From micro to macro:unravelling the underlying mechanisms of Transcranial Magnetic Stimulation (TMS)

This PhD research investigated the underlying mechanisms of transcranial magnetic stimulation (TMS). TMS is a form of non-invasive brain stimulation, which is used both in research to alter brain activity and in the clinic where it is a treatment option for many neuropsychiatric disorders such as major depression. However, not a lot is known about how TMS actually works. This research took an interdisciplinary approach to better understand the mechanisms of TMS. On the microscopic level, it used human neurons which were grown in the lab and stimulated with TMS to look for changes in plasticity such as neuronal firing, gene expression, and morphology. On the macroscopic level, the researcher stimulated human participants and measured indirect outcomes of plasticity, using multimodal setups such as combined TMS-EEG and TMS-EEG-fMRI. Better understanding the mechanisms of TMS is very important. If we fully understand how TMS works, we can optimize stimulation protocols, promoting increased responsiveness and better treatment outcomes in the clinic

Numerical modelling of plasticity induced by transcranial magnetic stimulation

We use neural field theory and spike-timing dependent plasticity to make a simple but biophysically reasonable model of long-term plasticity changes in the cortex due to transcranial magnetic stimulation (TMS). We show how common TMS protocols can be captured and studied within existing neural field theory. Specifically, we look at repetitive TMS protocols such as theta burst stimulation and paired-pulse protocols. Continuous repetitive protocols result mostly in depression, but intermittent repetitive protocols in potentiation. A paired pulse protocol results in depression at short (∼ 100 ms) interstimulus intervals, but potentiation for mid-range intervals. The model is sensitive to the choice of neural populations that are driven by the TMS pulses, and to the parameters that describe plasticity, which may aid interpretation of the high variability in existing experimental results. Driving excitatory populations results in greater plasticity changes than driving inhibitory populations. Modelling also shows the merit in optimizing a TMS protocol based on an individual’s electroencephalogram. Moreover, the model can be used to make predictions about protocols that may lead to improvements in repetitive TMS outcomes

Using non-invasive brain stimulation to augment motor training-induced plasticity

Therapies for motor recovery after stroke or traumatic brain injury are still not satisfactory. To date the best approach seems to be the intensive physical therapy. However the results are limited and functional gains are often minimal. The goal of motor training is to minimize functional disability and optimize functional motor recovery. This is thought to be achieved by modulation of plastic changes in the brain. Therefore, adjunct interventions that can augment the response of the motor system to the behavioural training might be useful to enhance the therapy-induced recovery in neurological populations. In this context, noninvasive brain stimulation appears to be an interesting option as an add-on intervention to standard physical therapies. Two non-invasive methods of inducing electrical currents into the brain have proved to be promising for inducing long-lasting plastic changes in motor systems: transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS). These techniques represent powerful methods for priming cortical excitability for a subsequent motor task, demand, or stimulation. Thus, their mutual use can optimize the plastic changes induced by motor practice, leading to more remarkable and outlasting clinical gains in rehabilitation. In this review we discuss how these techniques can enhance the effects of a behavioural intervention and the clinical evidence to date

Modulation of human corticospinal excitability by paired associative stimulation

Paired Associative Stimulation (PAS) has come to prominence as a potential therapeutic intervention for the treatment of brain injury/disease, and as an experimental method with which to investigate Hebbian principles of neural plasticity in humans. Prototypically, a single electrical stimulus is directed to a peripheral nerve in advance of transcranial magnetic stimulation (TMS) delivered to the contralateral primary motor cortex (M1). Repeated pairing of the stimuli (i.e., association) over an extended period may increase or decrease the excitability of corticospinal projections from M1, in manner that depends on the interstimulus interval (ISI). It has been suggested that these effects represent a form of associative long-term potentiation (LTP) and depression (LTD) that bears resemblance to spike-timing dependent plasticity (STDP) as it has been elaborated in animal models. With a large body of empirical evidence having emerged since the cardinal features of PAS were first described, and in light of the variations from the original protocols that have been implemented, it is opportune to consider whether the phenomenology of PAS remains consistent with the characteristic features that were initially disclosed. This assessment necessarily has bearing upon interpretation of the effects of PAS in relation to the specific cellular pathways that are putatively engaged, including those that adhere to the rules of STDP. The balance of evidence suggests that the mechanisms that contribute to the LTP- and LTD-type responses to PAS differ depending on the precise nature of the induction protocol that is used. In addition to emphasizing the requirement for additional explanatory models, in the present analysis we highlight the key features of the PAS phenomenology that require interpretation

