THE COMBINED USE OF TRANSCRANIAL MAGNETIC STIMULATION AND ELECTROENCEPHALOGRAPHY IN THE INVESTIGATION OF REACTIVITY, CONNECTIVITY AND PLASTICITY OF THE PRIMARY MOTOR CORTEX

The present thesis comprises two main parts: one theoretical and one experimental. The first part, composed of two chapters, is an in-depth introduction to transcranial magnetic stimulation (TMS) and its simultaneous use with neuroimaging techniques (coregistration). The second part is composed of some of the studies I conducted during my PhD. I chose to include three studies representing the different aspects of my research in the last three years, mainly regarding the study and the application of TMS-EEG coregistration in research (study 1), clinics (study 2) and technical methodology (study 3). The first study (study 1), conducted at the Department of General Psychology of Padova, was aimed to investigate the neuromodulatory effects of an rTMS protocol on healthy volunteers. The second study (study 2) was conducted at the Institute of Neurology of University College London in the context of the international “TrackOnHD” longitudinal project aimed to investigate Huntington disease (HD) in a multimodal approach. The target of this study was to investigate possible TMS-EEG markers of inhibition deficits in Huntington patients. The third study (study 3), conducted in collaboration with the Department of Information Engineering of Padova, was aimed to develop an algorithm of correction to remove an artefact induced by TMS during EEG recordings. CHAPTER I – TRANSCRANIAL MAGNETIC STIMULATION In the last twenty years the development of new techniques able to investigate the brain function in vivo during cognitive and motor tasks lead to impressive advances in understanding the human brain. Transcranial magnetic stimulation (TMS) is a tool whose popularity has grown progressively thanks to its ability to stimulate the brain in a focal and non-invasive way (Barker et al., 1985), permitting to establish a causal link in the brain-cognition/motor-behaviour relationship (Pascual-Leone et al., 2000). In the first chapter of this thesis the possible applications of TMS in the field of cognition, physiology and rehabilitation are discussed. Specifically, the first part focuses on the operating mechanisms of TMS and on the different stimulation parameters that define the effects of the stimulation. In the second part of the first chapter, the three main TMS protocols are discussed: single-pulse TMS, which is used in the temporal and spatial characterization of cognitive processes, in the study of motor cortex reactivity, and in the investigation of the cortico-spinal tract functioning; paired-pulse TMS, that investigates the connectivity and the interaction of cerebral networks at rest or during a task performance; and repetitive TMS (rTMS), that explores the cerebral plasticity processes both in relation to cognitive processing and for rehabilitation treatments. CHAPTER II – THE SIMULTANEOUS USE OF TRANSCRANIAL MAGNETIC STIMULATION WITH EEG AND OTHER NEUROIMAGING TECHNIQUES Despite the widespread use of TMS in current research, its mechanism of action is still poorly understood (Miniussi et al., 2010). This lack in comprehension results from missing a firsthand “visible” marker of cortical response and a need for secondary measures of primary motor and visual cortex stimulation. In the last twenty years, thanks to the progressive improvements in neuroimaging technology, the first attempts to simultaneously use TMS with other neuroimaging techniques have been made possible (e.g. TMS-EEG, Ilmoniemi et al., 1997; TMS-PET, Paus et al., 1997). On one hand, the possibility to actively stimulate the brain with TMS allows to establish “causal” inferences in neuroimaging studies, in which, traditionally, only “correlational” inferences were possible. On the other hand, neuroimaging techniques potentially provide an important contribution through the spatial and temporal information of the neural activation evoked by TMS. In the second chapter of this thesis, the strong and the weak points of different TMS-neuroimaging coregistration approaches are depicted. Specifically, the middle part of the chapter focuses on the main topic of this thesis, i.e. the TMS-EEG coregistration. TMS-EEG, among the different approaches, is the most successful and widespread, thanks to its promising value in the investigation of brain dynamics. Indeed, EEG is able to record the post-synaptic potentials following the neuronal depolarization evoked by TMS at a high temporal resolution (Ilmoniemi et al., 1997). The analysis of the TMS-evoked EEG activity in terms of time, space, frequency and power, potentially provides important and accurate information in the local activation induced by the stimulation (cerebral reactivity), in the spread of such activation (cerebral connectivity), and in the long-lasting neuromodulatory effects following rTMS protocols (cerebral plasticity). On the other hand, the TMS-EEG coregistration, presents several technical difficulties mainly due to the different artefacts that electromagnetic stimulation induces in the EEG signal. These aspects are discussed thoroughly in the second chapter. Finally, the last part of the second chapter is dedicated to the other TMS coregistration approaches with magnetic resonance imaging (MRI), functional magnetic resonance imaging (fMRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT) and near-infrared spectroscopy (NIRS). CHAPTER III – STUDY 1: NEUROMODULATORY EFFECTS OF LOW-FREQUENCY RTMS: INSIGHTS FROM TMS-EEG The neuromodulatory effects of rTMS have been mostly investigated by means of peripheral motor-evoked potentials (MEPs). However, MEPs are an indirect measure of cortical excitability, also being affected by spinal excitability. The development of new TMS-compatible EEG systems allowed the direct investigation of the stimulation effects through the cortical responses evoked by TMS (TEPs). In this study, we investigated the effects of a repetitive TMS (rTMS) protocol delivered at low frequency (1 Hz), which is known to produce an inhibitory effect on cortical excitability (Chen et al., 1997). The protocol was applied over the primary motor cortex of 15 healthy volunteers and, as a control, over the primary visual cortex of 15 different healthy volunteers to examine the spatial specificity of the stimulation. The effects of the stimulation were analyzed in both groups through the single-pulse stimulation of the primary motor cortex, before and immediately after the rTMS protocol. Different measures were tested: MEPs, TEPs, local mean field power and scalp maps of the activity distribution. Results on MEPs amplitude showed a significant reduction following the rTMS over the primary motor cortex. Results on TEPs, showed a well-known TEPs pattern evoked by single-pulse stimulation of the motor cortex: P30, N45, P60 and N100. Following the motor cortex rTMS, we observed a significant increase of P60 and N100 amplitude, whose