Name: non-invasive assessment of changes in corticomotoneuronal transmission in humans
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Description: Watch this Scientific Journal Video about Non-invasive Assessment of Changes in Corticomotoneuronal Transmission in Humans at JoVE.com

This study was supported by a grant from the Swiss National Science Foundation (316030_128826).

This protocol was approved by the local ethics committee and the experiments are in accordance with the Declaration of Helsinki (1964).
1. Subject Preparation
NOTE: Subject instructions - Before starting with the experiment, instruct each subject about the purpose of the study and potential risk factors. For transcranial magnetic stimulation (TMS), medical risks include any history of epileptic seizure, mental implants in eyes and/or ...

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	<li>Lemon, R. N., Kirkwood, P. A., Maier, M. A., Nakajima, K., Nathan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+and+indirect+pathways+for+corticospinal+control+of+upper+limb+motoneurons+in+the+primate.">Direct and indirect pathways for corticospinal control of upper limb motoneurons in the primate.</a> Prog.Brain Res. , 263-279 (2004).</li><li>Jankowska, E., Padel, Y., Tanaka, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+of+pyramidal+tract+cells+to+alpha-motoneurones+innervating+hind-limb+muscles+in+the+monkey.">Projections of pyramidal tract cells to alpha-motoneurones innervating hind-limb muscles in the monkey.</a> J. 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The H-reflex conditioning procedure described here has been specifically addressed to assess acute changes in transmission over the corticomotoneuronal synapse following repetitive activation of the corticospinal pathway28. In this respect, H-reflex conditioning has highlighted that rTMS does not only affect excitability of cortical structures but also has an effect on the corticomotoneural transmission at the corticomotoneural synapse. However, this method may indeed have broader application as c...

In primates, the corticospinal tract constitutes the major descending pathway controlling voluntary actions1. The corticospinal pathway connects motor cortical areas to spinal &#945;-motoneurons via direct monosynaptic corticomotoneuronal connections and via indirect oligo- and polysynaptic connections2,3. Although the motor cortex can easily be excited non-invasively by Transcranial Magnetic Stimulation (TMS), the evoked electromyographic response to this stimulation is often difficult to interpret. The reason for this is that the compound Motor Evoked Potential (MEP) can be influenced by changes in the excitability of intracortical and corticospinal neurons, spinal interneurons and spinal &#945;-motoneurons4,5,6,7. Several noninvasive electrophysiological techniques and stimulation protocols aim at determining whether changes in corticospinal excitability and transmission are caused by changes at the cortical or spinal level. Commonly, changes in the amplitude of the electrically evoked H-reflex are used as &#39;indicative&#39; of alterations of excitability at the motoneuron pool. However, it was previously shown that the H-reflex depends not only on the excitability of the motoneuron pool but is also modulated by other factors such as presynaptic inhibition8,9 or homosynaptic post-activation depression5,10. Another limitation when comparing MEPs and H-reflexes is the disability to detect excitability changes at the interneuronal level11,12. In addition to these drawbacks, the motoneurons might be differently activated by peripheral nerve stimulation than with TMS so that changes in the motoneuronal excitability would affect these responses in a different kind of way compared to responses mediated via the corticospinal pathway13,14,15.
Another method used to separate spinal from cortical effects represents Transcranial Electrical Stimulation (TES) of the motor cortex16. Applied at low stimulation intensities, TES was argued to be unaffected by changes in cortical excitability. As both TES and TMS activate the &#945;-motoneurons via the corticospinal pathway, the comparison of magnetically and electrically evoked MEPs provides a more attractive method to draw conclusions on the cortical nature of changes in the size of the MEPs than the comparison between H-reflexes and MEPs. However, when stimulation intensity is increased, TES-evoked MEPs are also influenced by changes in cortical excitability17,18. This problem can be circumvented when electrical stimulation is not applied to the motor cortex but at the cervicomedullary junction. However, although electrical stimulation can evoke cervicomedullary motor evoked potentials (cMEPs) in upper limb and lower limb muscles, most subjects perceive electrical stimulation at the brainstem (and cortex) as extremely unpleasant and painful. A less painful alternative is to activate the corticospinal pathway at the cervicomedullary junction by use of magnetic stimulation at the inion19. It is generally accepted that Cervicomedullary Magnetic Stimulation (CMS) activates many of the same descending fibers as motor cortical TMS and that changes in cortical excitability can be detected by comparing MEPs with cMEPs19. Increases in the excitability of intracortical cells and corticomotoneuronal cells are thought to facilitate the cortically evoked MEP without a concurrent change in the cervicomedullary evoked MEP.
However, in most subjects it is impossible to obtain magnetically evoked cMEPs in the lower extremity at rest20,21. One approach to overcome this problem is to elevate the excitability of spinal motoneurons by voluntary precontracting the target muscle. However, it is well known that slight changes in contraction strength influence the size of the cMEP. Thus, it is difficult to compare different tasks. In addition, changes in the motoneuronal excitability due to pre-contraction will influence MEPs and cMEPs but not necessarily to the same extent. Finally, by comparing compound MEPs with compound cMEPs some information contained in the descending volleys is lost. This has been revealed by studies involving conditioning of the H-reflex of soleus, tibialis anterior, and carpi radialis muscles by magnetic motor cortical stimulation12,22. By combining peripheral nerve stimulation and TMS over the motor cortex with specific interstimulus intervals (ISI), it is possible to study facilitatory and inhibitory effects of the different descending volleys on the H-reflex. This technique is greatly inspired by the spatial facilitation technique used to determine transmission in neural pathways in animal experiments and may be seen as a non-invasive, indirect version of that technique23. While the H-reflex is not only important to differentiate between different fractions of the corticospinal pathway (fast versus slower corticospinal projections) it is also essential to elevate spinal excitability in a controlled and comparable way. Thus, at rest and during activity, this combination of stimulation techniques allows assessment of changes in different fractions of the corticospinal pathway with a high temporal resolution, i.e. in the fastest, presumably monosynaptic corticomotoneuronal connections and in slower oligo- and polysynaptic pathways12,22,24,25. Recently, this technique was extended by not only conditioning the H-reflex with TMS over the motor