Role of astrocytes in an in vitro model of ischemic stroke

Ischemic stroke (IS) is the leading cause of complex and serious long-term disability in developed countries, and after decades of effort there are no effective clinical treatments for IS, especially in the subacute and chronic phases. Currently, in these stages of the IS there is no alternative to promote the recovery of brain tissues affected by the ischemic injury. Most of the treatments (e.g., physical therapy, speech therapy, occupational therapy) are applied with the aim of reducing the sequelae left, or to controlling modifiable risk factors (e.g., hypertension, diabetes, coagulopathies). This leads to a need to develop new approaches to recover those areas, reduce the neurological deficits and, if possible, enhance the functions regulated by the affected brain regions. In this context, this work intends to explore two approaches that hypothetically could induce the recovery of the areas affected by ischemia. The first is related to the potent physiological effects of estrogens on central nervous system (CNS) and its participation in several processes such as, neurogenesis, the expression of neuroprotective factors and antioxidant mechanisms, through the evaluation of the potential beneficial effects induced by the selective activation of G protein–coupled estrogen receptor 1 (GPER or GPR30). The second, by evaluating the potential protective effects induced by high frequency repetitive magnetic stimulation (HF-rMS), an approach that has been described as having the ability to correct maladaptive brain plasticity and to enhance neuronal communication during rehabilitation. In both cases the ability to induce neuroprotection in neurodegenerative disorders, such as, Alzheimer´s disease, Parkinson’s disease, and mood disorders, was already demonstrated. To standardize the ischemic damage and evaluate the potential beneficial effects induced by these two approaches several in vitro models of ischemia were developed and characterized. Neuron-enriched, neuron-glia, and astrocyte-enriched primary cortical cultures subjected to oxygen and glucose deprivation (OGD) followed by a reperfusion period, were used as models. The evaluation of the effects induced by GPER activation and by HF-rMS was performed through the assessment of several parameters related cell survival and proliferation, GPER expression, calcium imaging, as well as neurite morphometric and synaptic modifications. Concerning the role of GPER on the ischemic injury, we observe that ischemia did not change the levels of GPER in neurons and astrocytes. Moreover, GPER selective activation had no impact in neuronal survival, whereas it induced the apoptosis of astrocytes, being this effect meditated by the activation of phospholipase C pathway, and the subsequent intracellular calcium rise. These data indicate a direct impact of GPER on the viability of astrocytes, and the coupling of GPER to different signaling pathways in astrocytes and neurons. Our data also shows that HF-rMS reduces the neuronal loss, the initial neurite degeneration and the loss of synaptic markers triggered by ischemia. Interestingly the protective effect triggered by HF-rMS required the presence of astrocytes. Taken together the data obtained suggests that HF-rTMS has the potential to be used as a therapeutic approach to reduce neuronal death and neuronal damage, by limiting neurite degeneration and enhance functional connectivity and synaptic plasticity in the areas affected by the ischemia. Furthermore, our results also suggest that astrocytes play a crucial role on ischemic injury. Astrocytes were more resistant to ischemic periods than neurons in all experiments performed and when they were present the injury was smaller, which indicate an active role in the neuronal protection against ischemia-induced injury. Taking into account their preponderant role in neuronal physiology and the fact that their presence is crucial for the observed beneficial effects induced by HF-rMS it seems evident that astrocytes could have a substantial impact on the protection and recovery of ischemia-induced lesion. Thereby, we hypothesize that astrocytes could be a potential therapeutic target for the treatment of cerebral ischemia and any methodology/approach that potentiate their beneficial effects may be a promising therapeutic approach.O Acidente vascular cerebral isquémico representa uma das principais causas de incapacidade em países desenvolvidos, e mesmo após décadas de investigação ainda não existem abordagens