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 head, any diseases of the cardiovascular system, and pregnancy. Exclude all subjects affirming to one of these risk factors. Furthermore, in the experiment testing healthy individuals, exclude all subjects with neurological and/or orthopedic disease.
<ol>
	<li>Subject Placement
	<ol>
		<li>Place the subject in a chair that supports legs, trunk, and head in place. Ensure that the legs are outstretched so that the knees are extended and the peripheral nerve is closer to the skin making the nerve easier and more reliably excitable by electrical stimulation.</li>
		<li>Make sure that the subject&#39;s head is flexed, resting on a stable support surface such as a table and is secured with cushions. Ensure that the neck and the atlanto-occipital are flexed to allow stimulation of the corticospinal pathway.</li>
		<li>Position the double cone magnetic coil so that its central portion is placed on or near the inion and the first derivative of the induced current is cranially directed 19,26. Use elastic straps at the head and the trunk to ensure that this position is maintained throughout the experiment.</li>
	</ol>
	</li>
	<li>Using surface electrodes measure the electrophysiological responses by Peripheral Nerve Stimulation (PNS) and TMS.
	<ol>
		<li>Prepare the skin over the muscle belly of soleus by shaving, disinfection with propanol, and light abrasion.
		<ol>
			<li>Place self-adhesive EMG electrodes on the skin over the muscle belly of m. soleus. Place a reference electrode on the skin over the bone, e.g., on the patella or the medial malleolus.</li>
			<li>Connect all electrodes to an EMG-amplifier and finally an analogue-digital converter. Amplify EMG signals (&#215;1000), bandpass-filter (10 - 1000 Hz) and sample at 4 kHz.</li>
		</ol>
		</li>
		<li>PNS
		<ol>
			<li>For H-reflex conditioning, record H-reflexes in the soleus muscle by stimulating the posterior tibial nerve in the popliteal fossa.Apply stimulation with square-wave pulses lasting 1 ms. For stimulation, fixate an anode of 5&#160;x 5 cm with tape on the anterior aspect of the knee just underneath the patella. 
			NOTE: Stable H-reflex amplitude is a prerequisite for successful H-reflex conditioning and the least variability of all muscles can be found when recording from the soleus muscle.</li>
			<li>Move the cathode in the popliteal fossa until the best position for stimulation is found. 
			NOTE: The best position refers to recording H-reflexes in the soleus muscle with minimum stimulation intensity, without a visible M-wave in the EMG recordings at these low stimulation intensities, and without receiving any response in the antagonist m. tibialis.</li>
			<li>Avoid responses in m. tibialis muscle as those may affect results by reciprocal inhibition from Ia afferents of n. peroneus communis to spinal motoneurons of the soleus muscle. After finding the optimal location, place a self-adhesive electrode on the skin and fix the electrode with tape to ensure consistent stimulation conditions.</li>
		</ol>
		</li>
		<li>TMS
		<ol>
			<li>Stimulate the motor cortical area of the contralateral hemisphere with TMS using a figure eight coil to elicit motor evoked potentials (MEPs) in the electromyographic recordings of the soleus muscle.</li>
			<li>In order to find the optimal stimulation spot, place the coil first over the vertex and 1 cm frontal. The handle of the coil should point backwards, evoking a posterior to anterior flux of the induced current in the center of the coil.</li>
			<li>Start stimulation with low intensities of around 20 - 30% of the maximal stimulator output so that subjects get accustomed to the magnetic stimulus. Choose the pause between successive stimuli to be 4 s.</li>
			<li>After a few trials, increase stimulation intensity to around 40 - 60% of the maximal stimulator output and move the coil in the frontal-rostral and medio-lateral direction in order to find the hotspot of m. soleus. The hotspot is defined as the position where MEPs in the m. soleus can be evoked with minimum stimulation intensity.</li>
			<li>After finding the soleus hotspot, determine the resting motor threshold (1.0 MT) as the minimum intensity required to evoke MEP peak-to-peak amplitudes in the EMG larger than 50 &#181;V in six out of ten consecutive trials30. In subjects in whom the background EMG is already around 50 &#181;V, use 100 &#181;V as threshold.</li>
		</ol>
		</li>
		<li>Fixation of the Coil
		<ol>
			<li>Place the subject&#39;s head on a table (see &#34;Subject placement&#34;) and use rigid foam to prevent head movements in all directions. Fixate the coil to a stand and the subject&#39;s head to the chair.</li>
			<li>Fixate the coil with Velcro strips to the head and use an image-guided TMS navigational system for monitoring coil and head position throughout the experiment. Avoid even small movements of the coil relative to the subject&#39;s head as this changes the recruitment of neurons by TMS.</li>
		</ol>
		</li>
		<li>Magnetic Stimulation at the Cervicomedullary Junction
		<ol>
			<li>Use a double-cone magnetic coil placed at the cervicomedullary junction to excite axons of the corticospinal tract.</li>
			<li>Position the coil so that the first derivative of the induced current is cranially directed and that its central portion is on or near the inion. Apply stimulation with maximum stimulator output (100%). 
			NOTE: Even with this high stimulation intensity, the stimulus is too weak to sufficiently recruit spinal motoneurons and activate the muscles of the lower leg (i.e. m. soleus and m. tibialis anterior) in most subjects. Thus, with cervicomedullary stimulation, there is no compound potential in the surface EMG of lower leg muscles. Therefore, combine cervicomedullary simulation with the H-reflex (see &#34;3.1) to raise the excitability of the spinal motoneurons.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Premeasurement
<ol>
	<li>Adjust the size of the H-reflex (peripheral nerve stimulation)
	<ol>
		<li>For H-reflex conditioning, adjust the size of the H-reflex to 20% of the maximum M-wave (Mmax) 31 by changing the stimulation intensity of the electrical stimulator. To obtain Mmax, record an H-reflex recruitment curve. For this purpose, apply stimuli with varying stimulation intensities. The pause between successive trials is 4 s.</li>
