This study was supported by Russian Science Foundation grant (contract number: 17-11-01273). Special thanks to Andrey Afanasov and colleagues from Multifunctional Innovation Centre for Television Technics (National Research University, Higher School of Economics, Moscow, Russian Federation) for video recording and video editing.

All procedures were approved by the local research ethics committee of the Higher School of Economics (HSE), Moscow, with consent from all participants.
NOTE: Participants must report no history of implanted metal devices, neurological or psychiatric disease, drug abuse or alcoholism. TMS is used according to the most recent safety guidelines20. Subjects must be fully informed of the nature of the research and sign an informed consent form before starting the experiment...

<ol>
	<li>Priori, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+polarization+in+humans:+a+reappraisal+of+an+old+tool+for+prolonged+non-invasive+modulation+of+brain+excitability.">Brain polarization in humans: a reappraisal of an old tool for prolonged non-invasive modulation of brain excitability.</a> Clin. Neurophysiol. 114 (4), 589-595 (2003).</li><li>Herrmann, C. S., Rach, S., Neuling, T., Struber, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcranial+alternating+current+stimulation:+a+review+of+the+underlying+mechanisms+and+modulation+of+cognitive+processes.">Transcranial alternating current stimulation: a review of the underlying mechanisms and modulation of cognitive processes.</a> Front Hum. Neurosci. 7, 279 (2013).</li><li>Frohlich, F., McCormick, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+electric+fields+may+guide+neocortical+network+activity.">Endogenous electric fields may guide neocortical network activity.</a> Neuron. 67 (1), 129-143 (2010).</li><li>Ozen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcranial+electric+stimulation+entrains+cortical+neuronal+populations+in+rats.">Transcranial electric stimulation entrains cortical neuronal populations in rats.</a> J. Neurosci. 30 (34), 11476-11485 (2010).</li><li>Helfrich, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entrainment+of+brain+oscillations+by+transcranial+alternating+current+stimulation.">Entrainment of brain oscillations by transcranial alternating current stimulation.</a> Curr. Biol. 24 (3), 333-339 (2014).</li><li>Bergmann, T. O., Karabanov, A., Hartwigsen, G., Thielscher, A., Siebner, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+non-invasive+transcranial+brain+stimulation+with+neuroimaging+and+electrophysiology:+Current+approaches+and+future+perspectives.">Combining non-invasive transcranial brain stimulation with neuroimaging and electrophysiology: Current approaches and future perspectives.</a> Neuroimage. 140, 4-19 (2016).</li><li>Feher, K. D., Morishima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+Electroencephalography+Recording+During+Transcranial+Alternating+Current+Stimulation+(tACS).">Concurrent Electroencephalography Recording During Transcranial Alternating Current Stimulation (tACS).</a> J. Vis. Exp. (107), e53527 (2016).</li><li>Antal, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparatively+weak+after-effects+of+transcranial+alternating+current+stimulation+(tACS)+on+cortical+excitability+in+humans.">Comparatively weak after-effects of transcranial alternating current stimulation (tACS) on cortical excitability in humans.</a> Brain Stimul. 1 (2), 97-105 (2008).</li><li>Struber, D., Rach, S., Neuling, T., Herrmann, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+possible+role+of+stimulation+duration+for+after-effects+of+transcranial+alternating+current+stimulation.">On the possible role of stimulation duration for after-effects of transcranial alternating current stimulation.</a> Front Cell Neurosci. 9, 311 (2015).</li><li>Feurra, M., Paulus, W., Walsh, V., Kanai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+specific+modulation+of+human+somatosensory+cortex.">Frequency specific modulation of human somatosensory cortex.</a> Front Psychol. 2, (2011).</li><li>Feurra, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency-dependent+tuning+of+the+human+motor+system+induced+by+transcranial+oscillatory+potentials.">Frequency-dependent tuning of the human motor system induced by transcranial oscillatory potentials.</a> J. Neurosci. 31 (34), 12165-12170 (2011).</li><li>Feurra, M., Paulus, W., Walsh, V., Kanai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+specific+modulation+of+human+somatosensory+cortex.">Frequency specific modulation of human somatosensory cortex.</a> Front Psychol. 2, (2011).</li><li>Feurra, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State-dependent+effects+of+transcranial+oscillatory+currents+on+the+motor+system:+what+you+think+matters.">State-dependent effects of transcranial oscillatory currents on the motor system: what you think matters.</a> J. Neurosci. 33 (44), 17483-17489 (2013).</li><li>Feurra, M., Galli, G., Pavone, E. F., Rossi, A., Rossi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency-specific+insight+into+short-term+memory+capacity.">Frequency-specific insight into short-term memory capacity.</a> J. Neurophysiol. 116 (1), 153-158 (2016).