origin has been linked to the GABAb-mediated inhibitory post-synaptic potentials (Ferreri et al., 2011; Premoli et al, 2014). Results on LMFP, showed an increase of general activity induced by the single-pulse stimulation of the motor cortex, starting from 90 ms after the TMS pulse. This latency actually corresponds to the peak of GABAb inhibition. No significant effects were detected after rTMS of the primary visual cortex. The results of this study are relevant in three main aspects: (1) we confirmed the inhibitory effect of 1-hz rTMS, also providing a central correlate of such effect (TEPs); (2) we defined the spatial specificity and the origin of the inhibitory effect of 1-Hz rTMS; (3) we confirmed the possible role of the TMS-evoked N100 as a cortical inhibitory marker. The present findings could be of relevance both for therapeutic purposes, especially for pathologies characterized by inhibitory deficits (e.g. Parkinson’s disease; Huntington’s disease); and for basic research, especially in studies aimed to correlate a behavioral performance to the amount of cerebral excitability. CHAPTER IV – STUDY 2: TMS-EEG MARKERS OF INHIBITORY DEFICIT IN HUNTINGTON’S DISEASE Recent studies have shown the potential value of combining TMS and EEG for clinical and diagnostic purposes. Several TMS-EEG measures in terms of evoked potentials (i.e. TEPs), brain sources analysis, oscillatory activity and global power has been used in the assessment of brain dynamics deficits in several pathologies, such as: schizophrenia (Ferrarelli et al., 2008); psychotic disorders (Hoppenbrouwers et al., 2008); depression (Kähkönen et al., 2005); awareness disorders (Massimini et al., 2005); epilepsy (Rotenborg et al., 2008) and autism (Sokhadze et al., 2012). For instance, the potential contribution of TEPs in the investigation of the cerebral facilitatory/inhibitory balance has been demonstrated, given their origin from different GABAergic neuronal populations (Ferreri et al., 2011; Premoli et al., 2014). In particular, the TMS-evoked N100 has been related to the amount of GABAergic inhibition, as shown by pharmacological (e.g. Kähkönen et al., 2003) and behavioral research (e.g. Bender et al., 2005; Bonnard et al., 2009) as well as studies in patients (e.g. Helfrich et al., 2013). As a part of the multi-site international “TrackOnHD” project, we used TMS-EEG to investigate the electrophysiological markers of motor cortex stimulation in Huntington patients. In Huntington’s disease (HD) the progressive degeneration of GABAergic neurons in the striatum lead to a strong reduction of inhibition, resulting in an excessive increase in glutamatergic excitability (i.e. excitoxicity). Our study compared a group of 12 HD patients with a group of 12 healthy volunteers over several different TMS-EEG, EMG, fMRI and clinical measures (in the chapter only the TMS-EEG results are reported). We found a specific and significant decrease of the N100 as assessed by the time point-by-time point permutation analysis of TEPs and from the analysis of the global activity from 90 to 104 ms after the TMS pulse. Scalp maps of the activity distribution showed a bilateral decrease of negativity, such effect was stronger over the site of stimulation. Event-related spectral perturbation and inter-trial coherence analysis showed a significant difference in the oscillatory activity of the two groups within the GABAb-ergic time window (i.e. 60-110 ms after the TMS pulse). We speculated that the observed results might be produced by the deficit in GABAergic inhibition as a consequence of the striatum neuronal degeneration in HD patients. Although preliminary, these results provided potentially useful TMS-EEG markers for inhibitory deficits in HD patients. Further analyses are needed to correlate the present findings with the other measures collected. CHAPTER V – STUDY 3: TMS-EEG ARTIFACTS: A NEW ADAPTIVE ALGORITHM FOR SIGNAL DETRENDING During EEG recording the discharge of the TMS coil may generate an artefact that can last for tens of milliseconds, known as “decay artefact” (Rogasch et al., 2014). This can represent a problem for the analysis of the TMS-evoked potentials (TEPs). So far, two main strategies of correction have been proposed involving the use of a linear detrend or independent component analysis (ICA). However, none of these solutions may be considered optimal: firstly, because in most of the cases the decay artefact shows a non-linear trend; secondly, because the ICA correction (1) might be influenced by individual researcher’s choices and (2) might cause the removal of physiological responses. Our aim is to verify the feasibility of a new adaptive detrend able to discriminate the different trends of the decay (linear or non-linear). Forty healthy volunteers were stimulated with 55 TMS pulses over the left M1. The TMS-EEG responses were compared among five conditions: RAW (no correction of the decay artefact was applied); INFOMAX29 (the decay components were extracted and removed by the INFOMAX ICA algorithm, using 31 electrodes); FASTICA (the decay components were extracted and removed by the fastICA ICA algorithm, using 31 electrodes); INFOMAX15 (the decay components were extracted and removed by the INFOMAX ICA algorithm, using 15 electrodes) and ALG (the decay artefact was corrected through the use of an adaptive algorithm). To assess whether the artefact correction significantly affected the physiological responses to TMS as well, we examined the differences in the -100 + 400 ms time window around the TMS pulse by means of a non-parametric, cluster-based, permutation statistical test. Then we compared the peak-to-peak TEPs amplitude within the detected time windows. The grand-averaged EEG response revealed five main peaks: P30, N45, P60, N100 and P180. Significant differences (i.e. Monte Carlo p-values < 0.05) were detected in a cluster nearby the TMS coil, and specifically over FC1, CP1, C3 and FC2. Repeated-measures ANOVA revealed a significant corruption of the peak-to-peak amplitude after INFOMAX29 (3 TEPs out of 8), FASTICA (4 TEPs out of 12), INFOMAX15 (5 TEPs out of 15) and ALG correction (2 TEPs out of 15), compared to the original signal. Furthermore, abnormal LMFP and TEPs scalp distribution were detected following the INFOMAX29 and FASTICA correction. When our algorithm was used, however, the TEPs amplitude, morphology and distribution was in line with the literature and not significantly different from the original signal. Also the decay artefact was correctly removed. The main contribution of this study is the proposal of a new adaptive algorithm to correct the decay artefact induced by TMS in the EEG signal. Our results demonstrated that the proposed adaptive detrend is a reliable solution for the correction of this artefact, especially considering that, contrary to ICA, (1) it is not dependent from the number of recording channels; (2) it does not affect the physiological responses and (3) it is completely independent from the experimenter’s choices

