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Transcranial (electro)magnetic stimulation (TMS) is currently the method of choice to non-invasively induce neural activity in the human brain. A single transcranial stimulus induces a time-varying electric field in the brain that may evoke action potentials in cortical neurons. The spatial relationship between the locally induced electric field and the stimulated neurons determines axonal depolarization. The induced electric field is influenced by the conductive properties of the tissue compartments and is strongest in the superficial parts of the targeted cortical gyri and underlying white matter. TMS likely targets axons of both excitatory and inhibitory neurons. The propensity of individual axons to fire an action potential in response to TMS depends on their geometry, myelination and spatial relation to the imposed electric field and the physiological state of the neuron. The latter is determined by its transsynaptic dendritic and somatic inputs, intrinsic membrane potential and firing rate. Modeling work suggests that the primary target of TMS is axonal terminals in the crown top and lip regions of cortical gyri. The induced electric field may additionally excite bends of myelinated axons in the juxtacortical white matter below the gyral crown. Neuronal excitation spreads ortho- and antidromically along the stimulated axons and causes secondary excitation of connected neuronal populations within local intracortical microcircuits in the target area. Axonal and transsynaptic spread of excitation also occurs along cortico-cortical and cortico-subcortical connections, impacting on neuronal activity in the targeted network. Both local and remote neural excitation depend critically on the functional state of the stimulated target area and network. TMS also causes substantial direct co-stimulation of the peripheral nervous system. Peripheral co-excitation propagates centrally in auditory and somatosensory networks, but also produces brain responses in other networks subserving multisensory integration, orienting or arousal. The complexity of the response to TMS warrants cautious interpretation of its physiological and behavioural consequences, and a deeper understanding of the mechanistic underpinnings of TMS will be critical for advancing it as a scientific and therapeutic tool., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Andrea Antal has received honoraria from NeuroCare GmbH, Germany and Savir GmbH, Germany. Peter T. Fox has received patents in multiple jurisdictions for various TMS technologies, including the cortical column cosine model (C3) and the delivery of transcranial magnetic stimulation in accordance with the C3 model in an image-guided or robotically controlled manner. These patents are assigned to the University of Texas Board of Regents and have been licensed for commercialization by the University of Texas to a private entity in which Dr. Fox has ownership interest. Mark Hallett is an inventor of patents held by NIH for an immunotoxin for the treatment of focal movement disorders and the H-coil for magnetic stimulation; in relation to the latter, he has received license fee payments from the NIH (from Brainsway). He is on the Medical Advisory Boards of CALA Health and Brainsway (both unpaid positions). He is on the Editorial Board of approximately 15 journals and receives royalties and/or honoraria from publishing from Cambridge University Press, Oxford University Press, Springer, Wiley, Wolters Kluwer, and Elsevier. He has research grants from Medtronic, Inc. for a study of DBS for dystonia and CALA Health for studies of a device to suppress tremor. Hartwig R. Siebner has received honoraria as speaker from Sanofi Genzyme, Denmark and Novartis, Denmark, as consultant from Sanofi Genzyme, Denmark, Lophora, Denmark, and Lundbeck AS, Denmark, and as editor-in-chief (Neuroimage Clinical) and senior editor (NeuroImage) from Elsevier Publishers, Amsterdam, The Netherlands. He has received royalties as book editor from Springer Publishers, Stuttgart, Germany and from Gyldendal Publishers, Copenhagen, Denmark. Yoshikazu Ugawa has received honoraria from Takeda Pharmaceutical Company Limited, Eisai Co., Ltd., FP Pharmaceutical Corporation, Otsuka Pharmaceutical Co., Ltd., Elsevier Japan K. K., Kyowa Hakko Kirin Co., Ltd., Dainippon Sumitomo Pharma Co., Ltd., Mitsubishi Tanabe Pharma Corporation, NIHON PHARMACEUTICAL Co., Ltd., and Novartis Pharma K.K. He has received royalties as journal editor from CHUGAI-IGAKUSHA, Igaku-Shoin Ltd, Medical View Co. Ltd., and Blackwell Publishing K.K. Walter Paulus has received honoraria as speaker from Philips, Medipark Clinic and as a consultant from Abott and Precisis AG. A.V. Peterchev has received research funding, travel support, patent royalties, consulting fees, equipment loans, hardware donations, and/or patent application support from Rogue Research, Tal Medical/Neurex, Magstim, MagVenture, Neuronetics, BTL Industries, and Advise Connect Inspire. Ulf Ziemann received grants from Janssen Pharmaceuticals NV and Takeda Pharmaceutical Company Ltd., and consulting fees from Bayer Vital GmbH, Pfizer GmbH and CorTec GmbH. All other authors have no conflict of interest to report., (Copyright © 2022 International Federation of Clinical Neurophysiology. Published by Elsevier B.V. All rights reserved.)

Early critical period of visual cortex is characterized by enhanced activity-driven neuronal plasticity establishing the specificity of neuronal connections required for optimal processing of sensory signals. Deprivation from visual input by dark rearing (DR) during this period leads to a lasting impairment of visual performance. Previously, we demonstrated that repetitive transcranial magnetic stimulation (rTMS) applied with intermittent theta-burst (iTBS) pattern during the critical period improved the visual performance of the DR rats. In this study, we describe that the excitability of the binocular part of the visual cortex (V1b), as measured in acute brain slices by input-output ratios of field excitatory synaptic potentials (fEPSPs), is lowered in DR rats compared to normal controls. Verum rTMS applied with the iTBS pattern during DR reversed this DR effect, while no rTMS effect was evident in the non-DR (nDR) rats. In addition, verum rTMS reduced the number of neurons expressing the 67 kD isoform of glutamic acid decarboxylase (GAD67), the calcium-binding protein calbindin (CB) and the zinc-finger transcription factor zif268/EGR1, as determined via immunohistochemistry, only in DR rats but not in nDR rats. Moreover, rTMS reduced the number of neurons expressing the calcium-binding protein parvalbumin (PV) only in nDR rats which showed more PV+ neurons compared to DR rats. This study confirms that iTBS-rTMS may be able to prevent or reverse the effects of DR on visual cortex physiology, likely through a modulation of the activity of inhibitory interneurons., (© 2020 The Authors. Developmental Neurobiology published by Wiley Periodicals LLC.)