		<li>Calculate H-reflexes and M-waves as peak-to-peak amplitudes in the EMG (in mV) online in the recording software. Take care that the size of the control H-reflex stays constant at 20% of Mmax throughout the experiment and check its size in each trial. When detecting a systematic deviation of the H-reflex size (control H-reflex is always smaller or larger as the target size), adjust the stimulation intensity just prior to the consecutive trial.</li>
	</ol>
	</li>
	<li>Adjust the stimulation intensity of TMS prior to the experiment.
	<ol>
		<li>For H-reflex conditioning at rest, set the stimulation intensity for TMS over the motor cortex to 90 - 100% of MT.Ensure that no MEP is seen in trials without PNS. 
		NOTE: The simulation intensity should be close to 100% of MT in order to ensure large effects on the conditioned H-reflex at rest so that the early facilitation can easily be detected.</li>
		<li>Adjust cervicomedullary stimulation intensity prior to the experiment. Unlike cortical stimulation, always adjust stimulation intensity for cervicomedullary stimulation to 100% of the maximum stimulator output.</li>
	</ol>
	</li>
	<li>Condition the H-reflex with magnetic stimulation over the motor cortex.
	<ol>
		<li>Apply TMS and PNS by varying the timing between the two stimuli (H-reflex conditioning) to allow assessment of changes in corticomotoneuronal transmission. To detect the early facilitation, start the conditioning protocol with an interstimulus interval (ISI) of -5 ms and alter ISIs in steps of milliseconds, from -5 - +1 ms (Figure 1B). 
		NOTE: Negative ISIs indicate that PNS is elicited before TMS, positive ISIs indicate the opposite.</li>
		<li>Vary the ISI between TMS and PNS randomly from stimulation trial to stimulation trial so that no bias due to a certain order of stimuli may arise. 
		NOTE: The &#34;early facilitation&#34; should occur around ISIs -4 ms to -2 ms when applying TMS over the motor cortex. This means that the fastest (monosynaptic corticospinal pathways) collide with the afferent volley by PNS at the spinal motoneurons at this time (see 5.2 for detecting the early facilitation).</li>
		<li>Set the pause between successive stimulation trials to 4 seconds.</li>
	</ol>
	</li>
	<li>Condition the H-reflex with magnetic stimulation over the cervicomedullary junction. 
	NOTE: Using cervicomedullary stimulation for conditioning, excitation of the corticospinal pathways is spatially closer to the spinal motoneurons than with stimulation of the motor cortex. Therefore, the ISI corresponding to the early facilitation is shifted by approximately 3 - 4 ms. As an example, the early facilitation with TMS over the primary motor cortex at -4 ms would correspond to an ISI between -7 - -8 ms with cervicomedullary stimulation.
	<ol>
		<li>Use ISIs between ISI -9 - -3 ms in steps of 1 ms for cervicomedullary conditioning. Apply ISIs for TMS over the motor cortex and TMS over the cervicomedullary junction always together in one trial and record a control H-reflex and a control MEP in this trial, too. Use the control H-reflex as a reference for the conditioned H-reflexes and the control MEP to ensure comparable stimulation conditions. Record (at least) ten trials in the pre-measurement.</li>
	</ol>
	</li>
	<li>Alternating Stimulation over the Motor Cortex and Cervicomedullary Junction
	<ol>
		<li>Apply conditioning of the SOL H-reflex by magnetic stimulation of the motor cortex (M1-conditioning; see 2.1) and by magnetic cervicomedullary stimulation (CMS-conditioning; see 2.2) in a random order during the same trial. 
		NOTE: It is recommended to alternately apply M1- and CMS-conditioning in one and the same trial in order to refer the conditioned H-reflexes to the same sample of control H-reflexes (see Figure 1).</li>
	</ol>
	</li>
</ol>
3. Intervention - Slow Repetitive TMS
<ol>
	<li>Set the stimulation intensity to 1.2 MT, which induces a long-lasting 32,33 suppression of corticospinal excitability required as H-reflex conditioning takes several minutes to accomplish. During the rTMS intervention, apply TMS over the primary motor cortex at 1 Hz for 20 min.</li>
</ol>
4. Postmeasurement
<ol>
	<li>Directly after the intervention, apply H-reflex conditioning with the same ISIs as used in the premeasurement.</li>
	<li>Use the same stimulation intensities for magnetic stimulation over M1 and the cervicomedullary junction than in the pre-measurement.</li>
	<li>Ensure that the control H-reflex has the same size as in the pre-measurement. If a systematic deviation is detected, adjust the stimulation intensity.</li>
</ol>
5. Data Processing
<ol>
	<li>Calculate all physiological responses such as H-reflexes, MEPs, and conditioned H-reflexes as peak-to-peak amplitudes of the unrectified EMG.
	<ol>
		<li>For each ISI, average ten conditioned H-reflexes for a) cortical and b) cervicomedullary stimulation. Additionally, average ten control (i.e.&#160;unconditioned) H-reflexes that serve as a reference (i.e.&#160;100%) for the conditioned H-reflexes.</li>
		<li>Consequently, express the mean amplitude of the conditioned H-reflexes for each ISI as a percentage of the mean amplitude of the control H-reflex in both the pre- and post-measurement. Take care when determining the early facilitation as this is of critical significance: 
		NOTE: As there is inter-individual variability in the occurrence of the onset of the early facilitation, determine the early facilitation in the premeasurement for each subject separately.</li>
	</ol>
	</li>
	<li>Use nonparametric Wilcoxon tests to determine the first rise of the conditioned H-reflex. For CMS-conditioning, start the tests at ISI -9 ms, for M1-conditioning search the early facilitation beginning at ISI -5 ms. Compare the amplitude of this early facilitation obtained in the pre-measurement with the amplitude of the early facilitation obtained in the post-measurement using the same ISI.</li>
	<li>Additionally, verify the early facilitation by visual inspection. 
	NOTE: After M1-conditioning, the early facilitation is most likely to occur around ISI -3 ms. Shortly after the first rise in the conditioned H-reflex, i.e.&#160;1 to 2 ms later, there is a decline in the conditioned H-reflex before it rises again. After CMS-conditioning, the early facilitation is likely to occur around ISI -7 ms, thus, around 4 ms earlier than after M1-conditioning.</li>
</ol>