Transcranial Direct Corrent stimulation (tDCS) of the anterior prefrontal cortex (aPFC) modulates reinforcement learning and decision-making under uncertainty: A doubleblind crossover study

Reinforcement learning refers to the ability to acquire information from the outcomes of prior choices (i.e. positive and negative) in order to make predictions on the effect of future decision and adapt the behaviour basing on past experiences. The anterior prefrontal cortex (aPFC) is considered to play a key role in the representation of event value, reinforcement learning and decision-making. However, a causal evidence of the involvement of this area in these processes has not been provided yet. The aim of the study was to test the role of the orbitofrontal cortex in feedback processing, reinforcement learning and decision-making under uncertainly. Eighteen healthy individuals underwent three sessions of tDCS over the prefrontal pole (anodal, cathodal, sham) during a probabilistic learning (PL) task. In the PL task, participants were invited to learn the covert probabilistic stimulusoutcome association from positive and negative feedbacks in order to choose the best option. Afterwards, a probabilistic selection (PS) task was delivered to assess decisions based on the stimulus-reward associations acquired in the PL task. During cathodal tDCS, accuracy in the PL task was reduced and participants were less prone to maintain their choice after positive feedback or to change it after a negative one (i.e., winstay and lose-shift behavior). In addition, anodal tDCS affected the subsequent PS task by reducing the ability to choose the best alternative during hard probabilistic decisions. In conclusion, the present study suggests a causal role of aPFC in feedback trial-by-trial behavioral adaptation and decision-making under uncertainty

Real-time cortical dynamics during motor inhibition

The inhibition of action is a fundamental executive mechanism of human behaviour that involve a complex neural network. In spite of the progresses made so far, many questions regarding the brain dynamics occurring during action inhibition are still unsolved. Here, we used a novel approach optimized to investigate real-time effective brain dynamics, which combines transcranial magnetic stimulation (TMS) with simultaneous electroencephalographic (EEG) recordings. 22 healthy volunteers performed a motor Go/NoGo task during TMS of the hand-hotspot of the primary motor cortex (M1) and whole-scalp EEG recordings. We reconstructed source-based real-time spatiotemporal dynamics of cortical activity and cortico-cortical connectivity throughout the task. Our results showed a task-dependent bi-directional change in theta/gamma supplementary motor cortex (SMA) and M1 connectivity that, when participants were instructed to inhibit their response, resulted in an increase of a specific TMS-evoked EEG potential (N100), likely due to a GABA-mediated inhibition. Interestingly, these changes were linearly related to reaction times, when participants were asked to produce a motor response. In addition, TMS perturbation revealed a task-dependent long-lasting modulation of SMA-M1 natural frequencies, i.e. alpha/beta activity. Some of these results are shared by animal models and shed new light on the physiological mechanisms of motor inhibition in humans

Feeling of ownership over an embodied avatar's hand brings about fast changes of fronto-parietal cortical dynamics

When we look at our body parts, we are immediately aware that they belong to us and we rarely doubt about the integrity, continuity, and sense of ownership of our body. Despite this certainty, immersive virtual reality (IVR) may lead to a strong feeling of embodiment over an artificial body part seen from a first-person perspective (1PP). Although such feeling of ownership (FO) has been described in different situations, it is not yet understood how this phenomenon is generated at neural level. To track the real-time brain dynamics associated with FO, we delivered transcranial magnetic stimuli over the hand region in the primary motor cortex (M1) and simultaneously recorded electroencephalography (EEG) in 19 healthy volunteers (11 male/8 female) watching IVR renderings of anatomically plausible (full-limb) versus implausible (hand disconnected from the forearm) virtual limbs. Our data show that embodying a virtual hand is temporally associated with a rapid drop of cortical activity of the onlookers' hand region in the M1 contralateral to the observed hand. Spatiotemporal analysis shows that embodying the avatar's hand is also associated with fast changes of activity within an interconnected fronto-parietal circuit ipsilateral to the brain stimulation. Specifically, an immediate reduction of connectivity with the premotor area is paralleled by an enhancement in the connectivity with the posterior parietal cortex (PPC) which is related to the strength of ownership illusion ratings and thus likely reflects conscious feelings of embodiment. Our results suggest that changes of bodily representations are underpinned by a dynamic cross talk within a highly-plastic, fronto-parietal network