Key Points: Partial sensory deprivation (deafferentation) by removing whiskers from the rat snout resulted in a reduced responsiveness of related cortical representations. Repetitive transcranial magnetic stimulation (three blocks of intermittent theta-burst) applied for 5 days in combination with sensory exploration restored the normal responsiveness level of the deafferented barrel cortex. However, intracortical inhibition (lateral and recurrent) appeared to be reduced after repetitive transcranial magnetic stimulation, probably as the cause of improved responsiveness. Repetitive transcranial magnetic stimulation also reduced the asymmetry of the lateral spread of sensory activity., Abstract: Repetitive transcranial magnetic stimulation (rTMS) modulates human cortical excitability. It has the potential to support recovery to normal cortical function when the excitation-inhibition balance is altered (e.g. after a stroke or loss of sensory input). We tested cortical map plasticity on the basis of sensory responses (local field potentials, LFPs) and expression of neuronal activity marker proteins within the barrel cortex of rats receiving either active or sham rTMS after selective unilateral deafferentation by whiskers plucking. Rats received daily rTMS [intermittent theta-burst (iTBS), active or sham] for 5 days before exploring an enriched environment. Our previous studies indicated a disinhibitory effect of iTBS on cortical activity. Therefore, we also expected disinhibitory effects if deafferentation causes depression of sensory responses. Deafferentation resulted in an acute general reduction of sensory responsiveness and enhanced expression of inhibitory activity markers (GAD67, parvalbumin) in the deafferented hemisphere. Active but not sham-iTBS-rTMS normalized these measures. The stronger caudal-to-frontal horizontal spread of activity across barrels was reduced after deafferentation but not restored after active iTBS, despite generally increased responses. Fitting the LFP data with a computational model of different strengths and types of excitatory and inhibitory connections further revealed an iTBS-induced reduction of lateral and recurrent inhibition as the most probable scenario. Whether the disinhibitory effect of iTBS for the restoration of normal cortical function in the acute phase of depression after deafferentiation is also beneficial in humans remains to be demonstrated. As recently discussed, disinhibition appears to be required to open a window for neuronal plasticity., (© 2019 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Background: Repetitive transcranial magnetic stimulation (rTMS) is able to modify cortical excitability. Rat rTMS studies revealed a modulation of inhibitory systems, in particular that of the parvalbumin-expressing (PV+) interneurons, when using intermittent theta-burst stimulation (iTBS)., Objective: The potential disinhibitory action of iTBS raises the questions of how neocortical circuits stabilize excitatory-inhibitory balance within a physiological range. Neuropeptide Y (NPY) appears to be one candidate., Methods: Analysis of cortical expression of PV, NPY and vesicular glutamate transporter type 1 (vGluT1) by immunohistochemical means at the level of cell counts, mean neuropil expression and single cell pre-/postsynaptic expression, with and without intraventricular NPY-injection., Results: Our results show that iTBS not only reduced the number of neurons with high-PV expression in a dose-dependent fashion, but also increased the cortical expression of NPY, discussed to reduce glutamatergic transmission, and this was further associated with a reduced vGluT1 expression, an indicator of glutamateric presynaptic activity. Interneurons showing a low-PV expression exhibit less presynaptic vGluT1 expression compared to those with a high-PV expression. Intraventricular application of NPY prior to iTBS prevented the iTBS-induced reduction in the number of high-PV neurons, the reduction in tissue vGluT1 level and that presynaptic to high-PV cells., Conclusions: We conclude that NPY, possibly via a global but also slow homeostatic control of glutamatergic transmission, modulates the strength and direction of the iTBS effects, likely preventing pathological imbalance of excitatory and inhibitory cortical activity but still allowing enough disinhibition beneficial for plastic changes as during learning., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: In recent years, many studies have evaluated the effects of noninvasive brain stimulation (NIBS) techniques for the treatment of several neurological and psychiatric disorders. Positive results led to approval of NIBS for some of these conditions by the Food and Drug Administration in the USA. The therapeutic effects of NIBS have been related to bi-directional changes in cortical excitability with the direction of change depending on the choice of stimulation protocol. Although after-effects are mostly short lived, complex neurobiological mechanisms related to changes in synaptic excitability bear the potential to further induce therapy-relevant lasting changes., Objective: To review recent neurobiological findings obtained from in vitro and in vivo studies that highlight molecular and cellular mechanisms of short- and long-term changes of synaptic plasticity after NIBS., Findings: Long-term potentiation (LTP) and depression (LTD) phenomena by itself are insufficient in explaining the early and long term changes taking place after short episodes of NIBS. Preliminary experimental studies indicate a complex scenario potentially relevant to the therapeutic effects of NIBS, including gene activation/regulation, de novo protein expression, morphological changes, changes in intrinsic firing properties and modified network properties resulting from changed inhibition, homeostatic processes and glial function., Conclusions: This review brings into focus the neurobiological mechanisms underlying long-term after-effects of repetitive transcranial magnetic stimulation (rTMS) and transcranial direct current stimulation (tDCS) recently obtained from in vitro and in vivo studies, both in animals and humans., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background/objectives: Over the last ten years, an increasing number of authors have used the theta burst stimulation (TBS) protocol to investigate long-term potentiation (LTP) and long-term depression (LTD)-like plasticity non-invasively in the primary motor cortex (M1) in healthy humans and in patients with various types of movement disorders. We here provide a comprehensive review of the LTP/LTD-like plasticity induced by TBS in the human M1., Methods: A workgroup of researchers expert in this research field review and discuss critically ten years of experimental evidence from TBS studies in humans and in animal models. The review also includes the discussion of studies assessing responses to TBS in patients with movement disorders., Main Findings/discussion: We discuss experimental studies applying TBS over the M1 or in other cortical regions functionally connected to M1 in healthy subjects and in patients with various types of movement disorders. We also review experimental evidence coming from TBS studies in animals. Finally, we clarify the status of TBS as a possible new non-invasive therapy aimed at improving symptoms in various neurological disorders., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Modulation of human cortical excitability by repetitive transcranial magnetic stimulation (rTMS) appears to be in part related to changed activity of inhibitory systems. Our own studies showed that intermittent theta-burst stimulation (iTBS) applied via rTMS to rat cortex primarily affects the parvalbumin-expressing (PV) fast-spiking interneurons (FSIs), evident via a strongly reduced PV expression. We further found the iTBS effect on PV to be age-dependent since no reduction in PV could be induced before the perineuronal nets (PNNs) of FSIs start to grow around postnatal day (PD) 30. To elucidate possible iTBS-induced changes in the electrical properties of FSIs and cortical network activity during cortical critical period, we performed ex vivo-in vitro whole-cell patch clamp recordings from pre-labeled FSIs in the current study. FSIs of verum iTBS-treated rats displayed a higher excitability than sham-treated controls at PD29-38, evident as higher rates of induced action potential firing at low current injections (100-200 pA) and a more depolarized resting membrane potential. This effect was absent in younger (PD26-28) and older animals (PD40-62). Slices of verum iTBS-treated rats further showed higher rates of spontaneous excitatory postsynaptic currents (sEPSCs). Based on these and previous findings we conclude that FSIs are particularly sensitive to TBS during early cortical development, when FSIs show an activity-driven step of maturation which is paralleled by intense growth of the PNNs and subsequent closure of the cortical critical period. Although to be proven further, rTMS may be a possible early intervention to compensate for hypo-activity related mal-development of cortical neuronal circuits.

