Electrophysiological recording and biocytine filling

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High intensity rTMS

The intensity of an individual’s rTMS is usually determined by the cortical motor threshold (MT), defined as the minimal intensity at which TMS over the M1 induces a reliable electromyography (EMG) response around 100µV or a visible muscle twitch response in the target muscle (Rossini et al., 1994; Westin et al., 2014) Although twitch-based MT evaluation is easier to carry out, it is related to a high intra- and inter-rater variability and MTs assessed visually are about 10% higher than EMG-recorded MTs (Westin et al., 2014). Suprathreshold stimulations inducing action potential firing in stimulated neurons are required to induce such EMG or muscle twitch responses (Pell et al., 2011). MEP amplitude is commonly used to assess modulation of cortical excitability promoted by rTMS, and increases with increasing stimulation intensity (Rothwell et al., 1987). Intrinsic differences in cortical excitability induce inter and intra-subject variability in the MEP amplitude. While physiological fluctuations cannot be avoided, other physiological and technical parameters should be kept constant, including baseline activity of the target muscle, arousal and attention levels, environmental noise, and coil position/orientation (Cuypers et al., 2014; Rossini et al., 2015).
The stimulation intensity needed to induce a response in a resting muscle is often expressed relative to the resting motor threshold (RMT) and represents the percentage of the stimulator output to reproducibly induce MEPs (Fitzgerald et al., 2006; Rothwell et al., 1987). Stimulation intensity in human rTMS is generally applied around 80-120% of the RMT and therefore is only characterized as a percentage of the stimulator output. This represents another factor of variability between studies since identical devices are not always used.

Low intensity magnetic stimulation

As for high intensity rTMS, low intensity magnetic fields are delivered by one or more coils in which time-variable current flows. Parameters of stimulation are usually in the microtesla to millitesla (µT-mT) range and generally 10-100 Hz (0-300Hz) frequency (Di Lazzaro et al., 2013). Waveforms also can be mono-phasic or biphasic. Extremly low frequency magnetic field (ELF-MF) studies use various waveforms that can be asymmetric, biphasic, quasi-rectangular, or quasi triangular (Bassett, 1989) although most ELF-MF sources of stimulation produce sinusoidal waveforms (Juutilainen and Lang, 1997). Pulsed electromagnetic fields (PEMF), a subset of ELF-MF, usually induces greater electrical current than sinusoidal waveforms due to their faster magnetic field rate of change (Tesla/seconds) (Di Lazzaro et al., 2013). Unlike rTMS which delivers central focal high intensity stimulation of a targeted brain region, ELF-MF generates a diffuse homogeneous magnetic field within the whole brain. Helmholtz coil-based exposure systems are the most commonly used and are made of two identical circular magnetic coils that deliver a nearly uniform magnetic field. ELF-MF experiments use a wide variety of stimulation devices and coils which induce a broad range of waveforms (sinusoidal or pulsed), intensities (µT-mT) and frequencies (0-300Hz) of stimulation, making comparisons between studies very difficult.

Potential mechanisms underlying high-intensity rTMS

As described above variability in the outcomes between studies on the effects of rTMS are partly due to different applied protocols (pulse shape, intensity, frequency pattern…) as well as intra and inter-individual variability due to different baseline level of cortical excitability and other individual biological factors. Altogether this will in turn specifically modify the intracellular pathways activated by the magnetic field and therefore the subsequent long-term changes. It is thus of major importance to search for neurophysiological mechanisms responsible for these long-term effects. An understanding of these mechanisms will help us to fine-tune our rTMS protocols in order to optimally activate those mechanisms, depending on the type of individual subject, the neural network targeted and the pathology implicated.

Synaptic plasticity: evidence from human studies

rTMS has the appealing potential to modulate cortical excitability beyond the simulation period (Pell et al., 2011) and in both directions, either excitatory or inhibitory. These after-effects are dependent on several parameters which were presented in the previous chapter. A main approach has been to explain those effects on the brain through LTP/LTD like mechanisms.
Long-term changes in synaptic strength like LTP (long-term potentiation) and LTD (long-term depression) are durable changes in synaptic efficacy (Malenka and Bear, 2004; Raymond, 2007). LTP results in potentiation of synaptic strength that may last for days, weeks or months. Brief high-frequency stimulations are used to induce this potentiation. LTD results in a long-lasting reduction in synaptic strength (Duffau, 2006). Synaptic plasticity obeys to key rules that are described in detail in different experimental models (Abraham, 2008; Cooke and Bliss, 2006; Malenka and Bear, 2004) Studies on rTMS and their effects on human brain have used several key concepts shared with classic synaptic plasticity which are summarized in figure 5. However it is essential to specify that LTP- and LTD-like mechanisms induced by rTMS differ from their classic form of synaptic plasticity, which use direct electric stimulation of synapses in vivo and in vitro (Malenka and Bear, 2004; Pell et al., 2011). A main difference is related to the different conditions of stimulation: TMS activates a wide number of axons at presynaptic and postsynaptic terminals simultaneously (Funke and Benali, 2011) This could explain the significantly lower stimulation frequencies that induce LTP in rTMS studies compared to classic electrophysiology synaptic plasticity protocols (10Hz vs 100Hz) which induce facilitation (Vlachos et al., 2012).
Cortical excitability is modulated by magnetic stimulation in a frequency dependent manner. Brain activity is also affected by the stimulation as indicated by regional cerebral blood flow (Lee et al., 2003; Rounis et al., 2005), EEG responses (Esser et al., 2006; Huber et al., 2007; Litvak et al., 2007), and blood-oxygen level-dependent (BOLD) activation patterns (Hubl et al., 2008). High frequency rTMS for 10-20 minutes preceding a task produces prolonged increases in attentional control, consolidation of new motor skills, and tactile discrimination (Boyd and Linsdell, 2009; Hwang et al., 2010; Tegenthoff et al., 2005). All of these effects outlasted the stimulation period, which is an important component of synaptic and network plasticity.

Direct evidence for synaptic plasticity induced by rTMS in animal models

In vitro studies of rMS on organotypic rodent hippocampal slice cultures have provided evidence to support the synaptic plasticity thought to occur after human rTMS. (rMS indicates that the magnetic field does not go through the cranium). Direct evidence of LTP induced by high-frequency rMS (10Hz) has been found (Lenz et al., 2015; Tokay et al., 2009; Vlachos et al., 2012). Those studies provide pivotal insight into underlying mechanisms of rMS which are consistent with long-term potentiation of synaptic transmission. A durable increase (2-6 hours post stimulation) in synaptic transmission mediated by the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid AMPA receptor was demonstrated, along with remodeling of small dendritic spine on CA1 pyramidal neurons (Vlachos et al., 2012). This was associated with increased AMPA receptor cluster size and number in an NMDA receptor dependent manner, consistent with modifications observed after classical LTP protocols. Another recent study from this group (Lenz et al., 2015) showed that this induced potentiation of excitatory synapses is located on proximal dendrites, and that it requires voltage-gated channels (sodium and calcium), NMDA receptors, and calcium.
Using a computational approach to test the possible cooperative depolarization of pre- and postsynaptic structures, Lenz and colleagues showed that rMS-induced anterograde action potentials (aAPs) will provoke release of glutamate from the presynaptic ending. Backward propagating action potentials (bAPs) will depolarize dendrites of the post-synaptic neuron, favoring removal of the magnesium block from NMDA-R (Lenz et al., 2015). They propose that at the proximal dendrite of CA1 pyramidal neurons these two phenomenon are additive and therefore induce accumulation of AMPA-R on the post synaptic cell resulting in a facilitated potentiation (Lenz et al., 2015). This hypothesis of rMS-induced bAP-aAP mediated cooperative plasticity could account for the induction of rMS induced LTP-like effects with frequencies much lower (i.e., 10Hz) than the 100Hz needed with direct electrical stimulation in classic LTP experiments. However, direct evidence for this “bAP-aAP theory” is currently missing. It has to be noted that there is no direct evidence of rMS induced LTP in cortical neurons.