<ol>
	<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. Physiol. 249, 637-667 (1975).</li><li>Maertens de Noordhout, 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=Corticomotoneuronal+synaptic+connections+in+normal+man:+an+electrophysiological+study.">Corticomotoneuronal synaptic connections in normal man: an electrophysiological study.</a> Brain. 122, 1327-1340 (1999).</li><li>Noordhout, M. D., Pepin, J. L., Gerard, P., Delwaide, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facilitation+of+responses+to+motor+cortex+stimulation:+effects+of+isometric+voluntary+contraction.">Facilitation of responses to motor cortex stimulation: effects of isometric voluntary contraction.</a> Ann.Neurol. 32, 365-370 (1992).</li><li>Nielsen, J., Morita, H., Baumgarten, J., Petersen, N., Christensen, L. O. <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+comparability+of+H-reflexes+and+MEPs.">On the comparability of H-reflexes and MEPs.</a> Electroencephalogr.Clin.Neurophysiol.Suppl. 51, 93-101 (1999).</li><li>Morita, H., 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=Differential+changes+in+corticospinal+and+Ia+input+to+tibialis+anterior+and+soleus+motor+neurones+during+voluntary+contraction+in+man.">Differential changes in corticospinal and Ia input to tibialis anterior and soleus motor neurones during voluntary contraction in man.</a> Acta Physiologica Scandinavica. 170, 65-76 (2000).</li><li>Ugawa, Y., Terao, Y., Hanajima, R., Sakai, K., Kanazawa, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facilitatory+effect+of+tonic+voluntary+contraction+on+responses+to+motor+cortex+stimulation.">Facilitatory effect of tonic voluntary contraction on responses to motor cortex stimulation.</a> Electroencephalogr.Clin.Neurophysiol. 97, 451-454 (1995).</li><li>Rudomin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selectivity+of+the+central+control+of+sensory+information+in+the+mammalian+spinal+cord.">Selectivity of the central control of sensory information in the mammalian spinal cord.</a> Adv.Exp.Med.Biol. 508, 157-170 (2002).</li><li>Eccles, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+inhibition+in+the+spinal+cord.">Presynaptic inhibition in the spinal cord.</a> Prog.Brain Res. 12, 65-91 (1964).</li><li>Hultborn, H., 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=On+the+mechanism+of+the+post-activation+depression+of+the+H-reflex+in+human+subjects.">On the mechanism of the post-activation depression of the H-reflex in human subjects.</a> Exp.Brain Res. 108, 450-462 (1996).</li><li>Burke, D., Gandevia, S. C., McKeon, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monosynaptic+and+oligosynaptic+contributions+to+human+ankle+jerk+and+H-reflex.">Monosynaptic and oligosynaptic contributions to human ankle jerk and H-reflex.</a> J Neurophysiol. 52, 435-448 (1984).</li><li>Nielsen, J., Petersen, N., Deuschl, G., Ballegaard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Task-related+changes+in+the+effect+of+magnetic+brain+stimulation+on+spinal+neurones+in+man.">Task-related changes in the effect of magnetic brain stimulation on spinal neurones in man.</a> J. Physiol. 471, 223-243 (1993).</li><li>Morita, H., Baumgarten, J., Petersen, N., Christensen, L. O., Nielsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+of+extensor-carpi-radialis+motor+units+by+transcranial+magnetic+stimulation+and+radial-nerve+stimulation+in+human+subjects.">Recruitment of extensor-carpi-radialis motor units by transcranial magnetic stimulation and radial-nerve stimulation in human subjects.</a> Exp Brain Res. 128, 557-562 (1999).</li><li>Hultborn, H., Nielsen, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H-reflexes+and+F-responses+are+not+equally+sensitive+to+changes+in+motoneuronal+excitability.">H-reflexes and F-responses are not equally sensitive to changes in motoneuronal excitability.</a> Muscle Nerve. 18, 1471-1474 (1995).</li><li>Awiszus, F., Feistner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+order+of+single+motor+units+of+the+anterior+tibial+muscle+in+man.">Recruitment order of single motor units of the anterior tibial muscle in man.</a> Electroencephalogr.Clin.Neurophysiol.Suppl. 51, 102-112 (1999).