cortex (M1-conditioning) but also by additional conditioning stimulation at the cervicomedullary junction (CMS-conditioning)26. By comparing effects between M1- and CMS-conditioning, this technique allows pathway specific differentiation with a high temporal resolution and it allows interpretations to be made on cortical versus spinal mechanisms. Furthermore and most importantly with respect to the current study, this technique allows assessment of transmission at the corticomotoneural synapse when considering the early facilitation. The early facilitation of the H-reflex is in all likelihood caused by activation of direct, monosynaptic corticomotoneural projections to the spinal motoneurons12,26. To test the fastest corticospinal pathways and thus, the early facilitation, the H-reflex has to be elicited 2 to 4 ms before the TMS. The reason for this is the slightly shorter latency of the MEP (around 32 ms; see27) compared to the H-reflex (around 34 ms; see25). Eliciting the H-reflex shortly before applying TMS, leads to convergence of the ascending and fastest descending excitations at the level of the spinal motoneurons. When TMS is applied over the cervicomedullary junction, the descending volley will arrive around 3 - 4 ms earlier at the spinal motoneuron pool than after stimulation over M1. For CMS-conditioning, peripheral nerve stimulation should therefore be evoked 6 - 8 ms before the magnetic pulse. A change of the early facilitation after CMS-conditioning indicates differential transmission at the synapse between the corticospinal tract and the &#945;-motoneuron28. In the current study, this recently developed technique was used to differentiate spinal from cortical effects following low frequency repetitive TMS (rTMS). More specifically, we hypothesized that if the early facilitation with M1-conditioning is reduced following the rTMS intervention but the early facilitation following CMS-conditioning is not, the effect should be purely cortical in origin. In contrast, if the early facilitation with CMS-conditioning also changes, this alteration should be related to mechanisms taking place at the spinal level. More specifically, as the early facilitation of the H-reflex is thought to be caused by activation of direct, corticomotoneuronal projections to the spinal motoneurones12,29, a change of the CMS-and M1-conditioned H-reflex at the time of the early facilitation should indicate an altered corticomotoneuronal transmission i.e. synaptic efficacy28.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Self-adhesive EMG electrodes</td>
			<td>Blue sensor N, Ambu, Ballerup, Denmark</td>
			<td></td>
			<td>Used to record EMG signals</td>
		</tr>
		<tr>
			<td>Electrical stimulator</td>
			<td>Digitimer DS7A, Hertfordshire, UK</td>
			<td></td>
			<td>Used to elicit the soleus H-reflex</td>
		</tr>
		<tr>
			<td>Stimulating electrode</td>
			<td>Blue sensor N, Ambu, Ballerup, Denmark</td>
			<td></td>
			<td>Used to elicit the soleus H-reflex</td>
		</tr>
		<tr>
			<td>Magnetic stimulator no1</td>
			<td>Magstim Rapid2 TMS stimulator, Magstim Company Ltd., Whitland, UK</td>
			<td></td>
			<td>Used to elicit contralateral motor evoked potentials in the soleus muscle</td>
		</tr>
		<tr>
			<td>Coil no1: 90 mm figure-of-eight coil&#160;</td>
			<td>Magstim Company Ltd., Whitland, UK</td>
			<td></td>
			<td>Used to elicit contralateral motor evoked potentials in the soleus muscle</td>
		</tr>
		<tr>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>Stimulator no1 and coil no1 were used in the original publication (Taube et al. 2014; Cerebral Cortex)</td>
		</tr>
		<tr>
			<td>Magnetic stimulator no2</td>
			<td>MagPro X100 with MagOption, MagVenture A/S, Farum, Denmark</td>
			<td></td>
			<td>Used to elicit contralateral motor evoked potentials in the soleus muscle</td>
		</tr>
		<tr>
			<td>Coil no2: 95-mm focal &#8220;butterfly-shaped&#8221; coil (D-B80)&#160;</td>
			<td>MagVenture A/S, Farum, Denmark</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Stimulator no2 and coil no2 were used in the video session</td>
		</tr>
		<tr>
			<td>Magnetic stimulator no3</td>
			<td>Magstim Company Ltd., Whitland, UK</td>
			<td></td>
			<td>Used to stimulate at the cervicomedullary junction</td>
		</tr>
		<tr>
			<td>Coil no3: double-cone magnetic coil</td>
			<td>Magstim Company Ltd., Whitland, UK</td>
			<td></td>
			<td>Used to stimulate at the cervicomedullary junction</td>
		</tr>
		<tr>
			<td>Image-guided TMS navigational system no1</td>
			<td>Brainsight 2, Rouge Research, Montreal, Canada</td>
			<td></td>
			<td>Used in the original publication (Taube et al. 2014; Cerebral Cortex) to monitor coil position throughout the experiment</td>
		</tr>
		<tr>
			<td>Image-guided TMS navigational system no2</td>
			<td colspan="2">TMS Navigator SW-Version 2.0, LOCALITE GmbH, Sankt Augustin, Germany</td>
			<td>Used for the video session</td>
		</tr>
		<tr>
			<td>Literature:&#160;</td>
			<td><a name="RANGE!B15"></a></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Taube et al. 2014</td>
			<td colspan="3">Taube, W., Leukel, C., Nielsen, J. B. &#38; Lundbye-Jensen, J. Repetitive Activation of the Corticospinal Pathway by Means of rTMS may Reduce the Efficiency of Corticomotoneuronal Synapses. Cerebral cortex, doi:10.1093/cercor/bht359 (2014).</td>
		</tr>
	</tbody>
</table>

non-invasive assessment of changes in corticomotoneuronal transmission in humans

The corticospinal pathway is the major pathway connecting the brain with the muscles and is therefore highly relevant for movement control and motor learning. There exists a number of noninvasive electrophysiological methods investigating the excitability and plasticity of this pathway. However, most methods are based on quantification of compound potentials and neglect that the corticospinal pathway consists of many different connections that are more or less direct. Here, we present a method that allows testing excitability of different fractions of the corticospinal transmission. This so called H-reflex conditioning technique allows one to assess excitability of the fastest (monosynaptic) and also polysynaptic corticospinal pathways. Furthermore, by using two different stimulation sites, the motor cortex and the cervicomedullary junction, it allows not only differentiation between cortical and spinal effects but also assessment of transmission at the corticomotoneural synapse. In this manuscript, we describe how this method can be used to assess corticomotoneural transmission after low-frequency repetitive transcranial magnetic stimulation, a method that was previously shown to reduce excitability of cortical cells. Here we demonstrate that not only cortical cells are affected by this repetitive stimulation but also transmission at the corticomotoneuronal synapse at the spinal level. This finding is important for the understanding of basic mechanisms and sites of neuroplasticity. Besides investigation of basic mechanisms, the H-reflex conditioning technique may be applied to test changes in corticospinal transmission following behavioral (e.g., training) or therapeutic interventions, pathology or aging and therefore allows a better understanding of neural processes that underlie movement control and motor learning.

Occurrence of the early facilitation after M1- and CMS-conditioning
H-reflex conditioning with TMS over M1 resulted in an early facilitation that occurred around ISI -3 &#38; -4 ms. The early facilitation after CMS-conditioning occurred around 3 ms earlier (ISI -6 &#38; -7 ms, respectively). Exemplary ISI-curves of one subject are displayed in Figure 1. In the present study, the early facil...

Watch this Scientific Journal Video about Non-invasive Assessment of Changes in Corticomotoneuronal Transmission in Humans at JoVE.com

Non-invasive Assessment of Changes in Corticomotoneuronal Transmission in Humans

The aim of the present study was to assess changes in transmission at the corticomotoneuronal synapses in humans after repetitive transcranial magnetic stimulation. For this purpose, an electrophysiological method is introduced that allows assessment of pathway specific corticospinal transmission, i.e. differentiation of fast, direct corticospinal pathways from polysynaptic connections.

The corticospinal pathway is the major pathway connecting the brain with the muscles and is therefore highly relevant for movement control and motor ...