Simultaneous transcranial electrical and magnetic stimulation boost gamma oscillations in the dorsolateral prefrontal cortex

Neural oscillations in the gamma frequency band have been identified as a fundament for synaptic plasticity dynamics and their alterations are central in various psychiatric and neurological conditions. Transcranial magnetic stimulation (TMS) and alternating electrical stimulation (tACS) may have a strong therapeutic potential by promoting gamma oscillations expression and plasticity. Here we applied intermittent theta-burst stimulation (iTBS), an established TMS protocol known to induce LTP-like cortical plasticity, simultaneously with transcranial alternating current stimulation (tACS) at either theta (theta tACS) or gamma (gamma tACS) frequency on the dorsolateral prefrontal cortex (DLPFC). We used TMS in combination with electroencephalography (EEG) to evaluate changes in cortical activity on both left/right DLPFC and over the vertex. We found that simultaneous iTBS with gamma tACS but not with theta tACS resulted in an enhancement of spectral gamma power, a trend in shift of individual peak frequency towards faster oscillations and an increase of local connectivity in the gamma band. Furthermore, the response to the neuromodulatory protocol, in terms of gamma oscillations and connectivity, were directly correlated with the initial level of cortical excitability. These results were specific to the DLPFC and confined locally to the site of stimulation, not being detectable in the contralateral DLPFC. We argue that the results described here could promote a new and effective method able to induce long-lasting changes in brain plasticity useful to be clinically applied to several psychiatric and neurological conditions

Familial hypercholesterolaemia in children and adolescents from 48 countries: a cross-sectional study

Background: Approximately 450 000 children are born with familial hypercholesterolaemia worldwide every year, yet only 2·1% of adults with familial hypercholesterolaemia were diagnosed before age 18 years via current diagnostic approaches, which are derived from observations in adults. We aimed to characterise children and adolescents with heterozygous familial hypercholesterolaemia (HeFH) and understand current approaches to the identification and management of familial hypercholesterolaemia to inform future public health strategies. Methods: For this cross-sectional study, we assessed children and adolescents younger than 18 years with a clinical or genetic diagnosis of HeFH at the time of entry into the Familial Hypercholesterolaemia Studies Collaboration (FHSC) registry between Oct 1, 2015, and Jan 31, 2021. Data in the registry were collected from 55 regional or national registries in 48 countries. Diagnoses relying on self-reported history of familial hypercholesterolaemia and suspected secondary hypercholesterolaemia were excluded from the registry; people with untreated LDL cholesterol (LDL-C) of at least 13·0 mmol/L were excluded from this study. Data were assessed overall and by WHO region, World Bank country income status, age, diagnostic criteria, and index-case status. The main outcome of this study was to assess current identification and management of children and adolescents with familial hypercholesterolaemia. Findings: Of 63 093 individuals in the FHSC registry, 11 848 (18·8%) were children or adolescents younger than 18 years with HeFH and were included in this study; 5756 (50·2%) of 11 476 included individuals were female and 5720 (49·8%) were male. Sex data were missing for 372 (3·1%) of 11 848 individuals. Median age at registry entry was 9·6 years (IQR 5·8-13·2). 10 099 (89·9%) of 11 235 included individuals had a final genetically confirmed diagnosis of familial hypercholesterolaemia and 1136 (10·1%) had a clinical diagnosis. Genetically confirmed diagnosis data or clinical diagnosis data were missing for 613 (5·2%) of 11 848 individuals. Genetic diagnosis was more common in children and adolescents from high-income countries (9427 [92·4%] of 10 202) than in children and adolescents from non-high-income countries (199 [48·0%] of 415). 3414 (31·6%) of 10 804 children or adolescents were index cases. Familial-hypercholesterolaemia-related physical signs, cardiovascular risk factors, and cardiovascular disease were uncommon, but were more common in non-high-income countries. 7557 (72·4%) of 10 428 included children or adolescents were not taking lipid-lowering medication (LLM) and had a median LDL-C of 5·00 mmol/L (IQR 4·05-6·08). Compared with genetic diagnosis, the use of unadapted clinical criteria intended for use in adults and reliant on more extreme phenotypes could result in 50-75% of children and adolescents with familial hypercholesterolaemia not being identified. Interpretation: Clinical characteristics observed in adults with familial hypercholesterolaemia are uncommon in children and adolescents with familial hypercholesterolaemia, hence detection in this age group relies on measurement of LDL-C and genetic confirmation. Where genetic testing is unavailable, increased availability and use of LDL-C measurements in the first few years of life could help reduce the current gap between prevalence and detection, enabling increased use of combination LLM to reach recommended LDL-C targets early in life

THE COMBINED USE OF TRANSCRANIAL MAGNETIC STIMULATION AND ELECTROENCEPHALOGRAPHY IN THE INVESTIGATION OF REACTIVITY, CONNECTIVITY AND PLASTICITY OF THE PRIMARY MOTOR CORTEX