</li><li>Hess, C. W., Mills, K. R., Murray, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+in+small+hand+muscles+from+magnetic+stimulation+of+the+human+brain.">Responses in small hand muscles from magnetic stimulation of the human brain.</a> J Physiol. 388, 397-419 (1987).</li><li>Day, B. L., Thompson, P. D., Dick, J. P., Nakashima, K., Marsden, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+sites+of+action+of+electrical+and+magnetic+stimulation+of+the+human+brain.">Different sites of action of electrical and magnetic stimulation of the human brain.</a> Neurosci.Lett. 75, 101-106 (1987).</li><li>Lemon, R. N., et al., Pascual-Leone, 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=.">.</a> Handbook of Transcranial Magnetic Stimulation. , 61-77 (2002).</li><li>Taylor, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+at+the+cervicomedullary+junction+in+human+subjects.">Stimulation at the cervicomedullary junction in human subjects.</a> J. Electromyogr. Kinesiol. 16, 215-223 (2006).</li><li>Oya, T., Hoffman, B. W., Cresswell, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corticospinal-evoked+responses+in+lower+limb+muscles+during+voluntary+contractions+at+varying+strengths.">Corticospinal-evoked responses in lower limb muscles during voluntary contractions at varying strengths.</a> J. Appl. Physiol. 105, 1527-1532 (2008).</li><li>Ugawa, Y., Uesaka, Y., Terao, Y., Hanajima, R., Kanazawa, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+stimulation+of+corticospinal+pathways+at+the+foramen+magnum+level+in+humans.">Magnetic stimulation of corticospinal pathways at the foramen magnum level in humans.</a> Ann.Neurol. 36, 618-624 (1994).</li><li>Nielsen, J., Petersen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+effect+of+magnetic+brain+stimulation+accompanying+voluntary+dynamic+contraction+in+man.">Changes in the effect of magnetic brain stimulation accompanying voluntary dynamic contraction in man.</a> J. Physiol. 484, 777-789 (1995).</li><li>Baldissera, F. H. H., Illert , A. I., Brooks, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Physiology. Section 1: The nervous system, vol II. Motor control. , 509-595 (1981).</li><li>Petersen, N., Christensen, L. O. D., Nielsen, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+transcranial+magnetic+stimulation+on+the+soleus+H+reflex+during+human+walking.">The effect of transcranial magnetic stimulation on the soleus H reflex during human walking.</a> J Physiol. 513, 599-610 (1998).</li><li>Taube, W., 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=Direct+corticospinal+pathways+contribute+to+neuromuscular+control+of+perturbed+stance.">Direct corticospinal pathways contribute to neuromuscular control of perturbed stance.</a> J Appl Physiol. 101, 420-429 (2006).</li><li>Taube, W., Lundbye-Jensen, J., Schubert, M., Gollhofer, A., Leukel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+the+cortical+motor+command+for+the+initiation+of+dynamic+plantarflexion+consists+of+excitation+followed+by+inhibition.">Evidence that the cortical motor command for the initiation of dynamic plantarflexion consists of excitation followed by inhibition.</a> PLoS.One. 6, e25657 (2011).</li><li>Petersen, N., Christensen, L. O., Morita, H., Sinkjaer, T., Nielsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+a+transcortical+pathway+contributes+to+stretch+reflexes+in+the+tibialis+anterior+muscle+in+man.">Evidence that a transcortical pathway contributes to stretch reflexes in the tibialis anterior muscle in man.</a> J. Physiol. 512, 267-276 (1998).</li><li>Taube, W., Leukel, C., Nielsen, J. B., Lundbye-Jensen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repetitive+Activation+of+the+Corticospinal+Pathway+by+Means+of+rTMS+may+Reduce+the+Efficiency+of+Corticomotoneuronal+Synapses.">Repetitive Activation of the Corticospinal Pathway by Means of rTMS may Reduce the Efficiency of Corticomotoneuronal Synapses.</a> Cerebral cortex. , (2014).</li><li>Nielsen, J., Petersen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+favouring+different+descending+pathways+to+soleus+motoneurones+activated+by+magnetic+brain+stimulation+in+man.">Evidence favouring different descending pathways to soleus motoneurones activated by magnetic brain stimulation in man.</a> J Physiol. 486, 779-788 (1995).