was observed as a result of nonassociative or heterosynaptic activity in adjacent neural pathways converging in the same postsynaptic neural populations, but later research showed that LTD could also be elicited as a result of a correlation stimuli applied to a conditioned pathway and a test pathway (Christie and Abraham, 1992). The conditioned pathway received high frequency trains in 200ms intervals whereas the test pathway received single pulses interleaved with the stimuli trains of the conditioned pathway). Because the activity in either pathway was not sufficient to elicit LTD, this was considered as associative in nature (Chattarji et al., 1989; Stanton and Sejnowski., 1989). What Christie and Abraham found was that you could induce both heterosynaptic and homosynaptic (associative) LTD in one complex protocol design, which they called associative LTD. They found that if you stimulate with hippocampal theta (which is exactly 5Hz) some minutes before a negatively correlated associative conditioning stimulation, they could enhance the overall depression of action potentials. Normally, in the dentate gyrus of rats, negative associative conditioning only triggered non-associative LTD (heterosynaptic) but with this theta priming they could also trigger associative LTD (homosynaptic), so there was a summatory effect of both types of LTD, giving as a result an enhanced form of LTD. The first priming stimulation experiment with rtms was the study of Iyer et al (2003) and after the original experiment has not been successfully replicated (Daskalakis et al., 2006). What Iyer et al proposed was that, based on the original paper by Christie and Abraham, rtms could be used to reproduce this effect on the human motor cortex. Iyer et al stimulated the motor cortex (M1) with a 6 Hz (rather than 5 Hz) pre-treatment protocol for 10 minutes followed by a 1Hz protocol for another 10 minutes, they reported a stronger depression than that achieved by the 1hz protocol alone. The reduction of MEP was a remarkable ~30-40% with an effect that lasted for more than 1 hour.

31 20 As the duration of treatment in Iyer s study was doubled by using the priming stimulation, we questioned whether there was any real benefit in using theta priming instead of simply a longer period of 1Hz rtms. To address this we designed a study to compare 1 Hz rtms with and without theta priming, in which the total period of stimulation was the same. Also of importance was to observe if, as argued by Iyer, there was an interaction between associative and non-associative LTD that causes the enhanced inhibition in MEP measurements. We predicted that the shorter duration of priming and 1 Hz TMS would result in a smaller effect, but essentially the same effect: enhancement of the cortical inhibition.

32 Chapter II- Methods and results 21

33 22 The aim of this study was to compare, in young healthy volunteers, neurophysiological and behavioural effects of treatment with 1 Hz rtms alone for 10 minutes, 5 minutes of 1 Hz rtms primed with 5 minutes theta stimulation and 10 minutes of sham rtms using a placebo coil. The hypothesis was that primed 1 Hz rtms would achieve stronger corticospinal inhibition within the same 10 minute time frame. We used a similar protocol to Iyer et al (2003), except for the use of 80% of RMT instead of 90 % in our study and the timing of the treatment: in that study 10 minutes of theta burst stimulation preceded 10 minutes of 1 Hz rtms and was shown to be superior to 10 minutes of 1 Hz rtms alone. To be efficient in a clinical setting, priming should enhance the inhibitory effect without taking extra time to administer. The concept of priming in rtms is based on animal work where priming of long term depression has been demonstrated in the dentate gyrus (Christie and Abraham, 1992). 2.1 PARTICIPANTS There were 11 healthy volunteer participants in our study, recruited by advertisement, aged between 18 and 45 (mean 27.6± 7.4, range 18-42), 6 females and one left handed (Table 1). One additional participant was excluded because she could not complete the first session because of nausea during the rtms. Some participants were taking medications (amitriptyline, bricantyl, diclofenac, metoprolol, orphenadrine, silbutramine, symbicort, and thyroxine). Exclusion criteria were: past or recent history of neurological condition or seizures, neurosurgery or pacemakers, metal plates or cochlear implants. These criteria were assessed in the initial interview. The participants also completed the Edinburgh Handedness Inventory (Oldfield, 1971) (Appendix A) and a safety questionnaire based on the Laboratory for Magnetic Brain Stimulation, Beth Israel Deaconess Medical Centre, Boston (Appendix B). This study was approved by the Lower South Regional Ethics Committee, New Zealand, and written informed consent was obtained.