Early cortical critical period resembles a state of enhanced neuronal plasticity enabling the establishment of specific neuronal connections during first sensory experience. Visual performance with regard to pattern discrimination is impaired if the cortex is deprived from visual input during the critical period. We wondered how unspecific activation of the visual cortex before closure of the critical period using repetitive transcranial magnetic stimulation (rTMS) could affect the critical period and the visual performance of the experimental animals. Would it cause premature closure of the plastic state and thus worsen experience-dependent visual performance, or would it be able to preserve plasticity? Effects of intermittent theta-burst stimulation (iTBS) were compared with those of an enriched environment (EE) during dark-rearing (DR) from birth. Rats dark-reared in a standard cage showed poor improvement in a visual pattern discrimination task, while rats housed in EE or treated with iTBS showed a performance indistinguishable from rats reared in normal light/dark cycle. The behavioral effects were accompanied by correlated changes in the expression of brain-derived neurotrophic factor (BDNF) and atypical PKC (PKCζ/PKMζ), two factors controlling stabilization of synaptic potentiation. It appears that not only nonvisual sensory activity and exercise but also cortical activation induced by rTMS has the potential to alleviate the effects of DR on cortical development, most likely due to stimulation of BDNF synthesis and release. As we showed previously, iTBS reduced the expression of parvalbumin in inhibitory cortical interneurons, indicating that modulation of the activity of fast-spiking interneurons contributes to the observed effects of iTBS., (© 2015 Wiley Periodicals, Inc.)

Background: Intermittent theta-burst stimulation (iTBS) applied via transcranial magnetic stimulation has been shown to increase cortical excitability in humans. In the rat brain it strongly reduced the number of neurons expressing the 67-kD isoform of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD67) and those expressing the calcium-binding proteins parvalbumin (PV) and calbindin (CB), specific markers of fast-spiking (FS) and non-FS inhibitory interneurons, respectively, an indication of modified cortical inhibition., Objective: Since iTBS effects in humans have been shown to be NMDA receptor sensitive, we wondered whether the iTBS-induced changes in the molecular phenotype of interneurons may be also sensitive to glutamatergic synaptic transmission mediated by NMDA receptors., Methods: In a sham-controlled fashion, five iTBS-blocks of 600 stimuli were applied to rats either lightly anesthetized by only urethane or by an additional low (subnarcotic) or high dose of the NMDA receptor antagonist ketamine before immunohistochemical analysis., Results: iTBS reduced the number of neurons expressing GAD67, PV and CB. Except for CB, a low dose of ketamine partially prevented these effects while a higher dose almost completely abolished the iTBS effects., Conclusions: Our findings indicate that iTBS modulates the molecular, and likely also the electric, activity of cortical inhibitory interneurons and that the modulation of FS-type but less that of non-FS-type neurons is mediated by NMDA receptors. A combination of iTBS with pharmacological interventions affecting distinct receptor subtypes may thus offer options to enhance its selectivity in modulating the activity of distinct cell types and preventing others from being modulated., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Repetitive transcranial magnetic stimulation (rTMS) is able to induce changes in neuronal activity that outlast stimulation. The underlying mechanisms are not completely understood. They might be analogous to long-term potentiation or depression, as the duration of the effects seems to implicate changes in synaptic plasticity. Norepinephrine (NE) has been shown to play a crucial role in neuronal plasticity in the healthy and injured human brain. Atomoxetine (ATX) and other NE reuptake inhibitors have been shown to increase excitability in different systems and to influence learning processes. Thus, the combination of two facilitative interventions may lead to further increase in excitability and motor learning. But in some cases homeostatic metaplasticity might protect the brain from harmful hyperexcitability. In this study, the combination of 60 mg ATX and 10 Hz rTMS over the primary motor cortex was used to examine changes in cortical excitability and motor learning and to investigate their influence on synaptic plasticity mechanisms., Results: The results of this double-blind placebo-controlled study showed that ATX facilitated corticospinal and intracortical excitability in motor cortex. 10 Hertz rTMS applied during a motor task was able to further increase intracortical excitability only in combination with ATX. In addition, only the combination of 10 Hz rTMS and ATX was capable of enhancing the total number of correct responses and reaction time significantly, indicating an interaction effect between rTMS and ATX without signs of homeostatic metaplasticity., Conclusion: These results suggest that pharmacologically enhanced NE transmission and 10 Hz rTMS exert a synergistic effect on motor cortex excitability and motor learning in healthy humans.