The present thesis comprises two main parts: one theoretical and one experimental. The first part, composed of two chapters, is an in-depth introduction to transcranial magnetic stimulation (TMS) and its simultaneous use with neuroimaging techniques (coregistration). The second part is composed of some of the studies I conducted during my PhD. I chose to include three studies representing the different aspects of my research in the last three years, mainly regarding the study and the application of TMS-EEG coregistration in research (study 1), clinics (study 2) and technical methodology (study 3). The first study (study 1), conducted at the Department of General Psychology of Padova, was aimed to investigate the neuromodulatory effects of an rTMS protocol on healthy volunteers. The second study (study 2) was conducted at the Institute of Neurology of University College London in the context of the international “TrackOnHD” longitudinal project aimed to investigate Huntington disease (HD) in a multimodal approach. The target of this study was to investigate possible TMS-EEG markers of inhibition deficits in Huntington patients. The third study (study 3), conducted in collaboration with the Department of Information Engineering of Padova, was aimed to develop an algorithm of correction to remove an artefact induced by TMS during EEG recordings. CHAPTER I – TRANSCRANIAL MAGNETIC STIMULATION In the last twenty years the development of new techniques able to investigate the brain function in vivo during cognitive and motor tasks lead to impressive advances in understanding the human brain. Transcranial magnetic stimulation (TMS) is a tool whose popularity has grown progressively thanks to its ability to stimulate the brain in a focal and non-invasive way (Barker et al., 1985), permitting to establish a causal link in the brain-cognition/motor-behaviour relationship (Pascual-Leone et al., 2000). In the first chapter of this thesis the possible applications of TMS in the field of cognition, physiology and rehabilitation are discussed. Specifically, the first part focuses on the operating mechanisms of TMS and on the different stimulation parameters that define the effects of the stimulation. In the second part of the first chapter, the three main TMS protocols are discussed: single-pulse TMS, which is used in the temporal and spatial characterization of cognitive processes, in the study of motor cortex reactivity, and in the investigation of the cortico-spinal tract functioning; paired-pulse TMS, that investigates the connectivity and the interaction of cerebral networks at rest or during a task performance; and repetitive TMS (rTMS), that explores the cerebral plasticity processes both in relation to cognitive processing and for rehabilitation treatments. CHAPTER II – THE SIMULTANEOUS USE OF TRANSCRANIAL MAGNETIC STIMULATION WITH EEG AND OTHER NEUROIMAGING TECHNIQUES Despite the widespread use of TMS in current research, its mechanism of action is still poorly understood (Miniussi et al., 2010). This lack in comprehension results from missing a firsthand “visible” marker of cortical response and a need for secondary measures of primary motor and visual cortex stimulation. In the last twenty years, thanks to the progressive improvements in neuroimaging technology, the first attempts to simultaneously use TMS with other neuroimaging techniques have been made possible (e.g. TMS-EEG, Ilmoniemi et al., 1997; TMS-PET, Paus et al., 1997). On one hand, the possibility to actively stimulate the brain with TMS allows to establish “causal” inferences in neuroimaging studies, in which, traditionally, only “correlational” inferences were possible. On the other hand, neuroimaging techniques potentially provide an important contribution through the spatial and temporal information of the neural activation evoked by TMS. In the second chapter of this thesis, the strong and the weak points of different TMS-neuroimaging coregistration approaches are depicted. Specifically, the middle part of the chapter focuses on the main topic of this thesis, i.e. the TMS-EEG coregistration. TMS-EEG, among the different approaches, is the most successful and widespread, thanks to its promising value in the investigation of brain dynamics. Indeed, EEG is able to record the post-synaptic potentials following the neuronal depolarization evoked by TMS at a high temporal resolution (Ilmoniemi et al., 1997). The analysis of the TMS-evoked EEG activity in terms of time, space, frequency and power, potentially provides important and accurate information in the local activation induced by the stimulation (cerebral reactivity), in the spread of such activation (cerebral connectivity), and in the long-lasting neuromodulatory effects following rTMS protocols (cerebral plasticity). On the other hand, the TMS-EEG coregistration, presents several technical difficulties mainly due to the different artefacts that electromagnetic stimulation induces in the EEG signal. These aspects are discussed thoroughly in the second chapter. Finally, the last part of the second chapter is dedicated to the other TMS coregistration approaches with magnetic resonance imaging (MRI), functional magnetic resonance imaging (fMRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT) and near-infrared spectroscopy (NIRS). CHAPTER III – STUDY 1: NEUROMODULATORY EFFECTS OF LOW-FREQUENCY RTMS: INSIGHTS FROM TMS-EEG The neuromodulatory effects of rTMS have been mostly investigated by means of peripheral motor-evoked potentials (MEPs). However, MEPs are an indirect measure of cortical excitability, also being affected by spinal excitability. The development of new TMS-compatible EEG systems allowed the direct investigation of the stimulation effects through the cortical responses evoked by TMS (TEPs). In this study, we investigated the effects of a repetitive TMS (rTMS) protocol delivered at low frequency (1 Hz), which is known to produce an inhibitory effect on cortical excitability (Chen et al., 1997). The protocol was applied over the primary motor cortex of 15 healthy volunteers and, as a control, over the primary visual cortex of 15 different healthy volunteers to examine the spatial specificity of the stimulation. The effects of the stimulation were analyzed in both groups through the single-pulse stimulation of the primary motor cortex, before and immediately after the rTMS protocol. Different measures were tested: MEPs, TEPs, local mean field power and scalp maps of the activity distribution. Results on MEPs amplitude showed a significant reduction following the rTMS over the primary motor cortex. Results on TEPs, showed a well-known TEPs pattern evoked by single-pulse stimulation of the motor cortex: P30, N45, P60 and N100. Following the motor cortex rTMS, we observed a significant increase of P60 and N100 amplitude, whose origin has been linked to the GABAb-mediated inhibitory post-synaptic potentials (Ferreri et al., 2011; Premoli et al, 2014). Results on LMFP, showed an increase of general activity induced by the single-pulse stimulation of the motor cortex, starting from 90 ms after the TMS pulse. This latency actually corresponds to the peak of GABAb inhibition. No significant