34 23 Participant Age Sex Handedness Handedness Medications Score 1 37 F R 100 silbutramine 2 21 M R 41 None 3 35 M R 90 metoprolol 4 22 M R 90 None 5 23 M R 100 None 6 42 F R 79 diclofenac, amitryptiline, orphenadrine 7 27 M R 58 bricanyl, symbicort 8 23 F R 70 None 9 18 F L None F R 100 None F R 68 thyroxine Table 2.1 Participants. The demographic features, handedness and medications of the 11 participants is shown. 2.2 TESTS Strength testing Testing of strength was performed with a handgrip and pinchgrip dynamometers from Sammons Jammar. The pinch grip testing was performed by placing the dynamometer between the metacarpo-phalangeal joint and proximal inter-phalangeal joint of the index finger of each hand. The index finger was flexed to 90º at the metacarpo-phalangeal joint. Thumb was flexed and adducted to press the transducer medially on to the index finger. Maximum voluntary contraction was determined on each side by recording the peak force produced during 3 consecutive maximal contractions, using only the highest force out of the three measurements for analysis Motor reaction times Reaction times were measured unimanually and bimanually, right and left, using E- Prime software (Schneider W, Eschman A et al. 2002) on a computer with millisecond timing to produce visual stimuli and measure reaction times (Hammond-Tooke et al., 2010). A 1cm 2 white fixation cross was displayed on a black background and replaced by the imperative stimulus, a green circle of 45mm diameter, after a time-delay varying randomly between 500 and 1000 milliseconds (msec) after the last response. Responses

35 24 were recorded via a computer keyboard with separate keys assigned to the right and left index fingers. The stimulus disappeared as soon as the first key was pressed and was replaced by the fixation cross. Three blocks of 36 responses were recorded for the left, right and bimanual reaction times to total 9 blocks and the order was counterbalanced across subjects. Emphasis was placed on speed of responding, but participants were also urged not to anticipate the green circle s appearance. If a stimulus was anticipated and a response occurred before the stimulus, the green circle persisted until the key was pressed again and the participant was instructed once more to avoid anticipation. Responses faster than 150 ms or more than 3 SD longer than the mean response (calculated for each individual separately) were discarded in the analysis Electromyography Electromyography was recorded using a gold-plated disc electrodes connected to a Sapphire EMG system (Medelec) and placed with the active electrode over the muscle belly of the left first dorsal interosseus muscle (FDI), reference electrode on the lateral surface of the 2nd metacarpo-phalangeal joint, and a ground electrode on the dorsum of the left hand. A timebase of 200 milliseconds (msec) and sensitivity of 2mV/cm was used for recordings Transcranial Magnetic Stimulation A Magstim Rapid 2 magnetic stimulator with an air-cooled 70 mm figure of eight coil or placebo coil was used for all transcranial magnetic stimulation. The coil was held by a stand with movable arm, which facilitated stable coil fixation over the target area. Subjects were seated in a comfortable chair facing a blank wall. The coil was placed tangentially over the right motor cortex with the handle projecting posteriorly. The optimal site for evoking a response in the FDI was determined using cm adjustments around the initial placement and a Lycro swim cap was used to mark the optimal stimulation site. This was defined as the scalp position yielding the largest MEP amplitude using a suprathreshold stimulus. Resting and active motor thresholds (RMT and AMT) were determined and resting and active MEP (RMEP and AMEP) were recorded in accordance with recommendations of the IFCN Committee (Rossini et al., 1994). RMT was defined at the optimal site as the lowest stimulus