Using a rat model to study the cellular effects of repetitive transcranial magnetic stimulation (rTMS) with regard to changes in cortical excitability, we previously described opposite effects of continuous and intermittent theta-burst stimulation (cTBS, iTBS) on the expression of the calcium-binding proteins (CaBP) parvalbumin (PV), calbindin (CB) and calretinin (CR) in Dark Agouti rats (DA). While iTBS significantly reduced the number of cortical PV+ cells but did not affect the CB+ cells, cTBS resulted in a decrease in CB+ cells with no effects on PV+ cells. We concluded that activity of these classes of cortical interneurons is differently modulated by iTBS and cTBS. When testing two further rat strains, Sprague-Dawley (SD) and Long Evans (LE), we obtained deviating results. In SD, iTBS reduced PV and CB expression, while cTBS only reduced PV expression. In contrast, reanalysed DA showed reduced CB expression after cTBS and reduced PV expression after iTBS, while LE shows an intermediate reaction. CR expression was unaffected in any case. Interestingly, we found significantly different basal expression patterns of the CaBPs for the strains, with DA and LE showing much higher numbers of PV+, CB+ and CR+ cells than SD, and with particularly higher number of CB+ and CR+ cells in DA compared to the other two strains. These findings demonstrate that inhibitory systems may be either differently developed in rats belonging to diverse strains or show different basal levels of activity and CaBP expression and may therefore be differently sensitive to the rTMS protocols.

Background: Theta Burst stimulation (TBS) applied via transcranial magnetic stimulation (TMS) effectively modulates human neocortical excitability but repeated applications of the same TBS protocol at short intervals may be not simply accumulative., Objective: Our aim was to investigate the impact of multiple blocks of either intermittent (iTBS) or continuous TBS (cTBS) on the expression of neuronal activity marker proteins in rat cortex., Methods: Up to four iTBS- or cTBS-blocks of 600 stimuli were applied to urethane-anesthetized rats followed by immunohistochemical and Western blot analyses., Results: The effects of iTBS and cTBS were similar but slightly differed with regard to the number of stimuli applied. The expression of the 65-kD isoform of glutamic acid decarboxylase (GAD65) increased with each stimulation block, while that of the 67-kD isoform (GAD67), and that of the calcium-binding proteins (CaBP) Parvalbumin (PV) and Calbindin (CB) and that of the immediate early gene c-Fos progressively decreased. Both TBS protocols increased the expression of the vesicular glutamate transporter 1 (VGLUT1) with 1200-1800 stimuli but then decreased them after the 4th block., Conclusion: Our findings indicate that repeated TBS elicits no simple accumulative dose-dependent effect for all activity-markers but distinct profiles with threshold characteristics and a waxing-and-waning effect especially for the markers of inhibitory activity CB and GAD67. Interestingly, somatic activity markers, such as c-Fos for mainly excitatory and GAD67, CB and PV for inhibitory neurons, decreased with repeated stimulation while synaptic activity markers mainly increased which could be a result of the artificial stimulation of axons., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In a rat model of transcranial magnetic stimulation we could recently show that intermittent theta-burst stimulation (iTBS) affects the neocortical expression of the immediate early gene products c-Fos and zif268 as well as that of the two glutamic acid decarboxylase isoforms GAD65 and GAD67 and that of the calcium-binding proteins calbindin (CB) and parvalbumin (PV), known as markers of excitatory and inhibitory activity. We now analyzed in more detail the time course of changes in the expression of these proteins at 10, 20, 40, 80 and 160min following a single block of iTBS consisting of 600 stimuli. Initial increase in c-Fos, zif268 and GAD65 (20min) signals transient activation of excitatory and inhibitory neurons, thereafter first followed by a decrease in markers of activity of inhibitory neurons (GAD67, PV, CB: 20-80min) and then by a late decrease in c-Fos and GAD65 expression (160min). The results demonstrate that one iTBS block may have an after-effect of at least two different phases, an early phase with increased neuronal activity (c-Fos, zif268) but also the likelihood of increased GABA-release (GAD65), followed by a late phase (>40min) of reduced neuronal activity in excitatory and inhibitory systems which may indicate a state of reduced excitability., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Transcranial magnetic stimulation (TMS) has become a popular method to non-invasively stimulate the human brain. The opportunity to modify cortical excitability with repetitive stimulation (rTMS) has especially gained interest for its therapeutic potential. However, details of the cellular mechanisms of the effects of rTMS are scarce. Currently favoured are long-term changes in the efficiency of excitatory synaptic transmission, with low-frequency rTMS depressing it, but high-frequency rTMS augmenting. Only recently has modulation of cortical inhibition been considered as an alternative way to explain lasting changes in cortical excitability induced by rTMS. Adequate animal models help to highlight stimulation-induced changes in cellular processes which are not assessable in human rTMS studies. In this review article, we summarize findings obtained with our rat models which indicate that distinct inhibitory cell classes, like the fast-spiking cells characterized by parvalbumin expression, are most sensitive to certain stimulation protocols, e.g. intermittent theta burst stimulation. We discuss how our findings can support the recently suggested models of gating and homeostatic plasticity as possible mechanisms of rTMS-induced changes in cortical excitability.