effects were detected after rTMS of the primary visual cortex. The results of this study are relevant in three main aspects: (1) we confirmed the inhibitory effect of 1-hz rTMS, also providing a central correlate of such effect (TEPs); (2) we defined the spatial specificity and the origin of the inhibitory effect of 1-Hz rTMS; (3) we confirmed the possible role of the TMS-evoked N100 as a cortical inhibitory marker. The present findings could be of relevance both for therapeutic purposes, especially for pathologies characterized by inhibitory deficits (e.g. Parkinson’s disease; Huntington’s disease); and for basic research, especially in studies aimed to correlate a behavioral performance to the amount of cerebral excitability. CHAPTER IV – STUDY 2: TMS-EEG MARKERS OF INHIBITORY DEFICIT IN HUNTINGTON’S DISEASE Recent studies have shown the potential value of combining TMS and EEG for clinical and diagnostic purposes. Several TMS-EEG measures in terms of evoked potentials (i.e. TEPs), brain sources analysis, oscillatory activity and global power has been used in the assessment of brain dynamics deficits in several pathologies, such as: schizophrenia (Ferrarelli et al., 2008); psychotic disorders (Hoppenbrouwers et al., 2008); depression (Kähkönen et al., 2005); awareness disorders (Massimini et al., 2005); epilepsy (Rotenborg et al., 2008) and autism (Sokhadze et al., 2012). For instance, the potential contribution of TEPs in the investigation of the cerebral facilitatory/inhibitory balance has been demonstrated, given their origin from different GABAergic neuronal populations (Ferreri et al., 2011; Premoli et al., 2014). In particular, the TMS-evoked N100 has been related to the amount of GABAergic inhibition, as shown by pharmacological (e.g. Kähkönen et al., 2003) and behavioral research (e.g. Bender et al., 2005; Bonnard et al., 2009) as well as studies in patients (e.g. Helfrich et al., 2013). As a part of the multi-site international “TrackOnHD” project, we used TMS-EEG to investigate the electrophysiological markers of motor cortex stimulation in Huntington patients. In Huntington’s disease (HD) the progressive degeneration of GABAergic neurons in the striatum lead to a strong reduction of inhibition, resulting in an excessive increase in glutamatergic excitability (i.e. excitoxicity). Our study compared a group of 12 HD patients with a group of 12 healthy volunteers over several different TMS-EEG, EMG, fMRI and clinical measures (in the chapter only the TMS-EEG results are reported). We found a specific and significant decrease of the N100 as assessed by the time point-by-time point permutation analysis of TEPs and from the analysis of the global activity from 90 to 104 ms after the TMS pulse. Scalp maps of the activity distribution showed a bilateral decrease of negativity, such effect was stronger over the site of stimulation. Event-related spectral perturbation and inter-trial coherence analysis showed a significant difference in the oscillatory activity of the two groups within the GABAb-ergic time window (i.e. 60-110 ms after the TMS pulse). We speculated that the observed results might be produced by the deficit in GABAergic inhibition as a consequence of the striatum neuronal degeneration in HD patients. Although preliminary, these results provided potentially useful TMS-EEG markers for inhibitory deficits in HD patients. Further analyses are needed to correlate the present findings with the other measures collected. CHAPTER V – STUDY 3: TMS-EEG ARTIFACTS: A NEW ADAPTIVE ALGORITHM FOR SIGNAL DETRENDING During EEG recording the discharge of the TMS coil may generate an artefact that can last for tens of milliseconds, known as “decay artefact” (Rogasch et al., 2014). This can represent a problem for the analysis of the TMS-evoked potentials (TEPs). So far, two main strategies of correction have been proposed involving the use of a linear detrend or independent component analysis (ICA). However, none of these solutions may be considered optimal: firstly, because in most of the cases the decay artefact shows a non-linear trend; secondly, because the ICA correction (1) might be influenced by individual researcher’s choices and (2) might cause the removal of physiological responses. Our aim is to verify the feasibility of a new adaptive detrend able to discriminate the different trends of the decay (linear or non-linear). Forty healthy volunteers were stimulated with 55 TMS pulses over the left M1. The TMS-EEG responses were compared among five conditions: RAW (no correction of the decay artefact was applied); INFOMAX29 (the decay components were extracted and removed by the INFOMAX ICA algorithm, using 31 electrodes); FASTICA (the decay components were extracted and removed by the fastICA ICA algorithm, using 31 electrodes); INFOMAX15 (the decay components were extracted and removed by the INFOMAX ICA algorithm, using 15 electrodes) and ALG (the decay artefact was corrected through the use of an adaptive algorithm). To assess whether the artefact correction significantly affected the physiological responses to TMS as well, we examined the differences in the -100 + 400 ms time window around the TMS pulse by means of a non-parametric, cluster-based, permutation statistical test. Then we compared the peak-to-peak TEPs amplitude within the detected time windows. The grand-averaged EEG response revealed five main peaks: P30, N45, P60, N100 and P180. Significant differences (i.e. Monte Carlo p-values < 0.05) were detected in a cluster nearby the TMS coil, and specifically over FC1, CP1, C3 and FC2. Repeated-measures ANOVA revealed a significant corruption of the peak-to-peak amplitude after INFOMAX29 (3 TEPs out of 8), FASTICA (4 TEPs out of 12), INFOMAX15 (5 TEPs out of 15) and ALG correction (2 TEPs out of 15), compared to the original signal. Furthermore, abnormal LMFP and TEPs scalp distribution were detected following the INFOMAX29 and FASTICA correction. When our algorithm was used, however, the TEPs amplitude, morphology and distribution was in line with the literature and not significantly different from the original signal. Also the decay artefact was correctly removed. The main contribution of this study is the proposal of a new adaptive algorithm to correct the decay artefact induced by TMS in the EEG signal. Our results demonstrated that the proposed adaptive detrend is a reliable solution for the correction of this artefact, especially considering that, contrary to ICA, (1) it is not dependent from the number of recording channels; (2) it does not affect the physiological responses and (3) it is completely independent from the experimenter’s choices.La presente tesi si compone di due parti principali: una teorica e una sperimentale. La prima parte, suddivisa in due capitoli, è un approfondimento teorico sullo strumento stimolazione magnetica transcranica (TMS) e sul suo utilizzo simultaneo (ossia, in coregistrazione) con le tecniche di neuroimaging. La seconda parte comprende alcuni degli studi condotti durante il mio dottorato. Nello specifico, si tratta di tre studi che coprono i diversi aspetti applicativi delle ricerche che ho condotto in questi tre anni, ossia lo studio e l’utilizzo della