36 25 intensity required to produce a MEP of at least 50 microvolts (mv) in greater than 50% of trials. AMT was defined as the lowest stimulus intensity required to produce a MEP of at least 500 mv in 50% of trials. AMT was measured while the subject maintained a pinch grip of 15% of maximum force with the left hand, as measured with a pinchgrip dynamometer. For RMEP and AMEP, 10 consecutive responses, separated by at least 5 seconds, were recorded using a stimulus intensity of 110% of RMT. Peak to peak MEP amplitudes were measured and averaged. Cortical silent period (CSP) was defined as the time from stimulus until resumption of 20% of pre-stimulus EMG activity using a stimulus intensity of 110% of RMT. 2.3 EXPERIMENTAL PROCEDURES Procedures In each session, reaction times were recorded and muscle contraction force was measured in each hand by dynamometry, after which approximately 10 RMEP and 10 AMEP were recorded using single pulses of TMS at 110% of resting motor threshold at least 5 seconds apart. Stimulation or sham stimulation was then performed for 10 minutes, the rtms protocol being counterbalanced between participants. The RMEP and AMEP were then repeated (in that order), followed by dynamometry and then reaction times. The structure of a typical session is presented in Figure rtms protocols Three rtms protocols were used in the experiment: 1. 1Hz rtms at 110% of RMT for 10 minutes 2. Theta burst priming at 80% RMT for 5 minutes followed by 1HZ repetitive stimulation at 110% of RMT for 5 minutes. Theta burst priming consisted of 5 second trains separated by 25 seconds. 3. Sham stimulation at 1 Hz using a placebo coil for 10 minutes. Only one protocol was used per session, yielding a total of three sessions of about 75 minutes each, at least one week apart. The order of protocols was counterbalanced across subjects.

37 26 Experimental procedure Reaction times Strength testing MEP measuring 10 minute sham 10 minute 1Hz rtms 5 min 5Hz+ 5 min 1Hz rtms Reaction times Strength testing MEP measuring Figure-2.1 Structure of a typical experimental session Data analysis The effects of the three sessions on neurophysiological and behavioral variables were analyzed using a mixed linear model with the post-test values as the dependent variable and pre-test values as covariates and participant as a random variable. Estimated marginal means were determined and presented graphically for variables which showed statistical significance. All analyses were carried out using SPSS statistical software RESULTS Side effects of TMS Apart from the one participant who was excluded because of nausea, there were no side effects of TMS. Neurophysiological variables The results for the neurophysiological variables are shown in Table 2.2. There was a significant effect of rtms protocol on both RMEP and AMEP amplitude, p<0.05. Both were significantly lower after 1Hz rtms compared to sham, as shown in Figures 2.2 and 2.3. RMEP and AMEP amplitudes were not significantly changed compared to sham rtms after the priming protocol (Figures 2.2 and 2.3). There was no significant effect of unprimed or primed rtms on resting or active latencies, thresholds or CSP.

38 27 Behavioural variables The 1Hz protocol as well as the theta priming significantly reduced right unimanual reaction time compared to sham (Table 2.3). There was no effect on the contralateral (left) hand or on bimanual cost in either hand. There was no significant effect of protocol on muscle strength as tested by dynamometry. (Table 2.4). Figure 2.2 Resting motor evoked potential (RMEP) amplitudes. Values indicate the marginal means of the RMEP amplitudes following treatment with the rtms protocols. Error bars represent standard errors of the mean. * = p <0.005, compared to sham stimulation. V = microvolts.

39 28 Figure 2.3 Active motor evoked potentials. Active motor evoked potential (RMEP) amplitudes recorded during contraction at 15% of maximal voluntary contraction. Values indicate the marginal means of the AMEP amplitudes following treatment with the rtms protocols. Error bars represent standard errors of the mean. * = p=0.001, compared to sham stimulation. V = microvolts.