</li><li>Kanai, R., Paulus, W., Walsh, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcranial+alternating+current+stimulation+(tACS)+modulates+cortical+excitability+as+assessed+by+TMS-induced+phosphene+thresholds.">Transcranial alternating current stimulation (tACS) modulates cortical excitability as assessed by TMS-induced phosphene thresholds.</a> Clin. Neurophysiol. 121 (9), 1551-1554 (2010).</li><li>Polania, R., Moisa, M., Opitz, A., Grueschow, M., Ruff, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+precision+of+value-based+choices+depends+causally+on+fronto-parietal+phase+coupling.">The precision of value-based choices depends causally on fronto-parietal phase coupling.</a> Nat. Commun. 6, 8090 (2015).</li><li>Santarnecchi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency-dependent+enhancement+of+fluid+intelligence+induced+by+transcranial+oscillatory+potentials.">Frequency-dependent enhancement of fluid intelligence induced by transcranial oscillatory potentials.</a> Curr. Biol. 23 (15), 1449-1453 (2013).</li><li>Santarnecchi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+differences+and+specificity+of+prefrontal+gamma+frequency-tACS+on+fluid+intelligence+capabilities.">Individual differences and specificity of prefrontal gamma frequency-tACS on fluid intelligence capabilities.</a> Cortex. 75, 33-43 (2016).</li><li>Dayan, E., Censor, N., Buch, E. R., Sandrini, M., Cohen, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+brain+stimulation:+from+physiology+to+network+dynamics+and+back.">Noninvasive brain stimulation: from physiology to network dynamics and back.</a> Nat. Neurosci. 16 (7), 838-844 (2013).</li><li>Rossi, S., Hallett, M., Rossini, P. M., Pascual-Leone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety,+ethical+considerations,+and+application+guidelines+for+the+use+of+transcranial+magnetic+stimulation+in+clinical+practice+and+research.">Safety, ethical considerations, and application guidelines for the use of transcranial magnetic stimulation in clinical practice and research.</a> Clin. Neurophysiol. 120 (12), 2008-2039 (2009).</li><li>Nasseri, P., Nitsche, M. A., Ekhtiari, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+framework+for+categorizing+electrode+montages+in+transcranial+direct+current+stimulation.">A framework for categorizing electrode montages in transcranial direct current stimulation.</a> Front Hum. Neurosci. 9, 54 (2015).</li><li>Rossini, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+electrical+and+magnetic+stimulation+of+the+brain,+spinal+cord,+roots+and+peripheral+nerves:+Basic+principles+and+procedures+for+routine+clinical+and+research+application.+An+updated+report+from+an+I.F.C.N.+Committee.">Non-invasive electrical and magnetic stimulation of the brain, spinal cord, roots and peripheral nerves: Basic principles and procedures for routine clinical and research application. An updated report from an I.F.C.N. Committee.</a> Clin.Neurophysiol. 126 (6), 1071-1107 (2015).</li><li>Guerra, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+Dependency+of+the+Human+Primary+Motor+Cortex+and+Cholinergic+Inhibition+Cancelation+During+Beta+tACS.">Phase Dependency of the Human Primary Motor Cortex and Cholinergic Inhibition Cancelation During Beta tACS.</a> Cereb. Cortex. 26 (10), 3977-3990 (2016).</li><li>Fertonani, A., Ferrari, C., Miniussi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+do+you+feel+if+I+apply+transcranial+electric+stimulation?+Safety,+sensations+and+secondary+induced+effects.">What do you feel if I apply transcranial electric stimulation? Safety, sensations and secondary induced effects.</a> Clin. Neurophysiol. 126 (11), 2181-2188 (2015).</li><li>Feurra, M., Galli, G., Rossi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcranial+alternating+current+stimulation+affects+decision+making.">Transcranial alternating current stimulation affects decision making.</a> Front Syst.Neurosci. 6, 39 (2012).</li><li>Marshall, L., Helgadottir, H., Molle, M., Born, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boosting+slow+oscillations+during+sleep+potentiates+memory.">Boosting slow oscillations during sleep potentiates memory.</a> Nature. 444 (7119), 610-613 (2006).</li><li>Sela, T., Kilim, A., Lavidor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcranial+alternating+current+stimulation+increases+risk-taking+behavior+in+the+balloon+analog+risk+task.">Transcranial alternating current stimulation increases risk-taking behavior in the balloon analog risk task.</a> Front Neurosci. 6, (2012).</li><li>Goldsworthy, M. R., Vallence, A. M., Yang, R., Pitcher, J. B., Ridding, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+transcranial+alternating+current+stimulation+and+continuous+theta+burst+stimulation:+a+novel+approach+for+neuroplasticity+induction.">Combined transcranial alternating current stimulation and continuous theta burst stimulation: a novel approach for neuroplasticity induction.</a> Eur. J. Neurosci. 43 (4), 572-579 (2016).</li><li>Bestmann, S., Krakauer, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+uses+and+interpretations+of+the+motor-evoked+potential+for+understanding+behaviour.">The uses and interpretations of the motor-evoked potential for understanding behaviour.</a> Exp. Brain Res. 233 (3), 679-689 (2015).</li></ol>