coregistrazione TMS-EEG in ricerca (studio 1), in ambito clinico (studio 2) e per aspetti tecnico-metodologici (studio 3). Il primo studio (studio 1), condotto nel Dipartimento di Psicologia Generale di Padova, era volto all’analisi degli effetti neuromodulatori di un protocollo rTMS su volontari sani. Il secondo studio (study 2) è stato condotto all’Istituto di Neurologia dello University College London (Londra, Regno Unito) all’interno del progetto internazionale “TrackOnHD”, uno studio longitudinale avente come obiettivo l'indagine approfondita della Malattia di Huntington (HD) attraverso un approccio multimodale. L’obiettivo di questo studio era la ricerca di potenziali marker TMS-EEG che riflettessero il deficit di inibizione cerebrale che caratterizza questa patologia. Il terzo studio (study 3), svolto in collaborazione col Dipartimento di Ingegneria dell’Informazione di Padova, aveva l’obiettivo di sviluppare un algoritmo di correzione in grado di rimuovere un artefatto indotto dalla TMS durante la registrazione EEG. CAPITOLO I - LA STIMOLAZIONE MAGNETICA TRANSCRANICA (TMS) Negli ultimi anni lo sviluppo di nuove tecniche in grado di analizzare l’attivazione cerebrale durante processi cognitivi e motori, ha portato ad un avanzamento progressivo delle conoscenze sul cervello umano. La stimolazione magnetica transcranica (TMS) è stata uno degli strumenti la cui popolarità è cresciuta in questi ultimi anni, grazie alla possibilità di stimolare, in modo focale e non invasivo, il cervello in vivo (Barker et al., 1985). Tale capacità ha consentito, per la prima volta, la straordinaria possibilità di inferire delle relazioni causali tra cervello, processi cognitivi e motori, e comportamento (Pascual-Leone et al., 2000). Nel primo capitolo della presente tesi vengono passate in rassegna tutte le possibili applicazioni della TMS in campo cognitivo, fisiologico e riabilitativo. Nello specifico, la prima parte è dedicata ai meccanismi di funzionamento della TMS e ai parametri di stimolazione che ne definiscono i diversi effetti sul cervello. Nella seconda parte vengono invece passati in rassegna i tre principali protocolli di stimolazione: la TMS a singolo impulso, utilizzata per la caratterizzazione spaziale e temporale dei processi cognitivi, per analizzare la reattività della corteccia motoria primaria, e per verificare l’integrità del tratto cortico-spinale; la TMS a doppio impulso, per studiare la connettività e l’interazione di network cerebrali a riposo e durante lo svolgimento di un task; e la TMS ripetitiva (rTMS), utilizzata per analizzare i fenomeni di plasticità cerebrale sia durante processi cognitivi, sia in relazione a trattamenti riabilitativi. CAPITOLO II - L’UTILIZZO SIMULTANEO DELLA TMS CON L’EEG ED ALTRE TECNICHE DI NEUROIMAGING Nonostante la grande popolarità che la TMS ha conosciuto negli ultimi anni, molti aspetti del suo meccanismo d’azione sono ancora poco chiari (Miniussi et al., 2010). Tale ambiguità è dovuta al fatto che, fatta eccezione per la corteccia motoria e visiva primaria, la stimolazione TMS non fornisce dei marker “visibili” di eccitabilità corticale. Negli ultimi anni, grazie al miglioramento tecnologico degli strumenti di indagine neuroscientifica, si è iniziato a utilizzare simultaneamente (in coregistrazione) la TMS con diverse tecniche di neuroimaging. Ciò ha consentito di trarre delle inferenze di tipo “causale” e non più solo “correlazionale” (come nei tradizionali studi di neuroimaging) grazie alle informazioni spaziali e temporali sull’effetto della TMS che le tecniche di neuroimaging offrono. Nel secondo capitolo della presente tesi, vengono trattati dettagliatamente le potenzialità e i limiti delle diverse coregistrazioni TMS-neuroimaging. In particolare, nella parte centrale del capitolo è dato ampio spazio all’argomento centrale di questa tesi, ossia la coregistrazione TMS-EEG. L’approccio TMS-EEG, tra i vari metodi di coregistrazione, è stato quelli che negli ultimi anni ha riscontrato maggiore successo e diffusione, dovuto all’enorme potenzialità che questo metodo garantisce nello studio delle dinamiche cerebrali. L’EEG, infatti, è in grado di registrare, ad altissima risoluzione temporale, i potenziali post-sinaptici indotti dalla depolarizzazione neuronale evocata dalla TMS (Ilmoniemi et al., 1997). L’analisi dell’attività EEG indotta dalla TMS - in termini di tempo, spazio, frequenza e potenza - è in grado di fornire delle preziose informazioni sia sull’attivazione locale indotta dalla stimolazione (reattività cerebrale), sia su quella distale (connettività cerebrale), sia sulle modificazioni a seguito di protocolli di stimolazione ripetitiva (plasticità cerebrale). D’altra parte, la coregistrazione TMS-EEG presenta numerose difficoltà di tipo tecnico, dovuto ai numerosi artefatti che la stimolazione elettromagnetica induce sul segnale EEG (così come sui segnali delle altre tecniche di neuroimaging), questi aspetti sono trattati in maniera dettagliata all’interno del capitolo. Infine, l’ultima parte del capitolo è dedicata agli altri metodi di coregistrazione TMS con risonanza magnetica (MRI), risonanza magnetica funzionale (fMRI), tomografia a emissione di positroni (PET), tomografia a emissione di fotone singolo (SPECT) e spettroscopia del vicino infrarosso (NIRS). CAPITOLO III – STUDIO 1: EFFETTI NEUROMODULATORI DELLA RTMS A BASSA FREQUENZA: EVIDENZE DALL’APPROCCIO TMS-EEG Tradizionalmente gli effetti neuromodulatori della rTMS sono stati studiati attraverso l’analisi dei potenziali motori evocati (MEP). Tuttavia, come noto, i MEP sono una misura indiretta dell’eccitabilità corticale avendo una forte componente anche spinale. Con lo sviluppo di nuovi sistemi EEG compatibili con la TMS, è stato possibile analizzare gli effetti della stimolazione in modo più diretto, tramite l’analisi dei potenziali corticali evocati dalla TMS (TEPs). In questo studio abbiamo analizzato l’effetto di un protocollo di TMS ripetitiva (rTMS) a bassa frequenza (1 Hz) molto noto, soprattutto in ambito riabilitativo, per sortire un effetto di inibizione dell’eccitabilità corticale. Il protocollo è stato applicato sulla corteccia motoria primaria di quindici volontari sani e sulla corteccia visiva primaria di altri quindici volontari sani, assunti come gruppo di controllo per analizzare la specificità spaziale della stimolazione. Gli effetti della stimolazione ripetitiva sono stati testati su diverse misure elettrofisiologiche evocate da una stimolazione a singolo impulso della corteccia motoria, prima e subito dopo il protocollo rTMS, ossia: MEP, TEPs, local mean field power (LMFP) e distribuzione dell’attività sullo scalpo. I risultati sui MEP hanno mostrato una diminuzione significativa dell’ampiezza a seguito del protocollo rTMS sulla corteccia motoria. I risultati sui TEP hanno mostrato un pattern noto composto di quattro principali picchi: P30, N45, P60 e N100. A seguito del protocollo rTMS sulla corteccia motoria si è osservato un incremento significato dell’ampiezza dei TEP P60 e N100, la cui origine è legata all’attività dei potenziali post-sinaptici inibitori GABAb (Ferreri et al., 2011; Premoli et al., 2014). I risultati sul LMFP hanno mostrato un incremento di attiv