</li><li>Kujirai, T., 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=Corticocortical+inhibition+in+human+motor+cortex.">Corticocortical inhibition in human motor cortex.</a> J Physiol. 471, 501-519 (1993).</li><li>Crone, C., 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=Sensitivity+of+monosynaptic+test+reflexes+to+facilitation+and+inhibition+as+a+function+of+the+test+reflex+size:+a+study+in+man+and+the+cat.">Sensitivity of monosynaptic test reflexes to facilitation and inhibition as a function of the test reflex size: a study in man and the cat.</a> Exp.Brain Res. 81, 35-45 (1990).</li><li>Maeda, F., Keenan, J. P., Tormos, J. M., Topka, H., 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=Interindividual+variability+of+the+modulatory+effects+of+repetitive+transcranial+magnetic+stimulation+on+cortical+excitability.">Interindividual variability of the modulatory effects of repetitive transcranial magnetic stimulation on cortical excitability.</a> Exp.Brain Res. 133, 425-430 (2000).</li><li>Fitzgerald, P. B., Brown, T. L., Daskalakis, Z. J., Chen, R., Kulkarni, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intensity-dependent+effects+of+1+Hz+rTMS+on+human+corticospinal+excitability.">Intensity-dependent effects of 1 Hz rTMS on human corticospinal excitability.</a> Clin.Neurophysiol. 113, 1136-1141 (2002).</li><li>Nielsen, J., Petersen, N., Ballegaard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Latency+of+effects+evoked+by+electrical+and+magnetic+brain+stimulation+in+lower+limb+motoneurones+in+man.">Latency of effects evoked by electrical and magnetic brain stimulation in lower limb motoneurones in man.</a> J. Physiol. 484, 791-802 (1995).</li><li>Leukel, C., 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=Changes+in+corticospinal+transmission+following+8weeks+of+ankle+joint+immobilization.">Changes in corticospinal transmission following 8weeks of ankle joint immobilization.</a> Clin neurophysiol. , (2014).</li><li>Schubert, 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=Balance+training+and+ballistic+strength+training+are+associated+with+task-specific+corticospinal+adaptations.">Balance training and ballistic strength training are associated with task-specific corticospinal adaptations.</a> Eur J Neurosci. 27, 2007-2018 (2008).</li><li>Taube, W., 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=Cortical+and+spinal+adaptations+induced+by+balance+training:+correlation+between+stance+stability+and+corticospinal+activation.">Cortical and spinal adaptations induced by balance training: correlation between stance stability and corticospinal activation.</a> Acta Physiol (Oxf). 189, 347-358 (2007).</li><li>Di Lazzaro, V., 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=Effects+of+voluntary+contraction+on+descending+volleys+evoked+by+transcranial+stimulation+in+conscious+humans.">Effects of voluntary contraction on descending volleys evoked by transcranial stimulation in conscious humans.</a> J. Physiol. 508, 625-633 (1998).</li><li>Edgley, S. A., Eyre, J. A., Lemon, R. N., Miller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+activation+of+corticospinal+neurons+and+spinal+motor+neurons+by+magnetic+and+electrical+transcranial+stimulation+in+the+lumbosacral+cord+of+the+anaesthetized+monkey.">Comparison of activation of corticospinal neurons and spinal motor neurons by magnetic and electrical transcranial stimulation in the lumbosacral cord of the anaesthetized monkey.</a> Brain. 120 (Pt 5), 839-853 (1997).</li><li>Di Lazzaro, V., 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=Effects+of+voluntary+contraction+on+descending+volleys+evoked+by+transcranial+electrical+stimulation+over+the+motor+cortex+hand+area+in+conscious+humans.">Effects of voluntary contraction on descending volleys evoked by transcranial electrical stimulation over the motor cortex hand area in conscious humans.</a> Exp.Brain Res. 124, 525-528 (1999).</li><li>Taube, W., 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=Brain+activity+during+observation+and+motor+imagery+of+different+balance+tasks:+An+fMRI+study.">Brain activity during observation and motor imagery of different balance tasks: An fMRI study.</a> Cortex. 64, 102-114 (2015).</li></ol>