40 Figure 2.4 Right unimanual reaction times. Values indicate the marginal means of the reaction following treatment with the rtms protocols. Error bars represent standard errors of the mean. * p<0.02, ** p = compared to sham stimulation. msec = milliseconds 29

41 30 Variable Post-sham rtms Post-theta priming + 1Hz rtms Post- 1Hz rtms df F Significance Resting threshold (% RMT) 81.3± ± ±2.5 2, NS Active threshold (% RMT) 73.4± ± ±2.7 2, NS Resting latency (msec) 22.9± ± ±0.2 2, NS Active latency (msec) 21.2± ± ±0.4 2, NS Resting MEP amplitude (microv) 2230 ± ± ±436 2, p<0.05 Active MEP amplitude (microv) 4697 ± ± ±407 2, p<0.005 Cortical Silent Period (msec) 138 ± ± ±15 2, NS Table 2.2. Neurophysiological measures. Marginal estimates ± standard errors are shown for parameters following treatment with each protocol. F values and significance are shown for the test of fixed effects in the mixed linear model. Significant differences were noted for active latency and resting and active MEP. microv = microvolts; msec = milliseconds; RMT = resting motor threshold; NS = not significant; df = degrees of freedom.

42 31 Variable Post-sham rtms Post-theta priming + 1Hz rtms Post- 1Hz rtms df F Significance Left unimanual RT (msec) 236 ±6 232 ±7 227 ±6 2, NS Left Bimanual RT (msec) 244 ±5 239 ±6 240 ±6 2, NS Left BC (msec) 7.00 ± ± ±3.63 2, NS Right Unimanual RT (msec) 239 ±4 235 ±5 234 ±4 2, p<0.05 Right Bimanual RT (msec) 250±6 250 ±7 250 ±6 2, NS Right BC (msec) 9.70 ± ± ±4.14 2, NS Table 2.3 Reaction Times. Marginal estimates ± standard errors are shown for parameters following treatment with each protocol. F values and significance are shown for the test of fixed effects in the mixed linear model. There was a significant effect of protocol for right unimanual reaction times. RT = reaction times; msec = milliseconds; NS = not significant; df = degrees of freedom.

43 32 Variable Post-sham rtms Post-theta priming + 1Hz rtms Post- 1Hz rtms df F Significance Left Pinchgrip (Kg) 10.0± ± ±0.5 2, NS Right Pinchgrip (Kg) 10.8± ± ±0.4 2, NS Left Handgrip (Kg) Right Handgrip (Kg) 33.5± ± ±0.9 2, NS 35.9± ± ±1.2 2, NS Table 2.4 Strength Tests. Marginal estimates for dynamometer pinch and grip strength ± standard error of the mean are shown. There was no significant difference between the three protocols. Kg = kilograms; NS = not significant; df = degrees of freedom.

44 Chapter III- Discussion 33

45 DISCUSSION The purpose of this study was to investigate the use of theta priming with 1 Hz rtms to the motor cortex in order to induce corticospinal inhibition in healthy volunteers. The ultimate aim of this was to develop rtms further as a therapy for disease, as the effects of 1 Hz rtms on its own are inconsistent. In contrast to Iyer et al (2003), we found that instead of enhancing the inhibitory effect of 1Hz rtms, theta priming abolished or interfered with it. The main difference between the two studies was that we gave 5 minutes of theta priming followed by 5 minutes of 1Hz rtms and compared this with 10 minutes of rtms (keeping the total period of treatment the same), whereas Iyer and his colleagues added 10 minutes of theta priming to 10 minutes of the 1 Hz theta and compared it to 10 minutes of 1 Hz theta alone. As the duration of the treatment in Iyer s study was doubled by using the priming stimulation, the main question that motivated our experiment was whether there was any real benefit in using theta priming instead of simply a longer period of 1Hz rtms. We wished to determine whether theta priming could achieve the same or greater inhibition when given in the same time frame as 1 Hz rtms alone. We not only found lack of enhancement of the inhibitory effect of 1 Hz rtms, but the inhibitory effect was abolished. It is possible that 5 minutes of priming is insufficient or that priming would have been effective if 1 Hz rtms was given for at least 10 minutes. The duration of priming and rtms that it is designed to enhance may therefore be quite critical. Another difference in the two studies is that Iyer studied MEP continuously for 10 to 60 minutes after the treatment, using stimuli every 10 seconds. We studied the early effect on MEP (about 5 minutes after treatment) and did not look for late effects. Furthermore, we used 80% of RMT as the stimulus intensity for the priming protocol, rather than 90% RMT as used in their study. Other studies have failed to replicate those of Iyer et al (2003). Daskalakis et al (2006) also used theta priming in combination with 1 Hz (but other frequencies as well). They did not find any significant change in corticospinal excitability for any of the protocols they used, including priming. In another study, theta priming of a rtms protocol was applied to the temporal lobe to treat tinnitus (Langguth et al., 2007). Like the Iyer study, the total period of treatment was different in the theta priming and 1 Hz rtms arms of the trial. A protocol of 2000 stimuli at 1 Hz and an intensity of 110% motor threshold over the left auditory cortex was compared with 1040 stimuli at 1 Hz preceded by priming stimulation of 960 stimuli at 6 Hz and an intensity of 90% motor threshold (6