This approach represents a unique opportunity to directly test online effects of tACS of the primary motor cortex by measuring corticospinal output through MEPs recording. However, the placement of the TMS coil over the tACS electrode represents a critical step that should be accurately performed. Therefore, we would firstly suggest experimenters find a target point by single pulse TMS, then mark it on the scalp and, only after that, place the tACS electrode over the hotspot. Moreover, the availability of a neuronavigati...

Transcranial Electrical Stimulation (tES) is a neuromodulatory technique which allows the modification of neuronal states through different current waveforms1. Among different types of tES, transcranial Alternating Current Stimulation (tACS) enables the delivery of sinusoidal external oscillatory potentials in a specific frequency range and the modulation of physiological neural activity underlying perceptual, motor and cognitive processes2. Using tACS, it is possible to investigate potential causal links between endogenous oscillatory activity and brain processes.
In vivo, it has been shown that spiking neural activity is synchronized at different driving frequencies, suggesting that neuronal firing can be entrained by electrically applied fields3. In animal models, weak sinusoidal tACS entrains the discharged frequency of the widespread cortical neuronal pool4. In humans, tACS combined with online Electroencephalography (EEG) allows the induction of the so-called &#34;Entrainment&#34; effect on endogenous oscillatory activity by interacting with brain oscillations in a frequency-specific manner5. However, combining tACS with neuroimaging methods for a better understanding of the online mechanisms is still questionable because of AC-induced artifacts6. In addition, it is not possible to directly record the EEG signal over the stimulated target area without using a ring-like electrode which is a questionable solution7. Thus, there is a lack of systematic studies on this topic.
So far, there is no clear evidence about the lasting effects of tACS after stimulation cessation. Only a few studies have shown weak and unclear after-effects of tACS on the motor system8. Moreover, EEG evidence is still not clear about the after-effects of tACS9. On the other hand, most tACS studies showed prominent online effects10,11,12,13,14,15,16,17,18, which are difficult to measure at a physiological level because of technical constraints. Thus, the overall goal of our method is to provide an alternative approach to test online and frequency-dependent effects of tACS on the motor cortex (M1) by delivering single pulse Transcranial Magnetic Stimulation (TMS). TMS allows researchers to &#34;probe&#34; the physiological state of the human motor cortex19. Moreover, by recording Motor Evoked Potentials (MEP) on the subject&#39;s contralateral hand, we can investigate the effects of the ongoing tACS11. This approach lets us accurately monitor changes in corticospinal excitability by measuring MEP amplitude during online electrical stimulation delivered at different frequencies in an artifact-free fashion. In addition, this approach can also test online effects of any other waveform of tES.
To demonstrate the combined tACS-TMS effects, we will show the protocol by applying 20 Hz AC stimulation over the primary motor cortex (M1) while online neuronavigated single pulse TMS is delivered interspersed by random intervals from 3 to 5 s in order to test M1 cortical excitability.