terapêuticas eficazes, especialmente nas fases subaguda e crónica da doença. Atualmente, nestes estadios da patologia, não existe uma alternativa que promova a recuperação dos tecidos cerebrais que foram afetados pela isquemia. A maior parte dos tratamentos (fisioterapia, terapia da fala, terapia ocupacional, etc.) são aplicados com o objetivo de reduzir as sequelas ou de controlar os fatores de risco modificáveis (hipertensão, diabetes, coagulopatias, etc.). O que leva a que exista uma necessidade de desenvolver novas abordagens que possibilitem a recuperação desses tecidos, diminuam os défices neuronais e, se possível, promovam a melhoria das funções que são reguladas pelas regiões cerebrais afetadas. Tendo isto em consideração, este trabalho tem como principal objetivo explorar a ação de duas abordagens distintas na recuperação de lesões isquémicas. A primeira está relacionada com os potentes efeitos fisiológicos do estrogénio no sistema nervoso central e a sua participação em diversos processos como a neurogénese, promoção da expressão de fatores neuroprotetores e ativação de mecanismos antioxidantes, mais precisamente através da avaliação dos potenciais efeitos benéficos induzidos pela ativação seletiva do recetor de estrogénio acoplado à proteína G (GPER). A segunda será através da avaliação dos efeitos induzidos pela estimulação magnética repetitiva de alta frequência (HF-rMS), uma abordagem que já foi descrita como tendo a capacidade de corrigir distúrbios ao nível da neurotransmissão e de melhorar a comunicação neuronal durante o processo de recuperação. Ambas as abordagens já foram descritas como tendo a capacidade de induzir neuroprotecção em patologias neurodegenerativos, como é o caso das doenças de Alzheimer e Parkinson e de perturbações de humor. De forma a padronizar a lesão isquémica e avaliar os efeitos induzidos por estas duas abordagens, vários modelos in vitro foram desenvolvidos e caracterizados. Foram utilizados três tipos de culturas primárias do córtex (cultura de astrócitos, cultura de neurónios e cultura de neurónios e células gliais), as quais foram submetidas à privação de oxigénio e glucose, seguindo-se um período de reperfusão. A avaliação dos efeitos induzidos por estas duas abordagens foi feita através de vários parâmetros relacionados com a sobrevivência e proliferação celular, avaliação do cálcio intracelular, assim como da análise morfométrica das neurites e de modificações sinápticas. Em relação ao papel do GPER na lesão isquémica, observamos que a privação de oxigénio e glucose não alterou os níveis de expressão deste recetor, nem em neurónios nem em astrócitos. A ativação seletiva do GPER não teve impacto na sobrevivência neuronal mas promoveu a morte astrocitária através de um mecanismo que envolve a ativação da via da fosfolipase C e o subsequente aumento dos níveis de cálcio intracelular. Estes dados mostram um impacto direto do GPER na viabilidade dos astrócitos e que a ativação do GPER está associada a diferentes vias de sinalização em astrócitos e neurónios. Os nossos resultados indicam também a HF-rMS reduz alguns dos efeitos negativos desencadeados pela lesão isquémica, tais como a morte neuronal, a degeneração inicial das neurites e a diminuição de marcadores sinápticos. Curiosamente, o efeito protetor da HF-rMS apenas é observável na presença de astrócitos. Estes dados sugerem que a HF-rTMS tem potencial para poder ser utilizada como uma abordagem terapêutica para reduzir a morte neuronal e os danos neuronais, limitando a degeneração das neurites e melhorando a conectividade funcional e a plasticidade sináptica nas áreas afetadas pela isquemia. Os nossos resultados sugerem também que os astrócitos desempenham um papel crucial na lesão isquémica. Para além de serem mais resistentes a períodos de isquemia do que os neurónios, todos os dados experimentais obtidos mostraram que quando os astrócitos estavam presentes a lesão foi menor, o que indica um papel ativo na proteção neuronal contra a lesão induzida pela isquemia. Tendo em consideração o seu papel preponderante na fisiologia neuronal e o fato de a sua presença ser obrigatória para os efeitos benéficos induzidos pela HF-rMS, parece evidente que os astrócitos podem ter um impacto substancial na proteção e recuperação da lesão induzida por isquemia. Como tal os astrócitos devem ser encarados como potenciais alvos terapêuticos para o tratamento da isquemia cerebral e qualquer metodologia/abordagem que potencialize os seus efeitos protetores pode ser uma abordagem terapêutica bastante promissora