46 35 trains lasting 10 seconds separated by 20 seconds). Thus the standard treatment lasted 33 minutes and the theta priming protocol lasted 25 minutes. There was no significant difference in the effect of the two protocols; both resulted in clinical improvement within 10 days. Thus there was no effect of priming in that study, as in ours LIMITATIONS OF THE STUDY One limitation of our study is blinding, there is a serious limitation of TMS placebo coils: they are very easy to distinguish from real TMS, so patients and experimenters are often aware of the type of coil being used. The Magstim placebo coil provides the clicking sound of real coil, but no significant magnetic pulse. Nevertheless some of the participants would have noted the lack of effect on the target muscle when sham rtms was administered. We did not attempt to blind the investigator performing the study, but the participant could not see which coil was being used and was not told what protocol was being used. We did not use the theta stimulation in the placebo treatment which was carried out at 1 Hz only. More importantly we did not explore different durations of the priming and 1 Hz stimulation, which would have been invaluable, given the result we obtained. The effect of priming by itself was not studied, it is possible that it might have produce an effect by itself, in which case the 1 Hz stimulation may have produced depotentiation of a prior facilitatory effect of the theta burst stimulation. Another shortcoming was that we did not test MEP at later time points,, so we could not rule out the possibility of late effects of rtms. Finally, we did not use neuronavigation, in which the position of the coil is matched to the participants neuroimaging. This can improve the consistency of stimulation, ensuring that the same specific area is stimulated on each trial (Bashir et al, 2010). 3.3 EFFECTS OF PROTOCOLS We demonstrated that the inhibitory effect of 1 Hz rtms was abolished when combined with theta priming. One interpretation of this is that the priming protocol (theta stimulation) interfered with the second protocol (1Hz), and canceled out its inhibitory influence on the cortical tissue. We suggest that this phenomenon be referred to as priming interference. Another explanation of our results would be that the priming theta stimulation produced corticospinal facilitation and 1 Hz rtms abolished this by a

47 36 mechanism of de-potentiation analogous to studies in plasticity in the rat brain (Larsen et al., 1993). This has been described in previous rtms studies. Huang et al. (2010) reversed the excitatory effect of intermittent TBS (itbs) and the depressive effect of ctbs using a short form of continuous TBS given 1 min after itbs, whereas the depressive effect of continuous TBS (ctbs) was successfully abolished by a short form of itbs (itbs150). The authors suggested that the reversal was due to depotentiation. Iezzi et al. (2011) found that pre-treatment with 5 Hz rtms was capable of interfering with LTP and LTD induced by TBS. This was thought to be the result of short-term potentiation (STP) induced by the 5 Hz protocol causing a rise in calcium ion levels, impairing further calcium increase caused by TBS protocols and thus interfering with the second protocol. Nevertheless it is important to consider the fact that there might be several mechanisms of interference, just as there are several mechanisms that can produce plasticity. Because 5 Hz rtms on its own left corticospinal unchanged, this would be better described as priming interference rather than de-potentiation. In our study, we did not test the effects of the priming protocol alone, and so we are uncertain if it had a measurable effect on MEP parameters. Low frequency rtms is thought to work via induction of LTD. Iyer et al based their priming protocol on the work of Christie and Abraham (1992) who described a summatory effect of 5 Hz-induced associative LTD and 1 Hz-induced non-associative LTD (Christie and Abraham, 1992). Nevertheless in our study theta priming reversed the effects of the subsequent rtms. Christie and Abraham speculated that there is a possibility that 5 Hz associative LTD could interfere with non-associative LTD formation. In my view this is unlikely since both LTD types seem to perfectly overlap during successful priming experiments. It is more likely thus that interference is the result of producing associative LTP instead of associative LTD (further explanation below), causing an interference. LTP depotentiation at single synapses in the hippocampus can be induced by 1 Hz protocols, as has been shown in animal experiments (Abraham and Tate, 1997). Nonassociative LTD is obtained in a test neural pathway (T-Path) as a result of a stimulation protocol in adjacent conditioned pathway that induces LTP in the conditioned pathway (C-Path) (see Figure 3.1a). This type of LTD is heterosynaptic (via a group of