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			<td height="42" style="height:42px;width:213px;">BrainStim, high-resolution transcranial stimulator</td>
			<td style="width:136px;">E.M.S., Bologna, Italy</td>
			<td style="width:123px;">EMS-BRAINSTIM</td>
			<td style="width:157px;"></td>
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			<td height="63" style="height:63px;width:213px;">Pair of 1,5m cables for connection of conductive silicone electrodes</td>
			<td style="width:136px;">E.M.S., Bologna, Italy</td>
			<td style="width:123px;">EMS-CVBS15</td>
			<td style="width:157px;"></td>
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			<td height="42" style="height:42px;width:213px;">Reusable conductive silicone electrodes 50x50mm</td>
			<td style="width:136px;">E.M.S., Bologna, Italy</td>
			<td style="width:123px;">FIA-PG970/2</td>
			<td style="width:157px;"></td>
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			<td height="42" style="height:42px;width:213px;">Reusable spontex sponge for electrode 50x100mm</td>
			<td style="width:136px;">E.M.S., Bologna, Italy</td>
			<td style="width:123px;">FIA-PG916S</td>
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			<td height="42" style="height:42px;width:213px;">Rubber belts &#8211; 75 cm</td>
			<td style="width:136px;">E.M.S., Bologna, Italy</td>
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			<td height="42" style="height:42px;width:213px;">Plastic non traumatic button</td>
			<td style="width:136px;">E.M.S., Bologna, Italy</td>
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			<td height="42" style="height:42px;width:213px;">Brainstim</td>
			<td style="width:136px;">E.M.S., Bologna, Italy</td>
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			<td height="42" style="height:42px;width:213px;">MagPro X100 MagOption - transcranial magnetic stimulator</td>
			<td style="width:136px;">MagVenture, Farum, Denmark</td>
			<td style="width:123px;">9016E0731</td>
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			<td height="42" style="height:42px;width:213px;">8-shaped coil MC-B65-HO-2</td>
			<td style="width:136px;">MagVenture, Farum, Denmark</td>
			<td style="width:123px;">9016E0462</td>
			<td style="width:157px;"></td>
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			<td height="42" style="height:42px;width:213px;">Chair with neckrest</td>
			<td style="width:136px;">MagVenture, Farum, Denmark</td>
			<td style="width:123px;">9016B0081</td>
			<td style="width:157px;"></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:213px;">Localite TMS Navigator - Navigation platform, Premium edition</td>
			<td style="width:136px;">Localite, GmbH, Germany</td>
			<td style="width:123px;">21223</td>
			<td style="width:157px;"></td>
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			<td height="84" style="height:84px;width:213px;">Localite TMS Navigator - MR-based software, import data for morphological MRI (DICOM, NifTi)</td>
			<td style="width:136px;">Localite, GmbH, Germany</td>
			<td style="width:123px;">10226</td>
			<td style="width:157px;"></td>
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			<td height="57" style="height:57px;width:213px;">MagVenture 24.8 coil tracker, Geom 1</td>
			<td style="width:136px;">Localite, GmbH, Germany</td>
			<td style="width:123px;">5221</td>
			<td style="width:157px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Electrode wires for surface EMG</td>
			<td style="width:136px;">&#160;EBNeuro, Italy&#160;</td>
			<td style="width:123px;">6515</td>
			<td style="width:157px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Surface Electrodes for EEG/EMG</td>
			<td style="width:136px;">&#160;EBNeuro, Italy&#160;</td>
			<td style="width:123px;">6515</td>
			<td style="width:157px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:213px;">BrainAmp ExG amplifier - bipolar amplifier&#160;</td>
			<td style="width:136px;">Brain Products, GmbH, Germany</td>
			<td style="width:123px;"></td>
			<td style="width:157px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:213px;">&#160;BrainVision Recorder 1.21.0004&#160;</td>
			<td style="width:136px;">Brain Products, GmbH, Germany</td>
			<td style="width:123px;"></td>
			<td style="width:157px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Nuprep Skin Prep Gel&#160;</td>
			<td style="width:136px;">Weaver and Company, USA</td>
			<td></td>
			<td style="width:157px;"></td>
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			<td height="21" style="height:21px;width:213px;">Syringes</td>
			<td style="width:136px;"></td>
			<td style="width:123px;"></td>
			<td style="width:157px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Sticky tape</td>
			<td style="width:136px;"></td>
			<td style="width:123px;"></td>
			<td style="width:157px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">NaCl solution</td>
			<td style="width:136px;"></td>
			<td style="width:123px;"></td>
			<td style="width:157px;"></td>
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effects of transcranial alternating current stimulation on the primary motor cortex by online combined approach with transcranial magnetic stimulation

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific frequency and in turn modulate ongoing cortical oscillatory activity. This neurotool allows the establishment of a causal link between endogenous oscillatory activity and behavior. Most of the tACS studies have shown online effects of tACS. However, little is known about the underlying action mechanisms of this technique because of the AC-induced artifacts on Electroencephalography (EEG) signals. Here we show a unique approach to investigate online physiological frequency-specific effects of tACS of the primary motor cortex (M1) by using single pulse Transcranial Magnetic Stimulation (TMS) to probe cortical excitability changes. In our setup, the TMS coil is placed over the tACS electrode while Motor Evoked Potentials (MEPs) are collected to test the effects of the ongoing M1-tACS. So far, this approach has mainly been used to study the visual and motor systems. However, the current tACS-TMS setup can pave the way for future investigations of cognitive functions. Therefore, we provide a step-by-step manual and video guidelines for the procedure.