The authors have nothing to disclose.

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><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 #1</td><td>Magstim Rapid&lt;sup&gt;2&lt;/sup&gt; 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 #1: 90 mm figure-of-eight coil&amp;nbsp;</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>&amp;nbsp; &amp;nbsp;</td><td>&amp;nbsp; &amp;nbsp;</td><td>&amp;nbsp; &amp;nbsp;</td><td>Stimulator #1 and coil #1 were used in the original publication (Taube et al. 2014; Cerebral Cortex)</td></tr><tr><td>Magnetic stimulator #2</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>Co#2: 95 mm focal &amp;ldquo;butterfly-shaped&amp;rdquo; coil (D-B80)&amp;nbsp;</td><td>MagVenture A/S, Farum, Denmark</td><td></td><td></td></tr><tr><td colspan="4">Stimulator no2 and coil no2 were used in the video session</td></tr><tr><td>Magnetic stimulator #3</td><td>Magstim Company Ltd., Whitland, UK</td><td></td><td>Used to stimulate at the cervicomedullary junction</td></tr><tr><td>Coil #3: 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 #1</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 #2</td><td>TMS Navigator SW-Version 2.0, LOCALITE GmbH, Sankt Augustin, Germany</td><td>Used for the video session</td><td></td></tr><tr><td>Literature:&amp;nbsp;</td><td>&lt;a name=&quot;RANGE!B15&quot;&gt;&lt;/a&gt;</td><td></td><td></td></tr><tr><td>Taube et al. 2014</td><td>Taube, W., Leukel, C., Nielsen, J. B. &amp;amp; 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><td></td><td></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 ...