The overall goal of this neurophysiological experiment is to assess changes in transmission at the Corticomotoneuronal synapses in humans after repetitive Transcranial Magnetic Stimulation. The H-Reflex conditioning technique can help and psyche questions in the field of neuroscience, such as, better to understand basic mechanisms and sites of neuroplasticity. We were especially interested in the plasticity taking place between the corticospinal tract and the motor neurone, the Corticomotoneuronal synapse.The main advantage of this technique, compared to measure and compound potentials, is that it is possible to measure synaptic input produced by distinct Corticospinal connections to spinal motor neurons. There are at least two further advantages of conditioned H-reflexes compared to compound potentials. First, it's possible to detect influences of even weak Corticospinal volleys and second, it's possible to assess effects of longer latency Corticospinal volleys.Prior to the experiment, first screen the subject for TMS medical risks such as, history of epileptic seizures, obtain written and informed consent and inform the subject about the purpose of the experiment. Prepare the skin for surface electrode placement over the soleus muscle by shaving, disinfecting with propanol and applying light skin abrasion. Then, place surface electrodes to measure electrophysiological responses.Check the EMG signals on the screen, while the subject performs plantar and dorsiflexion. For H-Reflex conditioning, first use tape to fixate a five by five centimeter anode on the anterior aspect of the knee, just underneath the patella. Apply stimulation with square wave pulses lasting one millisecond and move the cathode on the popliteal fossa, until the optimal position for stimulation is located.Then, place a self-adhesive electrode on the skin and fix in place with tape. Prior to the experiment, first record an HM recruitment curve. Calculate H-Reflexes and M-Waves as peak to peak EMG amplitudes online in the recording software.Adjust the size of the H-Reflex to 20%of the maximum M-Wave. First, perform the calibration procedure for the neuro-navigational system. Use a figure eight coil to stimulate the subject's motor cortical area, contralateral to the placed electrodes.To find the optimal stimulation spot, first place the coil one centimeter in front of the vertex. Point that handle of the coil backwards to evoke a posterior to anterior flux of induced current in the center of the coil. Identify the position where motor evoked potentials can be evoked with minimum stimulation intensity.At this point, place the subject's head on the table and use rigid foam to prevent motion. Use an image guided TMS navigational system for a monitoring coil and head position throughout the experiment. Then, fixate the coil to a stand and the subject's head to the chair and fixate the coil with Velcro strips to the head.Next, determine the minimum intensity required to evoke motor-evoked potentials with peak to peak EMG amplitudes larger than 50 micro-volts in six out of 10 consecutive trial. This is the subject's resting motor threshold. Also place a double cone magnetic coil at the cervicomedullary junction to excite axons of the corticospinal tract.Position the coil so that the first derivative of the induced current is cranially directed and that its central position is on or near the enium. To begin, first take care to ensure that the size of the control H-Reflex stays constant throughout the experiment. If a deviation is detected, adjust the stimulation intensity prior to the consecutive trial.Now, adjust the stimulation intensity of the TMS coil over the motor cortex to between 90%and 100%of the subject's motor threshold. Also set the cervicomedullary stimulation intensity to 100%of the maximum stimulator output. Begin by conditioning the H-Reflex with stimulation over the motor cortex at rest.Apply TMS and peripheral nerve stimulation by varying the timing between the two stimuli to allow assessment of changes in Corticomotoneuronal transmission. Start with an interstimulus interval of negative five milliseconds, meaning peripheral nerve stimulation is illicited five seconds before TMS, then alter this interval in steps of milliseconds from negative five milli seconds to one millisecond. Depending on the interstimulus interval, the H-Reflex can be shifted forward in relationship to the descending volley where it can be shifted to the right, so that the slower corticospinal pathways can be tested.Vary the interval randomly from trial to trial, to eliminate bias due to order of stimuli and set the pause between stimulation trials to four seconds. Early facilitation should occur around intervals of negative four to negative two milliseconds. Next, condition the H-Reflex using magnetic stimulation over the cervicomedullary junction.Use interstimulus intervals between negative nine and negative three milliseconds in steps of one millisecond. Apply these TMS intervals over both locations together in each trial and record the resulting control H-Reflex and motor-evoked potentials. Use the control H-Reflex as a reference for the conditioned H-Reflexes and the control motor-evoked potential to ensure comparable stimulation conditions.Once pre-measurement is completed, set the stimulation intensity to 1.2 times the subject's motor threshold and apply the slow repetitive TMS intervention. Stimulate over the primary motor cortex at one hertz for 20 minutes to induce long lasting suppression of corticospinal excitability. An H-Reflex conditioning curve after M1-conditioning is displayed here while the H-Reflex conditioning curve after CMS-conditioning is shown here.Early facilitation with TMS over the primary motor cortex occurs around interstimulus intervals of negative three milliseconds. Note that the early facilitation after CMS-conditioning is occurring approximately three to four milliseconds earlier at an interstimulus interval of around negative seven milliseconds. Here, the averages of 10 traces for one representative subject before and after the rTMS intervention are displayed.It can be seen that the conditioned H-Reflexes representing the early facilitation, are reduced after both M1 and CMS-conditioning. Whereas, the control H-Reflexes remain unchanged. And here, the mean of two subjects is displayed showing the same pattern reduction in both M1 and CMS-conditioned to H-Reflexes without any change in the control H-Reflex.Once mastered, this protocol can be done in around 90 to 120 minutes. While performing this procedure, it's important to keep the H-Reflex size constant. After watching this video, you should have a good understanding of how to apply conditioning stimuli over the primary motor cortex and the cervicomedullary junction.This is used to detect the early facilitation, which represents synaptic input to spinal motor neurons mediated by the fastest corticospinal connections.

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