The first evidence of a tACS/TMS combined approach was shown by Kanai et al. in 2010. In that study, the authors applied tACS over the primary visual cortex (V1) and demonstrated a frequency-specific modulation of the visual cortical excitability measured by online TMS-induced phosphene perception15. A more refined version of the protocol was adopted to investigate a physiological modulation of the motor cortex excitability by Feurra et al. in 201...

Watch this Scientific Journal Video about Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation at JoVE

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Transcranial Alternating Current Stimulation (tACS) allows the modulation of cortical excitability in a frequency-specific fashion. Here we show a unique approach which combines online tACS with single pulse Transcranial Magnetic Stimulation (TMS) in order to "probe" cortical excitability by means of Motor Evoked Potentials.

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

The overall goal of this procedure is to assess modulation of ongoing effects of transcranial alternated current stimulation by means of single path transcranial magnetic path stimulation in a novel online combined approach. Transcranial electrical stimulation is a neuromodulatory technique that affects neuronal states through different current waveform. It is commonly used in neuroscience to investigate brain motor and cognitive functions.The most used approaches are based on direct current stimulation, random noise, and alternating current stimulation, which are able to induce changes in neuron activity. Both inside and outside the neurons, causing alteration of resting membrane potential and consequently modifying neuronal synaptic efficiency. TACS allow us to deliver sinusoidal external oscillatory potential in a specific frequency range.By inducing the so-called entrainment effect on the endogenous oscillatory activity. Here we are introducing a novel method which combine TACS and single path CMS during ongoing stimulation of the primary motor cortex. The method allow us to test online alternating current stimulation effects on primary motor cortex by means of single pulse TMS induced motor evoked potential.We are going to show an entire set of equipment needed to run an online combined TACS-TMS protocol by stimulation of the primary motor cortex. The list of equipment includes an electrical stimulation device which can generate electrical alternated current, for example BrainStim. Connected by two-surface saline soaked sponge electrodes size five to seven centimeters.In order to minimize skin sensation, the electrodes are completely saturated with the saline solution to keep impedance below 10 kiloOhm throughout the whole stimulation session. The target electrode is placed over the scalp corresponding to the primary motor cortex hotspot by using specific elastic straps while the reference electrode is placed over the ipsilateral shoulder by using sticky tape in a so-called monopole montage. In humans, oscillations in different frequencies reflect different behavior and cognitive processes that underlie different states of neural network activity.As typically measured by encephalography or by magneto-encephalography. Oscillations in the human brain are ranging from ultra-slow to ultra-fast oscillations. There are usually simultaneously present and can modulate and interact with each other.However, the link between oscillatory activity and brain processes has always been established by correlational methods. Therefore the issue of whether brain oscillations directly reflect fundamental mechanism in cortical information processing, or just an epiphenomenon is still unresolved. By using TACS we can causally modulate brain oscillations in a frequency-specific fashion, and in turn influence physiological neuronal activity linked to resting state, perceptual and cognitive processes.To set up TCS protocol using the BrainStim device, first check the battery status. Then stimulation parameters are manipulated by our special software. Frequency, duration, shape of the current.And transferred to the device by Bluetooth connection. By using the software, open a new session and manage a new protocol or stimulation. For example, the protocol will be named as Beta.Set the frequency of stimulation in our case it will be 20Hz, tune the waveform as sinusoidal, and set the total duration of the stimulation protocol, for example 600 seconds. Finally set the intensity of stimulation at 1 milliAmp while offset, fade in, fade out, and fade is set at zero. Then when the Bluetooth function is activated on the device, upload the protocol from the software to the stimulator.Then proceed by checking whether the protocol has been successfully transferred to the device. Now move to the TMS part. First place electrodes for a surface EMG to measure motor lock potentials by single pulse TMS.Clean the skin using a cleaning scrub under all the electrodes to achieve a low skin impedance, below 10 kiloOhm. Here we took a first dorsal interressus muscle and used and used a bipolar belly tendon montage. Put active electrode on the muscle, reference electrode on the bone, two centimeters distally and ground electrode more proximally on the arm.Motor lock potentials are measured by argentum, argentum chloride electrodes connected to the BrainAmp DC amplifier by a connector box. We are using frameless TMS navigation system