Repetitive transcranial magnetic stimulation (rTMS) can modulate cortical excitability in a stimulus-frequency-dependent manner. Two kinds of theta burst stimulation (TBS) [intermittent TBS (iTBS) and continuous TBS (cTBS)] modulate human cortical excitability differently, with iTBS increasing it and cTBS decreasing it. In rats, we recently showed that this is accompanied by changes in the cortical expression of proteins related to the activity of inhibitory neurons. Expression levels of the calcium-binding protein parvalbumin (PV) and of the 67-kDa isoform of glutamic acid decarboxylase (GAD67) were strongly reduced following iTBS, but not cTBS, whereas both increased expression of the 65-kDa isoform of glutamic acid decarboxylase. In the present study, to investigate possible functional consequences, we applied iTBS and cTBS to rats learning a tactile discrimination task. Conscious rats received either verum or sham rTMS prior to the task. Finally, to investigate how rTMS and learning effects interact, protein expression was determined for cortical areas directly involved in the task and for those either not, or indirectly, involved. We found that iTBS, but not cTBS, improved learning and strongly reduced cortical PV and GAD67 expression. However, the combination of learning and iTBS prevented this effect in those cortical areas involved in the task, but not in unrelated areas. We conclude that the improved learning found following iTBS is a result of the interaction of two effects, possibly in a homeostatic manner: a general weakening of inhibition mediated by the fast-spiking interneurons, and re-established activity in those neurons specifically involved in the learning task, leading to enhanced contrast between learning-induced and background activity., (© 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Transcranial magnetic stimulation (TMS) can be used in two different ways to manipulate cortical information processing, either by applying a single pulse around the time point of expected task processing or by persistently shifting cortical excitability by repetitive stimulation (rTMS). Single pulses applied when specific cortical processing takes place always impair cortical function due to increased noise or enhanced inhibition, both resulting in decreased signal-to-noise ratio, while repetitive stimulation may allow to weaken or improve cortical processing depending on the type of stimulation. The opposite effects of low- ( approximately 1 Hz) and high-frequency rTMS (5-20 Hz), as well as the opposing effects of continuous versus intermittent theta-burst trains, lowering or raising cortical excitability respectively, have mainly been attributed to synaptic plasticity. As reviewed in this article, in a series of electrophysiological, immunohistochemical and molecular-biological animal experiments we obtained evidence for modulation of inhibitory cortical activity as a further reason of changing cortical excitability following rTMS.

Modified cortical excitability following repetitive transcranial magnetic stimulation (rTMS) may be related to short- or long-term synaptic plasticity of neuronal excitation but could also affect cortical inhibition. Therefore, in the rat we tested how three different rTMS protocols, intermittent and continuous theta-burst (iTBS, cTBS), and low-frequency 1 Hz stimulation, change the expression of GAD65, GAD67 and GAT-1 which are expressed in cortical inhibitory interneurons in an activity-dependent manner. Acutely (2 h), all protocols reduced the expression of GAD67 in frontal, motor, somatosensory and visual cortex but increased that of GAD65 and GAT-1 to different degree, with iTBS having the strongest acute effect. The initial decrease in GAD67 reversed after 1 day, leading to a strong increase in GAD67 expression for up to 7 days primarily in the frontal cortex in case of iTBS, cTBS and in all studied areas following 1 Hz rTMS. While also GAD65 and GAT-1 expression reversed after 1 day in case of iTBS and cTBS, 1 Hz rTMS induced a steady increase in GAD65 and GAT-1 expression during the 7 days investigated. Our data demonstrate that rTMS affects the expression of activity-dependent proteins of the cortical inhibitory interneurons. Besides common effects of low- (1 Hz) and high-frequency (TBS) stimulation on protein expression, differences in quantity and time course of changes point to differences in the contribution of possible neuronal subsystems. Further studies are needed to distinguish cell-type specific effects., (© Springer-Verlag 2009)

Repetitive transcranial magnetic stimulation (rTMS) has been shown to alter cortical excitability depending on the stimulus-frequency used, with high frequency (5 Hz and higher) increasing it but low frequency (usually 1 Hz or lower) reducing it. To determine the efficiency of different rTMS protocols in inducing cortical network activity, we tested the acute effect of one low-frequency rTMS protocol (1 Hz) and two different high-frequency protocols (10 Hz and intermittent theta-burst stimulation, iTBS) on the expression of the two immediate early gene (IEG) proteins c-Fos and zif268 in the rat brain. The cortical expression of both IEGs was specifically changed in an rTMS-dependent manner. One and 10 Hz rTMS enhanced c-Fos protein expression in all cortical areas tested, while iTBS was effective only in limbic cortices. Zif268 expression was increased in almost all cortical areas after iTBS, while 10 Hz rTMS was effective only in the primary motor and sensory cortices. One Hertz rTMS had no effect on cortical zif268 expression. Furthermore, sham-rTMS had no effect on zif268 expression but increased c-Fos in limbic cortices. This is the first study demonstrating that cortical zif268 and c-Fos expression can be specifically modulated by acute rTMS depending on the pattern of stimulation applied.

Repetitive transcranial magnetic stimulation (rTMS) has been shown to alter cortical excitability that lasts beyond the duration of rTMS application itself. High-frequency rTMS leads primarily to facilitation, whereas low-frequency rTMS leads to inhibition of the treated cortex. However, the contribution of rTMS train duration is less clear. In this study, we investigated the effects of nine different rTMS protocols, including low and high frequencies, as well as short and long applications (1, 3 and 10 Hz applied for 1, 5 and 20 min), on visual cortex excitability in anaesthetized and paralysed cats by means of visual evoked potential (VEP) and electroencephalography (EEG) recordings. Our results show that 10 Hz rTMS applied for 1 and 5 min significantly enhanced early VEP amplitudes, while 1 and 3 Hz rTMS applied for 5 and 20 min significantly reduced them. No significant changes were found after 1 and 3 Hz rTMS applied for only 1 min, and 10 Hz rTMS applied for 20 min. EEG activity was only transiently (<20 s) affected, with increased delta activity after 1 and 3 Hz rTMS applied for 1 or 5 min. These findings indicate that the effects of rTMS on cortical excitability depend on the combination of stimulus frequency and duration (or total number of stimuli): short high-frequency trains seem to be more effective than longer trains, and low-frequency rTMS requires longer applications. Changes in the spectral composition of the EEG were not correlated to changes in VEP size.