Membrane potential and spontaneous activity

Synaptic plasticity is not the only potential mechanism for excitability changes. High intensity rTMS can cause depolarization of targeted neurons either directly, or indirectly via depolarization of interneurons (Pell et al., 2011), which may modify neural excitability. Alteration of membrane excitability induced by modification of the resting membrane potential, the action potential threshold (AP) and ion channels properties could be an alternative mechanism leading to modulation of excitability (Wagner et al., 2009)
Whole-cell patch-clamp recordings in acute rat brain slices showed that at voltages ranging from -65 mV to -25 mV each magnetic pulse led to transient influx of current into neurons (Banerjee et al., 2017). This current flow is sensitive to the membrane voltage and requires the activation of Voltage Gated Sodium Channels (VGSCs) in the soma of cortical neurons, thereby leading to an increased steady state current of the neurons, induction of action potentials, and modulation of spike timing in these neurons. 10s of stimulation increased the intracellular calcium concentration 70s after the stimulation, potentially due to the cumulative effect of rTMS induced depolarization of the neuron (Banerjee et al., 2017). Combining optical imaging with voltage-sensitive dye (VSD), researchers were able to record gradual changes of membrane potential induced by each TMS pulse in cat neurons, providing crucial information on neuronal population dynamics (Kozyrev et al., 2014) A single pulse of high intensity TMS induced a brief peak of activity in cortical neurons followed immediately by decreased activity below baseline, characteristic of suppression (Kozyrev et al., 2014). The authors suggest that TMS preferentially affects inhibitory (Werhahn et al., 1999) parvalbumin positive neurons (Benali et al., 2011; Funke and Benali, 2011), potentially because of their axonal and dendritic morphology (Chung et al., 2013; McAllister et al., 2009), and that it results in strong synchronized inhibition of the targeted cortical network (Pascual-Leone et al., 2000). When TMS pulses were applied at 10Hz the first pulse induced suppression and the consecutive pulses produces a progressive increase of activity in a large population of neurons reaching a level concordant with high spiking activity, weakening inhibitory action and stimulating excitatory circuits through NMDA receptors (Huang et al., 2008b). Similarly, spontaneous single-unit spiking activity in cerebellar slices showed that short term inhibition was induced by 1Hz rTMS whereas 20Hz stimulation produces excitation and although the intensity of stimulation regulated the level of modulation it did not influence the direction of this modulation (ie inhibition or excitation) (Tang et al., 2016c). In the rat brain, stimulation with an iTBS pattern depolarized neuronal membrane potential, increased sEPSCs and the rate of action potential firing, of fast spiking interneurons (FSIs) at postnatal day (PD) 29-38, but neither before nor after these ages (Hoppenrath et al., 2016). In accordance with previous finding showing that the reduction of PV expression could not be induced before PD 30 but gradually increased between PD32 and PD37 (Mix et al., 2014) these results suggest that FSIs are particularly sensitive to iTBS during critical cortical development time windows.

Table of contents :