48 37 indirectly related synaptic targets different to the concerned pathways) and happens when the test pathway is inactive (Levy and Stewart, 1979). This depotentiation facilitates heterosynaptic (non-associative) LTD (Christie and Abrahm, 1992). Associative LTD is the result of homosynaptic activity (via interconnected synapses of the concerned pathways) as demonstrated by stimulating a conditioned and a test pathway of the rat hippocampus (Stanton and Sejnowksi, 1989) (see Figure 3.1b). A similar approach was used by Christie and Abraham (1992), who found that if 5 hz trains of stimuli were applied in the test pathway prior to the induction of a negative correlation protocol in the rat dentate gyrus, associative LTD was obtained and the overall LTD was enhanced (see Fig 3.1c). It seems that both group of researchers achieved associative LTD, but the latter also found a way to enhance associative LTD through priming. Nevertheless, there is an interesting case scenario: if both pathways were stimulated in a positively correlated fashion (in phase) then the result in the test pathway should be associative LTP, depending on the timing of the stimuli (see Figure 3.1d) (Stanton and Sejnowksi, 1989) Mechanisms of associative LTP/LTD It seems that the mechanisms of associative LTD or LTP are the result of the interaction of states of polarization in which the concerned pathways are in a specific moment. For example, if the C-Path activity is out of phase with the T-Path activity, the postsynaptic neurons in the C-Path are hyperpolarized as a result of inhibitory postsynaptic potentials when the stimulus in the T-Path arrives. It appear that postsynaptic hyperpolarization in one path when coupled with presynaptic activity in the other, triggers LTD (ibid). In associative LTP, stimuli in both C-Path and T-Path are simultaneous, causing an increase in AP peak amplitude, this is possibly the result of a resonant activity in both pathways, causing potentiation of the test pathway input synapses, triggering a fast high rise in calcium levels activity and the resulting LTP in the T-Path.

49 38 Figure 3.1 stimulation paradigms. A) non-associative LTD, as proposed by Christie and Abraham, B) associative LTD as proposed by Stanton et al, C) Priming as proposed by Christie and Abraham, D) Associative LTP as proposed by Stanton and Sejnowksi (1989). These paradigms utilize stimuli with a periodicity in the range of 5Hz hippocampal rhythmical slow-wave activity (RSA).

50 Extrapolation to rtms In order to understand the phenomenon of priming interference, the interaction between the 5 Hz priming protocol and the subsequent 1Hz must first be explained for the situation when it is successful in achieving enhanced inhibition and then when interference develops. Successful priming of inhibition as described above is the result of a combination of pre-treatment and a negatively correlated homosynaptic activity in two or more interconnected neural pathways. However, the difference between animal experiments and rtms is that magnetic stimulation is a non-invasive technique that targets populations of neurons instead of a couple of pathways, this means that there is no way to control the coupling relationship between postsynaptic hyperpolarization and presynaptic activity in adjacent pathways, so there is no way of knowing if the inherent firing rates of stimulated pathways are negatively or positively correlated. Thus, with current priming protocols is impossible to control whether LTD or LTP will develop. Nevertheless when priming is successful, as in the case of the Iyer experiment, it seems logical to assume that the target population was working with a degree of negative correlation in their firing rates amongst pathways before the priming protocol, achieving thus enhanced associative LTD (see figure 3.1c). The second protocol (1Hz) then induces non- associative LTD, producing an overall increment in inhibition. It is beyond the scope of this thesis to speculate on the exact reasons for a negative or positive correlation trend of activity in naturally behaving neural populations, but it has been speculated that synaptic plasticity must have a mechanism to avoid synaptic saturation (a theoretical supposition where synapses cannot encode more information due to the inability to be weakened, not just strengthened). Such balance mechanisms are speculated to be constrained to regular fluctuations in neural networks of positive and negative correlation of firing rates among neurons (von der Malsburg, 1973; Uttley, 1976; Sejnowski, 1977). In the case of unsuccessful priming one may speculate that a positive correlation in naturally behaving neural pathways is set (see Figure 3.1d) inducing associative LTP before priming stimulation takes place. In this scenario, priming probably enhances associative LTP, and then the second protocol (1Hz) induces the non-associative form of LTD in the entire target population. It is then likely that a functional interference