Plasticity induced by non-invasive transcranial brain stimulation: A position paper

Several techniques and protocols of non-invasive transcranial brain stimulation (NIBS), including transcranial magnetic and electrical stimuli, have been developed in the past decades. Non-invasive transcranial brain stimulation may modulate cortical excitability outlasting the period of non-invasive transcranial brain stimulation itself from several minutes to more than one hour. Quite a few lines of evidence, including pharmacological, physiological and behavioral studies in humans and animals, suggest that the effects of non-invasive transcranial brain stimulation are produced through effects on synaptic plasticity. However, there is still a need for more direct and conclusive evidence. The fragility and variability of the effects are the major challenges that non-invasive transcranial brain stimulation currently faces. A variety of factors, including biological variation, measurement reproducibility and the neuronal state of the stimulated area, which can be affected by factors such as past and present physical activity, may influence the response to non-invasive transcranial brain stimulation. Work is ongoing to test whether the reliability and consistency of non-invasive transcranial brain stimulation can be improved by controlling or monitoring neuronal state and by optimizing the protocol and timing of stimulation

Understanding the Effects of Repetitive Transcranial Magnetic Stimulation on Neuronal Circuits

Despite the widespread use of repetitive transcranial magnetic stimulation (rTMS) in both research and clinical settings, there is a paucity of evidence regarding the effects of its application on neural activity. Studies investigating the effects of rTMS on human participants (Huang et al., 2005) have shown that patterned trains of rTMS can be used to modulate the sensitivity of motor pathways for a period outlasting the stimulation itself. These changes are often attributed to an rTMS-induced increase in neural plasticity or a change in excitability of the motor pathway. Evidence that rTMS can modify the strength of motor pathways has led to its introduction into stroke rehabilitation research. It is hypothesized that post-stroke, rTMS can enhance plasticity induction within the brain and, when combined with manual therapy, can facilitate surviving neurons assuming the function of those lost to the stroke (Hsu et al., 2012). In practice however, despite a multitude of studies investigating this approach, there remains no convincing evidence that rTMS is capable of promoting sustained long-term improvement in recovery, above the effects of rehabilitation alone (Hsu et al., 2012; Lefaucheur et al., 2014). We are of the opinion that a lack of advancement within the field is due to an incomplete understanding of the effects of TMS on neural elements. Here we discuss some of the existing evidence and propose experimental approaches that may enhance the human application of rTMS

Resting state fMRI study of brain activation using rTMS in rats

Background and purpose: Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive neuromodulation technique used to treat many neurological and psychiatric conditions. However, not much is known about the mechanisms underlying its efficacy because human rTMS studies are mostly non-invasive while most animal studies are invasive. Invasive animal studies allow for cellular and molecular changes to be detected and hence, have been able to show that rTMS may alter synaptic plasticity in the form of long-term potentiation. This is the first rodent study using non-invasive resting state functional magnetic resonance imaging (rs-fMRI) to examine the effects of low-intensity rTMS (LI-rTMS) in order to provide a more direct comparison to human studies. Methods: rs-fMRI data were acquired before and after 10 minutes of LI-rTMS intervention at one of four frequencies—1 Hz, 10 Hz, biomimetic high frequency stimulation (BHFS) and continuous theta burst stimulation (cTBS)—in addition to sham. We used independent component analysis to uncover changes in the default mode network (DMN) induced by each rTMS protocol. Results: There were considerable rTMS-related changes in the DMN. Specifically, (1) the synchrony of resting activity of the somatosensory cortex was decreased ipsilaterally following 10 Hz stimulation, increased ipsilaterally following cTBS, and decreased bilaterally following 1 Hz stimulation and BHFS; (2) the motor cortex showed bilateral changes following 1 Hz and 10 Hz stimulation, an ipsilateral increase in synchrony of resting activity following cTBS, and a contralateral decrease following BHFS; and (3) in the hippocampus, 10 Hz stimulation caused an ipsilateral decrease while 1 Hz and BHFS caused a bilateral decrease in synchrony. There was no change in the correlation of the hippocampus induced by cTBS. Conclusion: The present findings suggest that LI-rTMS can modulate functional links within the DMN of rats. LI-rTMS can induce changes in the cortex, as well as in remote brain regions such as the hippocampus when applied to anaesthetised rats and the pattern of these changes depends on the frequency used, with 10 Hz stimulation, BHFS and cTBS causing mostly ipsilateral changes in synchrony of activity in the DMN and 1 Hz stimulation causing bilateral changes in synchrony, with the contralateral changes being more prominent than ipsilateral changes. Hence, combined rTMS-fMRI emerges as a powerful tool to visualise rTMS-induced cortical connectivity changes at a high spatio-temporal resolution and help unravel the physiological processes underlying these changes in the cortex and interconnected brain regions