Abstract
Résumé
List of figures an tables
Abbreviations
Chapter I – Introduction
I) Magnetic stimulation: a non-invasive approach to enhance neuroplasticity?
I.1) What is neuroplasticity?
I.2) Neuroplasticity over the lifespan
I.3) What is rTMS?
I.4) Basic principle of rTMS
I.5) TMS and rTMS applications
I.6) rTMS parameters
I.6.1) Pulse shapes and coil types
I.6.2) Stimulation intensity
I.6.3) rTMS frequency
II) Potential mechanisms underlying high-intensity rTMS
II.1) Synaptic plasticity: evidence from human studies
II.2) Direct evidence for synaptic plasticity induced by rTMS in animal models
II.2.1) rMS induced synaptic plasticity at excitatory synapses
II.2.2) rTMS modulation of inhibitory networks
II.2.3) Neurobiological effects of rTMS underlying induced plasticity
III) Low intensity magnetic stimulation
III.1) A need for a defined terminology and reproducible parameters
III.2) ELF-MF effects on the human brain
III.2.1) Effects on cortical excitability and brain oscillations
III.2.2) Effects on human brain functions and potential therapeutic applications
III.3) ELF-MF effects on animal models and underlying mechanisms
III.3.1) Magnetoreception: the cryptochrome radical-pair mechanism hypothesis
III.3.2) Biological events induced by ELF-MF
III.4) LI-rTMS effects on the rodent brain
III.5) Conclusion
IV) The cerebellum as a model to study LI-rTMS induced plasticity
IV.1) Cerebellum
IV.1.1) Cerebellar circuitry
IV.1.2) Cerebellar afferents
IV.2) The olivocerebellar pathway (OCP)
IV.2.1) The inferior olivary nucleus
IV.2.2) Climbing fibers and the OCP
IV.3) Development of the OCP
IV.3.1) Morphological PC differentiation
IV.3.2) Synaptogenesis of the olivocerebellar pathway and refinement of CF Projections
IV.4) Plasticity of the OCP following lesion
IV.4.1) Developmental plasticity of the OCP
IV.4.2) Plasticity in the mature OCP
IV.5) Ageing of the cerebellum
V) Summary and aims
Chapter II – Article1
Abstract
Introduction
Materials and Methods
Animals
Administration of Magnetic Stimulation
Behavioural analysis
Electrophysiological recording and biocytine filling
Morphological Analysis
Spine density
Sholl analysis
Statistical analysis
Results
Early postnatal PC dendritic development and synaptic maturation are not altered by LIrTMS
Mature Purkinje cell dendrites change little with age
Short-term treatment with LI-rTMS increases PC spine density and alters spine
morphology
Long-term treatment with LI-rTMS alters PC dendritic morphology
Handling and behavioural testing alone did not change dendritic morphology but
did alter spine density
Chronic LI-rTMS treatment does not alter motor function, but improves spatial
memory
Discussion
References
Figures and Legends
Chapter III – Article 2
Abstract
Introduction
Methods
Animals
In vivo olivocerebellar axonal transection (pedunculotomy)
Organotypic Cultures and cerebellar denervation
Magnetic stimulation
Immunohistochemistry
Histological analysis
qRT-PCR and gene analysis
Statistical analysis
Results
LI-rTMS induces CF reinnervation in vivo
LI-rMS induces PC reinnervation in vitro in a frequency dependent manner
LI-rMS induced reinnervation requires simultaneous stimulation of both pre-and postsynaptic
structures of the OCP
LI-rMS activates Purkinje cells and interneurons
LI-rMS modulates gene expression appropriately for PC reinnervation
Cryptochrome is required for LI-rMS-induced post-lesion repair
Discussion
Post-lesion repair depends on stimulation pattern, not numbers of pulses ………..
LI-rMS modifies gene expression in a frequency-dependent manner
LI-rMS potentially activates c-fos in PC and interneurons
Cryptochromes magnetoreceptors are key elements for the transduction of the
magnetic field into biological effect
Conclusions
References
Figures and Legends
Chapter IV– Article 3
Abstract
Introduction
Materials and Methods
Animals
Organotypic Cultures and cerebellar denervation
Genotyping
Administration of LI-rTMS
Behavioural analysis
Electrophysiological recording and biocytin filling
Immunohistochemistry and Histological analysis
qRT-PCR
Statistical analysis
Results
Post-lesion repair by LI-rMS is impaired in RORα haplodeficient hindbrain explants
Four weeks LI-rTMS and psychomotor activity moderately alters PC dendritic
morphology
Chronic LI-rTMS treatment does not alter motor or spatial behaviour of RORα+/
animals
RORα
PC responses to acute LI-rTMS treatment are small and differ with age
Discussion
References
Figures and Legends
Chapter V – General Discussion
V) Discussion
V.1) Mechanisms underlying the effectsof LI-rTMS
V.1.1) LI-rTMS pattern of frequency are crucial for the induction of biological effects
V.1.2) LI-rTMS requires cryptochromes to interact with biological tissue and potentially activate the transcription of signalling pathways
V.1.3) Intracellular calcium may be a second messenger mediating the biological effects of cryptoctochrome activation by LI-rMS
V.1.3.1) Intracellular levels and downstream signalling cascades
V.1.3.2) ROS production
V.1.4) RORα seems necessary to observe neural circuit plasticity and behavioural improvements induced by LI-rTMS
V.2) Biological effects of LI-rTMS
V.2.1) LI-rTMS targeting the cerebellum induces neuronal structural plasticity
and improvements in cerebellar related behaviour in adult mice
V.2.2) Reduced neural plasticity in aged mice may explain reduced LI-rTMS effects on cerebellar structural plasticity and behaviour
V.2.3) Further investigation into the effects of magnetic stimulation are required prior to eventual clinical use in young patients
VI Conclusions

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