Transcranial magnetic stimulation (TMS) has become a well established procedure for testing and modulating the neuronal excitability of human brain areas, but relatively little is known about the cellular processes induced by this rather coarse stimulus. In a first attempt, we performed extracellular single-unit recordings in the primary visual cortex (area 17) of the anaesthetised and paralysed cat, with the stimulating magnetic field centred at the recording site (2 x 70 mm figure-of-eight coil). The effect of single biphasic TMS pulses, which induce a lateral-to-medial electric current within the occipital pole of the right hemisphere, was tested for spontaneous as well as visually evoked activity. For cat visual cortex we found that a single TMS pulse elicited distinct episodes of enhanced and suppressed activity: in general, a facilitation of activity was found during the first 500 ms, followed thereafter by a suppression of activity lasting up to a few seconds. Strong stimuli exceeding 50 % of maximal stimulator output could also lead to an early suppression of activity during the first 100-200 ms, followed by stronger (rebound) facilitation. Early suppression and facilitation of activity may be related to a more or less direct stimulation of inhibitory and excitatory interneurons, probably with different thresholds. The late, long-lasting suppression is more likely to be related to metabotropic or metabolic processes, or even vascular responses. The time course of facilitation/inhibition may provide clues regarding the action of repetitive TMS application.

Animal TMS models are especially useful for studying the cellular and molecular effects related to the lasting effects achieved with prolonged stimulation of a particular pattern (rTMS). In translational terms, it is exceptionally challenging to realize stimulation conditions resembling those supposed to occur in the human brain. Due to the very different ratio of coil-to-brain sizes, a focal stimulation of small animal brains as can be achieved in humans using figure-of-eight coils is hardly realistic today. Attempts to develop small (e.g. rodent coils) are confronted with reduced field power or limited number of high-frequency train pulses due to heating problems. Nevertheless, animal TMS models will be helpful in terms of studying relationships between stimulation-induced changes in neuronal activity and plasticity and changes in behavioral phenotypes. Differences in rTMS effects found in Parkinson patients compared to healthy controls which appear to be related to disturbed synaptic plasticity could be replicated in a rat Parkinson disease model ( Hsieh et al., 2015 ) and beneficial stimulation effects related to modulation of striatal dopamine content have been reported ( Ghiglieri et al., 2012 ). Principally, global brain stimulation using rTMS appears adequate if studying neuromodulatory effects not confined to a particular system as is found with changes in extracellular levels of dopamine or glutamate, brain-derived neurotrophic factor (BDNF), pro- or anti-inflammatory mediators and changes in the excitatory-inhibitory balance ( Suppa et al., 2016 ). Nonfocal cortical and/or subcortical stimulation should be suitable to also answer the question if distinct types of neurons (including their genesis and developmental state) or their compartments are more sensitive to a particular stimulation protocol than others if analyzing neuronal activity and plasticity markers. Even if those changes have been achieved with limited focal stimulation in vivo, matched in vitro studies on isolated neuronal systems allow estimating the relevance of focal stimulation as recently demonstrated for the entorhino-hippocampal pathway in mice ( Lenz et al., 2016 ). Focal electric stimulation applied in a way to either mimic the timing of spatially distant evoked activity – as in case of interhemispheric stimulation ( Barry et al., 2014 )-, or to mimic the electric field distribution likely achieved with an imaginary focal coil could further help to evaluate the differences between focal and global stimulation. However, it has to be kept in mind that magnetic fields induced a different electric field distribution within the tissue as can be achieved with contact electrodes, both with regard to spatial current density and also with regard to extra- and intracellular induction of electric field gradients. In this respect, animal studies demonstrating neuronal effects with patterned magnetic stimulation of rather low field strength (10–100 mT) are of further interest, indicating modulation of molecular processes like intracellular calcium release and gene expression even in the absence of detectable electric responses ( Grehl et al., 2015 ). Even if animal models cannot imitate TMS of human cortex in a one-to-one fashion, they definitely offer new ways to investigate how magnetically induced electric field can interact with neuronal processes and which factors determine a variable result of stimulation. [ABSTRACT FROM AUTHOR]