51 40 between non-associative LTD and associative LTP develops. This is consistent with the Covariance model of synaptic plasticity (Sejnowski TJ 1977). This model explains that stimuli that do not produces covariance, produce no change in synaptic strength. (In this context, covariance is defined as change in the synaptic strength as a result of naturally occurring firing rates or stimulation.) No covariance means that a test pathway that receives the same number of positively correlated stimuli (in phase) and negatively correlated stimuli (out of phase) produces no net change in synaptic strength. In this scenario, LTP and LTD mechanisms are balanced, indicating a similarity of strength. In naturally behaving neural populations this could mean that if priming rtms stimulation is applied to a neural network, (supposing the trend of the activity at that specific moment is in phase and produces associative LTP), it is then correlated to a second stimuli train of stimulation applied at 1Hz that will trigger non-associative LTD, a counterbalance of effects can develop if strengths are similar, appearing as a very mild change in MEP measurement that is statistically non-relevant. In our experiment the protocols were balanced in duration: (5 minutes theta priming+5minutes 1Hz). Our intensities were 80% for the theta stimulation, and 110% for the 1Hz stimulation. Further experiments would help to understand what is the exact proportion of each variable (duration, frequency and intensity) that is necessary in each protocol to avoid an interference effect on motor inhibition OVERCOMING INTERFERENCE The main problem with current priming protocols used in rtms experiments is the lack of capacity to stimulate pathways with negative correlation. The original animal research proposes an associative conditioning stimulation that controls both paths to be conditioned; this is quite different to current rtms paradigms, which use the priming and the 1Hz protocol in the same tissue with no control over neural paths whatsoever. It is impossible to achieve with non-invasive rtms the same degree of specificity as observed in animal experiments. Nevertheless, it is possible to have more control if for example, a priming protocol is followed by an actual conditioning stimulation, which is what has been recently proposed by Delvendahl et al (2010) using Paired Associative Stimulation. However if we want to overcome the phenomenon of priming interference, a way must be found to induce a negative correlation in stimulated neural paths. One

52 41 possible solution could be a co-stimulation protocol resembling the experiments with negative correlation stimulation. To obtain inhibition in the left primary motor area M1 for example, the transcallosal communication pathway between contralateral M1 areas could be used to provide a conditioned right M1 stimulation and a test left M1 stimulation with a negative correlation. For this purpose two rtms coils could be placed on each M1. Previous to the conditioning protocol, a priming 5 Hz theta burst stimulation would applied to the left test area, then, in the conditioning protocol, both coils would stimulate with an interleaved protocol. The right coil would stimulate with a 25 Hz burst every 200 msec for 5seconds, with a 10 second resting period, the left coil would then stimulate with a single pulse in the middle of the 10 second resting period (see figure 3.2). A 5 minute protocol would apply 20 theta bursts to the right side and 20 pulses to the left. This would be very similar to the protocol used by Christie and Abraham (1992) in their animal experiments (figure 3.1c). It is predicted that this or similarly designed stimulation paradigms would achieve enhanced associative LTD in the left M1 area. Figure 3.2 Co-stimulation protocol with negative correlation. 5 Hz at 80% RMT is applied to the right M1 and 0.1Hz at 115% RMT to the left.