non-invasive-assessment-changes-corticomotoneuronal-transmission

Research

JoVE Journal

Neuroscience

7.8K Views.  University of Fribourg (Switzerland).  Het doel van de huidige studie was om veranderingen in transmissie bij de corticomotoneuronale synaptes in mensen na de herhalende transcraniale magnetische stimulatie te beoordelen. Hiervoor wordt een elektrofysiologische methode geïntroduceerd die het mogelijk maakt om de path-specifieke corticospinale transmissie te beoordelen, dwz de differentiatie van snelle, directe corticospinale pathes van polysynaptische verbindingen. 

Video: Non-invasive Assessment of Changes in Corticomotoneuronal Transmission in Humans

Corticospinal Excitability Modulation During Action Observation

This study used the transcranial magnetic stimulation/motor evoked potential (TMS/MEP) technique to pinpoint when the automatic tendency to mirror someone else's action becomes anticipatory simulation of a complementary act. TMS was delivered to the left primary motor cortex corresponding to the hand to induce the highest level of MEP activity from the abductor digiti minimi (ADM; the muscle serving little finger abduction) as well as the first dorsal interosseus (FDI; the muscle serving index finger flexion/extension) muscles. A neuronavigation system was used to maintain the position of the TMS coil, and electromyographic (EMG) activity was recorded from the right ADM and FDI muscles. Producing original data with regard to motor resonance, the combined TMS/MEP technique has taken research on the perception-action coupling mechanism a step further. Specifically, it has answered the questions of how and when observing another person's actions produces motor facilitation in an onlooker's corresponding muscles and in what way corticospinal excitability is modulated in social contexts.

Single-pulse transcranial magnetic stimulation over the primary motor cortex, neuronavigation, and registration of electromyographic activity of hand muscles were used in this study to explore corticospinal excitability while participants were observing action sequences.

This study used the transcranial magnetic stimulation/motor evoked potential (TMS/MEP) technique to pinpoint when the automatic tendency to mirror someone ...

Behavior

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Medicine

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques &#34;by threshold tracking&#34; expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.
The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.
Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

Bilateral Assessment of the Corticospinal Pathways of the Ankle Muscles Using Navigated Transcranial Magnetic Stimulation

Distal leg muscles receive neural input from motor cortical areas via the corticospinal tract, which is one of the main motor descending pathway in humans and can be assessed using transcranial magnetic stimulation (TMS). Given the role of distal leg muscles in upright postural and dynamic tasks, such as walking, a growing research interest in the assessment and modulation of the corticospinal tracts relative to the function of these muscles has emerged in the last decade. However, methodological parameters used in previous work have varied across studies making the interpretation of results from cross-sectional and longitudinal studies less robust. Therefore, use of a standardized TMS protocol specific to the assessment of leg muscles&#39; corticomotor response (CMR) will allow for direct comparison of results across studies and cohorts. The objective of this paper is to present a protocol that provides the flexibility to simultaneously assess the bilateral CMR of two main ankle antagonistic muscles, the tibialis anterior and soleus, using single pulse TMS with a neuronavigation system. The present protocol is applicable while the examined muscle is either fully relaxed or isometrically contracted at a defined percentage of maximum isometric voluntary contraction. Using each subject&#39;s structural MRI with the neuronavigation system ensures accurate and precise positioning of the coil over the leg cortical representations during assessment. Given the inconsistency in CMR derived measures, this protocol also describes a standardized calculation of these measures using automated algorithms. Though this protocol is not conducted during upright postural or dynamic tasks, it&#160;can be used to assess bilaterally any pair of leg muscles, either antagonistic or synergistic, in both neurologically intact and impaired subjects.

The present protocol describes the simultaneous, bilateral assessment of the corticomotor response of the tibialis anterior and soleus during rest and tonic voluntary activation using a single pulse transcranial magnetic stimulation and neuronavigation system.

Distal leg muscles receive neural input from motor cortical areas via the corticospinal tract, which is one of the main motor descending pathway in humans ...

In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological characteristics in the intact spinal network and holds significant advantages relative to classical in vitro or extracellular recording techniques. An advantage of in vivo intracellular recordings is that this method can be performed on adult animals with a fully mature nervous system, and therefore many observed physiological mechanisms can be translated to practical applications. In this methodological paper, we describe this procedure combined with externally applied constant current stimulation, which mimics polarization processes occurring within spinal neuronal networks. Trans-spinal direct current stimulation (tsDCS) is an innovative method increasingly used as a neuromodulatory intervention in rehabilitation after various neurological injuries as well as in sports. The influence of tsDCS on the nervous system remains poorly understood and the physiological mechanisms behind its actions are largely unknown. The application of the tsDCS simultaneously with intracellular recordings enables us to directly observe changes of motoneuron membrane properties and characteristics of rhythmic firing in response to the polarization of the spinal neuronal network, which is crucial for the understanding of tsDCS actions. Moreover, when the presented protocol includes the identification of the motoneuron with respect to an innervated muscle and its function (flexor versus extensor) as well as the physiological type (fast versus slow) it provides an opportunity to selectively investigate the influence of tsDCS on identified components of spinal circuitry, which seem to be differently affected by polarization. The presented procedure focuses on surgical preparation for intracellular recordings and stimulation with an emphasis on the steps which are necessary to achieve preparation stability and reproducibility of results. The details of the methodology of the anodal or cathodal tsDCS application are discussed while paying attention to practical and safety issues.