Introduction: Human cortical excitability can be modified by repetitive transcranial magnetic stimulation (rTMS) but the factors that determine the direction of change remain still elusive. Classical forms of synaptic plasticity and metaplastic processes like homeostatic synaptic scaling and priming-dependent synaptic plasticity are possible mechanistic scenarios. Although the activity of excitatory neurons finally represents the degree of cortical activation, a multitude of inhibitory cortical systems governs about magnitude and pattern of cortical activity and the degree of plasticity allowed without loosing essential excitation-inhibition balance. It is thus likely that rTMS-induced changes in cortical excitability are associated with changes in cortical inhibition and that classes of interneurons differently contribute. Objectives: We established a rat model of rTMS enabling to study cellular effects of rTMS in more detail and with possible relation to behavioural changes. To focus on changes in inhibitory cortical activity, we studied the expression of certain activity markers which are specific to inhibitory neurons in general, like the two isoforms of the glutamic acid decarboxylase GAD65 and GAD67, or relate to a certain class of interneurons, like the expression of the calcium-binding proteins parvalbumin (PV), calbindin (CB) and calretinin (CR). Our recent studies focus on the temporal dynamics of these activity markers which is of particular interest in case of combining rTMS with other artificial or natural interventions or repeated rTMS. Materials and methods: Adult male Sprague–Dawley rats (n =42.15weeks old) were trained to tolerate the procedure of manual restrain and rTMS without signs of stress before receiving a single block (600 pulses) of either verum or sham intermittent theta-burst stimulation protocol (iTBS) at an intensity just subthreshold for evoking motor responses (MagStim rapid, 70mm figure-of-eight coil). Then, rat were deeply anesthetized and perfused at different intervals post-rTMS (10, 20, 40, 80, 160min) for immunohistochemical analysis of neuronal activity marker expression (GAD65/67, PV, CB for inhibitory neurons; c-Fos and zif268 mainly for excitatory neurons). Results: Three phases of changes in neuronal activity marker expression could be distinguished: an early phase (10–20min) of strongly increased c-Fos and GAD65 expression, a following phase (40–80min) of reduced GAD67, PV and CB expression and a late phase (>160min) of reduced c-Fos and GAD65 expression. Conclusion: Early increased levels of c-Fos and GAD65 indicate a strong co-activation of excitatory and inhibitory neurons due to iTBS. The delayed decrease in GAD67, PV and CB expression indicates a secondary depression of activity of inhibitory neurons without signs of increased excitatory activity (c-Fos, zif268). The late decrease in c-Fos and GAD65 finally indicates a matched reduction in the activity of both excitatory and inhibitory activities. Obviously, excitation-inhibition balance is never violated but the phases of changed synaptic (GAD65) and somatic (GAD67, PV, CB) inhibitory neuron activity to not co-vary in time. This study has been supported by the Deutsche Forschungsgemeinschaft, DFG (FU 256/3-2, SFB 874, TP A4). [Copyright &y& Elsevier]

Questions Our previous studies showed that intermittent theta-burst stimulation (iTBS) applied via transcranial magnetic stimulation (TMS) to the rat brain strongly reduced the expression of the calcium-binding protein parvalbumin (PV) in fast-spiking inhibitory interneurons (FSI). This finding indicates that disinhibition and the associated increase in cortical excitability may be one way to open a window for plastic changes in the cortical network. The mechanisms leading to reduced PV expression are not known but are either related to a strong activation during iTBS or a subsequent reduction in glutamatergic excitation of FSI in a manner of Ltd. One well-described regulator of glutamate release at excitatory terminals is neuropeptide Y (NPY) which has been shown to be up-regulated after epileptic activity. Therefore, we tested if iTBS increases the expression of NPY in rat cortex, whether the reduced PV expression in FSI is associated with reduced activity of excitatory inputs and finally if intrathecal application of NPY prior to TMS can counteract the cortical iTBS effect. Methods In a first study, parallel changes in the cortical expression of PV and NPY in rats previously treated with one to five blocks of iTBS (600 pulses per block, intervals of 15 min) were analysed using immunohistochemistry. In a second study, we analysed the relationship between the level of PV expression of single FSI and the strength of glutamatergic input measured as the level of expression of the vesicular glutamate transporter type 1 (VGluT1) in cortical synapses terminating on FSI. Finally in a third study, rats received either 20 μ l NPY (24 nMol) or methylene blue as a control (MB) via intracranial injection to the left lateral ventricle (2 μ l/min) followed 60 min later by either verum or sham iTBS (3 blocks @ 15 min = 1800 pulses) to test if enhanced NPY level can influence the effect of iTBS on PV and vGluT1 expression. Results In a dose-dependent fashion, iTBS gradually decreased the number of PV+ neurons but also increased the level of NPY in the neuropil and the number of NPY+ neurons. Interneurons showing low PV expression had a significantly weaker labelling of glutamatergic cortical input (less vGluT1) than cells showing high PV level. NPY injected into the lateral ventricle of the left forebrain (no diffusion to the right hemisphere) prevented the reduction of PV+ cells (−40%) by iTBS in the left but not in the right hemisphere and further increased the number of PV+ cells. A slight increase in PV+ cells was also evident in sham stimulated animals when comparing NPY with MB injections. Conclusions As expected, iTBS increased the expression of NPY likely due to the strong and highly synchronous activation of cortical networks. Since strong excitation is especially harmful to FSI, the reduction in PV expression appears to signal a post-stimulation depression of excitatory inputs and subsequent hypoactivity of these interneurons. The finding that NPY prevents the strong reduction in PV expression indicates that it is able to prevent over-excitation of FSI. This study has been supported a grant of the Federal Ministry of Education and Research (BMBF): GCBS-WP1_Bochum ( 01EE1403B ) to K. Funke. [ABSTRACT FROM AUTHOR]

Question Gestational infection is a risk factor for psychiatric disorders. Accordingly, preclinical models of maternal immune activation (MIA) have been developed to study effects of gestational infection on neural and behavioral dysfunction in the offspring. Considering that early pre-symptomatic stages of schizophrenia may affect hippocampal circuits, we here established a hippocampal ‘organotypic in vitro MIA model’ based on the well-established polyinosinic-polycytidylic acid [Poly(I:C)] model of schizophrenia. We studied alterations in synaptic neurotransmission and tested whether repetitive magnetic stimulation (rMS) influences alterations in synaptic excitation/inhibition-balance in this model. Methods Entorhino-hippocampal slice cultures were prepared from litters of Poly(I:C) or vehicle-only injected pregnant dams. The effects of 10 Hz rMS on MIA associated alterations in synaptic properties were assessed in CA1 pyramidal neurons using whole-cell patch clamp recordings. Results Our experiments disclose an increase in inhibitory synaptic strength, while excitatory synapses on CA1 neurons are not changed in slice cultures prepared from Poly(I:C) offspring. This difference is also observed in cultures prepared from cross-fostered animals, thus confirming that gestational infection triggers the synaptic phenotype. Since our recent work has demonstrated that 10 Hz rMS can shift synaptic excitation/inhibition-balance toward excitation, we employed this rMS protocol to test whether we can influence the synaptic phenotype in the Poly(I:C) group. Indeed, 10 Hz rMS reversed the increased inhibitory synaptic strength without affecting excitatory synapses in MIA slice cultures. Conclusion These results suggest that rMS may exert positive effects under pathological conditions by restoring physiological synaptic excitation/inhibition-balance. We propose that ‘organotypic in vitro MIA models’ may serve as preclinical drug and intervention screening assays to evaluate and learn more about rTMS-based treatment strategies in psychiatric diseases associated with gestational infection. (Supported by Federal Ministry of Education and Research, Germany (BMBF, GCBS-WP1: 01EE1403B).). [ABSTRACT FROM AUTHOR]