Nuove e vecchie correnti sul cervello: l'avvento della TMS nello studio delle funzioni cognitive e nel trattamento dei disturbi psichici

TMS, ossia Stimolazione Magnetica Transcranica. Dietro a questo termine che potrebbe riportare alla mente le famose scene di \u201cQualcuno vol\uf2 sul nido del cuculo\u201d, si cela uno strumento di grande utilit\ue0 nel campo dello studio delle funzioni cognitive, da sempre oggetto di interesse della Psicologia. Il paragone fra TMS ed \u201cElettroshock\u201d si limita al solo fatto che entrambe le tecniche modulano l\u2019eccitabilita della corteccia cerebrale tramite un passaggio di corrente. Eppure, questa comune caratteristica, \ue8 stata sufficiente a provocare l\u2019annuncio di un presunto \u201critorno dell\u2019elettroshock\u201d da parte di alcuni quotidiani, palesando la necessit\ue0 di chiarire alcune distinzioni fra le vecchie e le nuove correnti (siano elettriche o di pensiero) che hanno attraversato e attraversano il cervello: tale \ue8 l\u2019intento in cui si cimenta questo articolo

No frequency effects when judging contrasts

In literature, experimental findings showed that absolute judgments are affected by contextual effects. In our experiment we tested if judgments of differences, in particular, judgments of brightness contrasts between two chips are affected by the skewing of frequency distributions of stimulus presentations. We asked participants to rate the degree of contrast by using two methods: the category rating method and the adjustment method. Experimental results show that frequency effects do not affect contrast judgments and that there is no difference between the subjective scales obtained with the two methods, except for the \u201cregression effect\u201d. Therefore, judgments of differences, contrarily to absolute judgments, are not affected by contextual effects