This protocol describes in vivo intracellular recording of rat lumbar motoneurons with simultaneous trans-spinal direct current stimulation. The method enables us to measure membrane properties and to record rhythmic firing of motoneurons before, during and after anodal or cathodal polarization of the spinal cord.

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological ...

Assessing Corticospinal Excitability During Goal-Directed Reaching Behavior

Reaching is a widely studied behavior in motor physiology and neuroscience research. While reaching has been examined using a variety of behavioral manipulations, there remain significant gaps in the understanding of the neural processes involved in reach planning, execution, and control. The novel approach described here combines a two-dimensional reaching task with transcranial magnetic stimulation (TMS) and concurrent electromyography (EMG) recording from multiple muscles. This method allows for the noninvasive detection of corticospinal activity at precise time points during the unfolding of reaching movements. The example task code includes a delayed response reaching task with two possible targets displayed &#177; 45&#176; off the midline. Single pulse TMS is delivered on the majority of task trials, either at the onset of the preparatory cue (baseline) or 100 ms prior to the imperative cue (delay). This sample design is suitable for investigating changes in corticospinal excitability during reach preparation. The sample code also includes a visuomotor perturbation (i.e., cursor rotation of &#177; 20&#176;) to investigate the effects of adaptation on corticospinal excitability during reach preparation. The task parameters and TMS delivery can be adjusted to address specific hypotheses about the state of the motor system during reaching behavior. In the initial implementation, motor evoked potentials (MEPs) were successfully elicited on 83% of TMS trials, and reach trajectories were recorded on all trials.

Reaching is a fundamental skill that allows humans to interact with the environment. Several studies have aimed to characterize reaching behavior using a variety of methodologies. This paper offers an open-source application of transcranial magnetic stimulation to assess the state of corticospinal excitability in humans during reaching task performance.

Reaching is a widely studied behavior in motor physiology and neuroscience research. While reaching has been examined using a variety of behavioral ...

Open-Source Real-Time Closed-Loop Electrical Threshold Tracking for Translational Pain Research

Nociceptors are a class of primary afferent neurons that signal potentially harmful noxious stimuli. An increase in nociceptor excitability occurs in acute and chronic pain conditions. This produces abnormal ongoing activity or reduced activation thresholds to noxious stimuli. Identifying the cause of this increased excitability is required for the development and validation of mechanism-based treatments. Single-neuron electrical threshold tracking can quantify nociceptor excitability. Therefore, we have developed an application to allow such measurements and demonstrate its use in humans and rodents. APTrack provides real-time data visualization and action potential identification using a temporal raster plot. Algorithms detect action potentials by threshold crossing and monitor their latency after electrical stimulation. The plugin then modulates the electrical stimulation amplitude using an up-down method to estimate the electrical threshold of the nociceptors. The software was built upon the Open Ephys system (V0.54) and coded in C++ using the JUCE framework. It runs on Windows, Linux, and Mac operating systems. The open-source code is available (<a data-saferedirecturl="https://www.google.com/url?q=https://github.com/Microneurography/APTrack&amp;source=gmail&amp;ust=1680789829274000&amp;usg=AOvVaw0nMk9J_EI2jUn8SlmC5JJm" href="https://github.com/Microneurography/APTrack" target="_blank">https://github.com/Microneurography/APTrack</a>). The electrophysiological recordings were taken from nociceptors in both a mouse skin-nerve preparation using the teased fiber method in the saphenous nerve and in healthy human volunteers using microneurography in the superficial peroneal nerve. Nociceptors were classified by their response to thermal and mechanical stimuli, as well as by monitoring the activity-dependent slowing of the conduction velocity. The software facilitated the experiment by simplifying the action potential identification through the temporal raster plot. We demonstrate real-time closed-loop electrical threshold tracking of single-neuron action potentials during&#160;in vivo&#160;human microneurography, for the first time, and during&#160;ex vivo&#160;mouse electrophysiological recordings of C-fibers and A&#948;-fibers. We establish proof of principle by showing that the electrical threshold of a human heat-sensitive C-fiber nociceptor is reduced by heating the receptive field. This plugin enables the electrical threshold tracking of single-neuron action potentials and allows the quantification of changes in nociceptor excitability.

APTrack is a software plugin developed for the Open Ephys platform that enables real-time data visualization and the closed-loop electrical threshold tracking of neuronal action potentials. We have successfully used this in microneurography for human C-fiber nociceptors and mouse C-fiber and A&#948;-fiber nociceptors.

Nociceptors are a class of primary afferent neurons that signal potentially harmful noxious stimuli. An increase in nociceptor excitability occurs in ...

Non-invasive Assessment of Changes in Corticomotoneuronal Transmission in Humans

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video