Questions High-frequency transcranial magnetic stimulation with theta-burst stimulation (TBS) induces strong and highly synchronous cortical network activity principally able to trigger epileptiform activity or to cause over-excitation of distinct neurons. Our previous studies showed that intermittent TBS (iTBS) strongly reduces the expression of the calcium-binding protein parvalbumin (PV) in fast-spiking inhibitory interneurons (FSI). Since PV is expressed in an activity-dependent manner, strong activation of these neurons during iTBS might have triggered mechanisms to prevent permanent over-excitation. One well-described process is the reduction of glutamate release from excitatory terminals targeting FSI via the action of neuropeptide Y (NPY). Therefore, we first tested if iTBS increases the expression of NPY in rat cortex and second, if application of NPY to the lateral ventricle can counteract the cortical iTBS effect of reducing PV expression. Methods In the first study, frontal sections of rat brains previously treated with one to five blocks of iTBS (600 pulses per block, intervals of 15 min) were immunostained for NPY and PV. In the second study, rats received either 20 μ l NPY (24 nMol) or methylene blue as a control (MB) via intracranial injection to the left lateral ventricle (2 μ l/min) followed 60 min later by either verum or sham iTBS (3 blocks @ 15 min = 1800 pulses). Further 60 min later rats were perfused for PV immunostaining of their brains. Results iTBS gradually increased the number of NPY+ neurons in a dose-dependent fashion from about 60 cells/mm 2 to about 130 cells/mm 2 after five iTBS blocks. The same stimulation protocol was previously found to decrease the number of PV+ neurons by about 40% after five iTBS blocks (Volz et al., Brain Stimulation 2013). NPY injected into the lateral ventricle of the left forebrain (no diffusion to the right hemisphere) prevented the reduction of PV+ cells (−40%) by iTBS in the left but not in the right hemisphere and further increased the number of PV+ cells. A slight increase in PV+ cells was also evident in sham stimulated animals when comparing NPY with MB injections. Conclusions As expected, iTBS increased the expression of NPY likely due to the strong and highly synchronous activation of cortical networks. Since strong excitation is especially harmful to FSI, the reduction in PV expression indicates a post-stimulation depression of excitatory inputs and subsequent hypoactivity of these interneurons. The finding that NPY prevents the strong reduction in PV expression indicates that it is able to prevent over-excitation of FSI. This study has been supported by grants of the Deutsche Forschungsgemeinschaft (DFG): SFB 874 TP A4 and the Federal Ministry of Education and Research (BMBF): GCBS-WP1_Bochum (01EE1403B) to K. Funke. [ABSTRACT FROM AUTHOR]

Introduction: Theta Burst stimulation (TBS) applied via transcranial magnetic stimulation (TMS) is an effective tool to modulate human neocortical excitability (Huang et al., 2005). Repeated application of the same TBS protocol or variation of the number of stimuli has been shown to alter the strength and direction of changes in cortical excitability compared to the standard TBS protocols (Gentner et al., 2008; Gamboa et al., 2010). TBS applied to rat cortex affected the expression of activity-dependent proteins related to the cortical inhibitory systems, suggesting altered cortical inhibition contributing to the TBS after-effects (Benali et al., 2011; Funke and Benali, 2011). Objectives: Our aim was to investigate the impact of varying numbers of TBS-stimuli applied as multiple blocks of intermittend TBS (iTBS) or continuous TBS (cTBS) on cortical protein expression in the rat, to further our insights in physiological mechanisms underlying TBS-induced changes in cortical excitability. Materials and methods: Nine groups of anesthetized rats (male Sprague Dawley, 400–600g) received TMS. Eight groups received a different number of iTBS/cTBS-blocks summing up to either 600, 1200, 1800, or 2400 stimuli, applied with breaks of 15min between blocks of 600 stimuli. Sham stimulation (coil more distant to the head) was applied to a control group. Rats were sacrificed for immunohistochemical analysis or western blotting focussing on frontal, motor, sensory and visual cortex. Results: In general, quite similar effects for iTBS and cTBS were observed. The expression of the 65-kDa isoform of glutamic acid decarboxylase (GAD65) increased, while that of the 67-kDa isoform (GAD67) and that of the calcium-binding proteins Parvalbumin (PV) and Calbindin (CB) progressively decreased. Also the expression of the immediate early gene c-Fos decreased with an increasing number of blocks. A more detailed analysis, however, revealed that the sensitivity of distinct proteins to stimulation varied with the number of stimuli and type of stimulation. Conclusion: Our findings show that both iTBS and cTBS affect the activity of inhibitory interneurons and indicate that repeated TBS elicits no simple accumulative dose-dependent effect for all activity-markers but distinct profiles with threshold characteristics and a waxing-and-waning effect especially for two markers of inhibitory activity, CB and GAD67. Thus, our data do not suggest fundamentally different modulation of the cortical inhibitory systems by iTBS and cTBS. Subtle differences in stimulation after-effects on different neuronal subsystems might contribute to the opposite impact on cortical excitability following iTBS and cTBS and the switching sign of effect with repeated stimulation. [Copyright &y& Elsevier]