LocalEye TMS navigator to achieve proper positioning of the TMS coil. First we place the tracking sensors on the front of the participant.For TMS stimulation we are using magnetic stimulator in a bi-phasic regime and an H-shaped 75 millimeter swing red stimulation coil. Then using the video of participant's structural T-one MRI, and perform a current illustration of the participant's head in the 3-D MRI head model by navigation system. After that, accurately position the coil over the primary motor area of the head, the so-called motor knob region.Then start to test single pass TMS motor lock potentials. Once an optimal hotspot for the studied hand model cortical representation is found, this location is marked for the subsequent TCS target electrode placement. Adjust carefully the first elastic strap on the head with respect to the head center's position.Then take the target electrode position with the second stripe. Once TCS electrodes are placed both on the skull and on the ipsilateral shoulder, proceed to connect them to the stimulator. Before starting the stimulation session, ensure that position of the target electrode is centered over the marked hotspot by visual inspection.Then place the TMS coil over the TCS electrodes by readjusting intensity and orientation using neuro-navigation in order to find the hotspot again. At this stage, method of the resting model threshold for this new combined TCS-TMS montage by taking into account the thickness of the target electrode. Set the intensity of stimulation along the experiment at 110%of their resting motor threshold.Most of the TCS studies showed prominent online effects, at the same time, there is still no clear evidence about long-lasting effects of TCS after stimulation cessation. Only few studies in the literature showed weak and unclear after-effects with respect to those ones who used online TCS. EG evidence is not clear about TCS after-effects either.In vivo it was shown that spike in activity is synchronized with different driving frequencies suggesting that neuronal firing can be entrained by electrically applied fields. Thus in animal models weak sinusoidal TCS entrains the discharged frequency of the widespread cortical-neuronal pulse. In humans recent evidence showed that online TCS can induce entrainment effect on the EG signature.However, the TCS EG approach is still questionable, firstly because of the AC-induced artifacts and secondly because it's not practically possible to directly capture the EG signal of the stimulated area. Such combined TCS-TMS montage allows to observe especially for the sensorimotor system the online frequency specific TCS effects on the cortical spinal output by using as a probe single pass TMS of the primary motor cortex. Thus, start the 20Hz electrical stimulation protocol while delivering online TMS single pulses interspersed by random intervals, from three to five seconds.For instance, we can show the increase of motor lock potential amplitudes during online 20Hz TCS with respect to 10Hz or sham stimulation. On this picture you can see an example of the experimental design where TMS coil is placed over the target TCS electrode. This experiment target red electrodes are placed on the left motor cortex and the right parietal cortex.The blue reference electrode is placed on the midline corresponding to PZ position of the 1020 international EG system. The coil is held on the sponge electrode placed over the left motor cortex. The human motor cortex addressed has an activity at about 20Hz.Frequent collaborators provided the crucial evidence of 20Hz activity at rest in humans by showing the online increase of the cortico-spinal excitability while stimulation was applied at 20Hz. On the graph the Y-axis represents the mean MEP amplitude as measured by TMS single pulse while the TCS stimulation was ongoing. The X-axis represents different stimulation frequencies.Only TCS delivered at 20Hz on the motor cortex increased the corticospinal output. Whereas there was no effect of the stimulation on other frequencies or other stimulation of the parietal cortex. This was the first evidence of an online frequency-specific effect of TCS on the motor cortex measured by TACS, TMS combined approach.The rationale of combining the use of TMS and TACS lies on different information that they convey. By recording motor evoked potentials on the contralateral hand to the primary motor cortex we can check the artifact-free effects of ongoing TCS. This allow us to accurately monitor changes in cortical excitability by motor evoked potentials during electrical stimulation delivered at different frequencies in an artifact-free fashion.This new approach can be used to test online effect of any other forms of transcranial electrical stimulation.

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8.9K 조회수.  National Research University Higher School of Economics. The overall goal of this procedure is to assess modulation of ongoing effects of transcranial alternated current stimulation by means of single path transcranial magnetic path stimulation in a novel online combined approach. Transcranial electrical stimulation is a neuromodulatory technique that affects neuronal states through different current waveform. It is commonly used in neuroscience to investigate brain motor and cognitive functions.The most used approaches are based on direct c...

비디오: Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation