Neurophysiological characteristics of human leg muscle action potentials evoked by transcutaneous magnetic stimulation of the spine

Bioelectromagnetics34:200^210 (2013)

NeurophysiologicalCharacteristicsofHumanLegMuscleActionPotentialsEvokedbyTranscutaneous

Magnetic Stimulationof theSpine

MariaKnikou1,2,3,4*1Department of PhysicalTherapy, College of Staten Island, Staten Island, NewYork

2GraduateCenter/CUNY, NewYork, NewYork3NorthwesternUniversityFeinbergSchoolofMedicine, Chicago, Illinois

4SensoryMotorPerformanceProgram,Rehabilitation Institute of Chicago,Chicago, Illinois

The objectives of this study were to establish the neurophysiological properties of the compoundmuscle action potentials (CMAPs) evoked by transcutaneous magnetic stimulation of the spine(tsMSS) and the effects of tsMSS on the soleus H-reflex. In semi-prone seated subjects with trunksemi-flexed, the epicenter of a figure-of-eight magnetic coil was placed at Thoracic 10 with thehandle on the midline of the vertebral column. The magnetic stimulator was triggered by mono-phasic single pulses of 1 ms, and the intensity ranged from 90% to 100% of the stimulator outputacross subjects. CMAPs were recorded bilaterally from ankle and knee muscles at the interstimu-lus intervals of 1, 3, 5, 8, and 10 s. The CMAPs evoked were also conditioned by posterior tibialand common peroneal nerve stimulation at a conditioning-test (C-T) interval of 50 ms. The soleusH-reflex was conditioned by tsMSS at the C-T intervals of 50, 20, �20, and �50 ms. The ampli-tude of the CMAPs was not decreased when evoked at low stimulation frequencies, excitationof group I afferents from mixed peripheral nerves in the leg affected the CMAPs in a non-somatotopical neural organization pattern, and tsMSS depressed soleus H-reflex excitability.These CMAPs are likely due to orthodromic excitation of nerve motor fibers and antidromicdepolarization of different types of afferents. The latency of these CMAPs may be utilized toestablish the spine-to-muscle conduction time in central and peripheral nervous system disordersin humans. tsMSS may constitute a non-invasive modality to decrease spinal reflex hyperexcit-ability and treat hypertonia in neurological disorders. Bioelectromagnetics 34:200–210, 2013.� 2012 Wiley Periodicals, Inc.

Key words: H-reflex; low-frequency depression; magnetic stimulation; spine; multisegmentalresponses; thoracolumbar region

INTRODUCTION

Transcranial magnetic stimulation (TMS) overthe primary motor cortex, a modality introducedmore than 20 years ago [Barker et al., 1985], hasbeen utilized extensively to study corticomotorexcitability in health and disease [Berardelli et al.,1991; Rothwell, 1991; Rossini et al., 1994; Edgleyet al., 1997; Di Lazzaro et al., 2008; Knikou, 2012].TMS produces compound muscle action potentials(CMAPs), known as motor evoked potentials(MEPs), in the contralateral limb muscles [Di Laz-zaro et al., 1998; Rothwell et al., 1999]. In additionto TMS of the primary motor cortex, TMS deliveredover the cervical or thoracic spine produces CMAPsin upper and lower limb muscles [Ugawa et al.,1989; Cros et al., 1990; Mills et al., 1993]. The laten-cy of these CMAPs remains unchanged when themagnetic coil moves rostrocaudally or away fromthe midline of the spine [Ugawa et al., 1989;

Chokroverty et al., 1991] and at increased stimula-tion intensities [Ugawa et al., 1989], suggesting thatthe same neural pathways are activated regardless of

Grant sponsor: Professional Staff Congress of the City Universityof New York (63159-00-41).

Conflict of interest: The author has no financial interest and noconflicts of interest to report.

*Correspondence to: Maria Knikou, Department of PhysicalTherapy, College of Staten Island/Graduate Center, City Univer-sity of New York, 2800 Victory Blvd, Bldg 5N-207, StatenIsland, NY 10314. E-mail: [email protected]

Received for review 17 January 2012; Accepted 13 October2012

DOI 10.1002/bem.21768Published online 28 November 2012 in Wiley Online Library(wileyonlinelibrary.com).

� 2012WileyPeriodicals,Inc.

the coil’s position and stimulation intensity strength.CMAPs in upper and lower limb muscles canalso be evoked following transcutaneous electricstimulation of the cervicothoracic and/or thoracolum-bar spine (tsESS) in humans while at rest [Millsand Murray, 1986; Maertens de Noordhout et al.,1988; Sabbahi and Sengul, 2011]. The site ofactivation by electric stimulation appears to be simi-lar to that of magnetic stimulation [Ugawa et al.,1989].

CMAPs induced by tsESS have been describedto be susceptible to neural phenomena and inhibitorymechanisms similar to those known for the mono-synaptic soleus Hoffmann (H)-reflex [Knikou, 2008].For example, the soleus H-reflex in humans isprofoundly depressed following Achilles tendonvibration and activation of antagonist motoneurons[Crone et al., 1987; Abbruzzese et al., 2001; Knikou,2008], while it is modulated in a phase-dependentpattern during human walking [Capaday and Stein,1987; Knikou et al., 2009, 2011]. Similarly, volun-tary contraction of the soleus muscle enhanced thesoleus CMAPs, while dorsi flexion of the footor vibration of the Achilles tendon at 50 Hzreduced their amplitude [Maertens de Noordhoutet al., 1988]. Further, electrically induced CMAPsincreased or decreased when the corresponding mus-cle was active or relaxed during human walking[Courtine et al., 2007].

The aforementioned support the idea that tsESScan potentially influence spinal neuronal circuitscritical for human movement and muscle tone.This means that these CMAPs could potentially beutilized for diagnostic and/or prognostic purposes inneurological disorders. However, the neurophysiolog-ical properties of these CMAPs are poorly under-stood and, to best of our knowledge, no study hasexamined their susceptibility to low-frequencydepression and how this type of stimulation affectsspinal reflex excitability. In the current study, weutilized transcutaneous magnetic stimulation of thespine (tsMSS) in order to eliminate the possibilityof electrons flowing in the wires of the stimulatingelectrodes, which are transferred to an ion flow atthe electrode tissue interface [Barker, 1991], andthus counteract a possible depression of the soleusH-reflex by skin afferents [Knikou, 2007]. Accord-ingly, the purpose of this study was to establish thebehavior of the CAMPs evoked by tsMSS at lowstimulation frequencies, the relationship of theirlatency and shape to motor responses (M-waves) andH-reflexes, and the interaction with sensory afferentsfrom the legs. Lastly, we established the effects oftsMSS on the soleus H-reflex.

MATERIALS AND METHODS

Subjects

The experimental protocol was approved by theGraduate Center of the City University of New York(New York, NY, USA) Institutional Review Boardand was conducted in compliance with the Declara-tion of Helsinki. Each subject signed an informedconsent form before participating in the study. Tenadults (7 male and 3 female) free of any neuromus-cular or orthopedic disorders and between the ages of21–42 years (28 � 7.4) participated in the study.

Electromyographic (EMG) Recordings

Following standard skin preparation, single dif-ferential bipolar surface EMG electrodes (Bagnoli-8system; Delsys, Boston, MA) were placed on thesoleus (SOL), tibialis anterior (TA), peroneus longus(PL), and rectus femoris (RF) muscles and weresecured with 3M Tegaderm transparent film (3M,St. Paul, MN). All EMG signals were filtered with acut-off frequency of 20–1000 Hz (1401 plus runningSpike2 software; Cambridge Electronic Design,Cambridge, UK).

Posterior Tibial Nerve Stimulation

The soleus H-reflex was evoked by stimulationof the right posterior tibial nerve at the popliteal fossawith a square pulse stimuli of 1 ms duration deliv-ered by a constant current stimulator (Model DS7A;Digitimer, Welwyn Garden City, Hertfordshire, UK)[Knikou and Taglianetti, 2006; Knikou, 2008]. Thecathode (stainless steel electrode of 4 cm diameter)was placed proximal to the patella. A stainless steel,hand-held, monopolar electrode was used as a probeto determine the most optimal stimulation site, whichcorresponded to the site at which primary musclespindle ending afferents (Ia) could selectively beexcited at low stimulation intensities with M-wavesbeing absent. Then, the monopolar electrode wasreplaced with a permanent electrode (N-10-A, Medi-cotest, Ølstykee, Denmark), which was held underconstant pressure throughout the experiment. Theposterior tibial nerve was stimulated at intensitiesthat ranged from 5.3 to 30.5 mA (17 � 2.2) acrosssubjects. These intensities correspond to soleusM-waves that ranged from 3% to 5% of the maximalM-wave (Mmax) across subjects.

Common Peroneal Nerve Stimulation

The right common peroneal nerve was stimulat-ed with a single shock of 1 ms duration, generated

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by a constant current stimulator (DS7A), and deliv-ered with a bipolar stainless steel electrode placeddistal to the head of the fibula [Knikou and Mummi-disetty, 2011]. The optimal stimulation site wasselected based on the following criteria: (1) the TAmotor threshold was lower than that of the peronealmuscles, and (2) at increased levels of stimulationintensities, ankle eversion and peroneus and extensordigitorum muscle activity were absent. When thestimulation site for the common peroneal nerve wasidentified, the bipolar electrode was stabilized andsecured with an athletic wrap. The stimulus was de-livered at 0.2 Hz at a TA motor threshold that wasdetermined by the presence of a small amplitude TAM-wave. The common peroneal nerve was stimulatedat intensities that ranged from 14.3 to 32 mA(21.8 � 2.1) across subjects; these intensities corre-sponded to the TA M-wave motor threshold.

Experimental Protocol

Subjects were seated semi-prone with the trunksemi-flexed1 on a Biodex accessory chair (Model870–170; Biodex Medical Systems, Shirley, NY),with hips at 110–1208, knees at 100–1258, ankles at908, and both feet and arms supported. All subjectswere asked to relax during the experiments, and notrotate their head or move their arms or legs. Initially,the Thoracic 9 (Th 9) spinous process was identifiedthrough palpation. The epicenter of a figure-of-eightcoil with a diameter of 70 mm, connected to aMagstim 200 stimulator (Magstim, Carmarthenshire,UK), was placed at the intervertebral space betweenthe Th 9 and Thoracic 10 (Th 10) spinous processesso the current would flow in an anti-clockwise direc-tion (Fig. 1A). The placement of the magnetic coilwith the current flowing in a clockwise direction didnot induce a difference in the amplitude of theCMAPs. The handle of the figure-of-eight coil wasabove and parallel with the midline of the vertebralcolumn. The epicenter of the coil, in which the cur-rent density is maximal compared to other parts ofthe coil, was aligned exactly with the midline of thevertebral column. Because the trunk was semi-flexed,potential existence of lower-trunk lordosis was coun-teracted and the epicenter of the coil was always incontact with the skin of the spine while its outeredges were in contact with the paraspinal muscles.The position of the coil was maintained by the

experimenter, and the coil was placed in the sameorientation in all subjects.

The magnetic stimulator was triggered by ananalog-to-digital acquisition system with customizedscripts written in Spike2 with single monophonicpulses of 1 ms duration at 0.2 Hz. With the figure-of-eight coil positioned as previously described, thedevice stimulation output was set at 50% and wasincreased by 5% to establish the stimulation intensitythat CMAPs could be induced in most of the legmuscles. At 90% of the device stimulation output,CMAPs were evoked only in few leg muscles, so themagnetic coil was moved caudally by one vertebra(i.e., from Th 9 to Th 10) while maintaining theexact position. For all subjects, CMAPs followingtsMSS were recorded with the epicenter of thefigure-of-eight coil placed at Th 10; tsMSS was setat 90–100% of the stimulator device output acrosssubjects. No pain or discomfort was reported by thesubjects upon stimulation.

After the stimulation sites were established,CMAPs were recorded randomly at the interstimulusintervals of 1, 3, 5, 8, and 10 s with subjects seatedsemi-prone in order to establish their susceptibilityto low-frequency stimulation. At each interstimulusinterval, 10 CMAPs were recorded. When the sus-ceptibility of CMAPs to low-frequency stimulationwas not examined, tsMSS was always delivered at0.2 Hz. The CMAPs evoked by tsMSS were condi-tioned by low-threshold stimulation of the commonperoneal and/or posterior tibial nerves at the condi-tioning-test (C-T) interval of 50 ms [Deletis et al.,1992] (Fig. 1A,Bii). Shorter C-T intervals could notbe tested because the appearance of CMAPs on theEMG was contaminated by the stimulus artifact fromthe conditioning peripheral nerve stimulation.

Lastly, the Mmax of the soleus muscle wasevoked, measured online as the peak-to-peak ampli-tude, and saved for offline analysis. The stimulusintensity was adjusted to evoke control H-reflexes onthe ascending part of the soleus H-reflex recruitmentcurve that ranged from 20% to 40% of the Mmax

across subjects [Knikou, 2008]. Twenty reflexeselicited at 0.2 Hz were recorded at this stimulationintensity. The soleus H-reflex was conditioned bytsMSS at C-T intervals of 50, 20, �20, and �50 mswith subjects seated semi-prone (Fig. 1Biii). Nega-tive C-T intervals denoted that tsMSS was deliveredafter posterior tibial nerve stimulation.

Data Analysis

All recorded responses were measured as thearea under the full-wave rectified waveform. Foreach subject, CMAPs at the interstimulus intervals of

1Trunk flexion removes pressure from the intervertebral foramenspace, while CMAPs in the TA after L5 nerve root stimulation areenhanced when compared to those observed during trunk extension[Morishita et al., 2006].

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1, 3, 5, and 8 s were expressed as a percentage of themean amplitude of the associated CMAP evoked ev-ery 10 s. The CMAPs were then grouped across sub-jects based on the interstimulus interval and muscle,and a multiple analysis of variance (MANOVA) wasconducted to establish statistically significant differ-ences of the CMAPs across the interstimulus inter-vals tested. For each subject, the conditioned CMAPswith low-threshold common peroneal and posteriortibial nerve stimulation at the C-T interval of 50 mswere expressed as a percentage of the associatedcontrol CMAPs. The conditioned CMAPs were thengrouped across subjects based on the muscle, and aMANOVA was applied to the pooled data.

The soleus H-reflex conditioned by tsMSS wasexpressed as a percentage of the mean size of thecontrol H-reflex, for each subject separately. The

mean amplitude of the conditioned soleus H-reflexfrom each subject was grouped based on the C-T in-terval, and a one-way ANOVA along with Bonferronitests were conducted to establish statistically signifi-cant differences of the conditioned H-reflexes acrossC-T intervals. This analysis was also conducted forthe corresponding M-waves, which were expressedas a percentage of the Mmax. Mean amplitudes arereported along with the standard error of means(SEM).

RESULTS

In Figure 2A,B, representative non-rectifiedwaveform averages (n ¼ 10 at 0.2 Hz) of CMAPsrecorded from two subjects are indicated. Note thatthe shape of the CMAPs recorded from the right (R)

Fig. 1. A: Top schema indicates the position of the figure-of-eight magnetic coil with respect tothevertebralcolumn. tsMSSwasdeliveredalone or in combinationwith the stimulationof commonperoneal or posterior tibial nerves at low intensities, evoking monosynaptic excitation of soleusand tibialis anteriormotoneurons (bottom schema).B: Schematic illustration of all stimulation pro-tocols conducted in this study. Graph ii shows the relationship between the conditioning stimuluspulse delivered to theperipheralnerves and the timingof tsMSS (control stimulus).Graph iii showsthe relationship between the conditioning magnetic pulse delivered to the spine (tsMSS) and thetiming of posterior tibial nerve stimulation (control stimulus). CP, common peroneal; PT, posteriortibial; SOLMN, soleusmotoneurons;TAMN, tibialisanteriormotoneurons.

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and left (L) TA, SOL, and PL muscles was triphasic,while the shape of the CMAPs recorded from theR/L RF muscle was polyphasic. The first and secondcomponent of the CMAP had a mean latency of9.49 � 0.46 and 19.39 � 0.9 ms for the R RF,respectively, and 10.11 � 0.35 and 17.38 � 3.53 msfor the L RF, respectively. The relationship betweenthe SOL CMAP, soleus H-reflex, and maximal M-wave latency from two subjects is depicted inFigure 3A,B. The soleus H-reflex was prolonged by12 ms compared to the SOL CMAP latency in thesetwo subjects, and by 9.5 � 2.48 ms across all sub-jects. The mean latency of CMAPs estimated fromall subjects is indicated in Figure 2C. In this graph,the early component from the R/L RF was notincluded.

The overall (from all subjects) amplitude ofCMAPs recorded at the interstimulus intervals of 1,3, 5, and 8 s as a percentage of the mean amplitudeof the associated CMAP evoked every 10 s is indi-cated in Figure 4. The first component of the CMAPsin the R/L RF muscle were measured separately andwere not included in the normalized averages shownin Figure 4. A one-way ANOVA along with the posthoc Bonferroni test showed that the R SOL CMAPamplitude remained unchanged at different stimula-tion frequencies (F3,20 ¼ 0.033, P ¼ 0.9). The sameresult was found for CMAPs recorded from the R/L

RF, SOL, and TA, and R PL muscles. A repeatedMANOVA at 7 � 4 levels (7 muscles � 4 interstim-ulus intervals) showed that the amplitude of theCMAPs did not vary across the different C-T inter-vals tested (F ¼ 0.187, P ¼ 0.89).

The overall effects of posterior tibial and com-mon peroneal nerve stimulation on the amplitude ofthe CMAPs are indicated in Figure 5A,B, respective-ly. The conditioned CMAPs for all muscles are indi-cated for the C-T interval of 50 ms. Low-thresholdposterior tibial nerve stimulation significantly en-hanced the amplitude of the CMAPs recorded fromthe ipsilateral (right) R RF, R TA, and R PL muscles(Fig. 5A) when compared to control values(P < 0.05). In addition, the contralateral (left) TAresponse was enhanced. However, different effectswere observed following low-threshold commonperoneal nerve stimulation. The CMAPs increased inthe R RF, and decreased in the R TA and PL muscles(Fig. 5B).

The effects of tsMSS at the C-T interval of50 ms on the average soleus H-reflex recorded fromfour subjects (subjects 3, 4, 5, and 8) seated semi-prone is shown in Figure 6A. In all cases, the condi-tioned soleus H-reflex amplitude was significantlyreduced when compared to control reflex values.Note that the soleus H-reflex depression occurredwith stable M-waves under control conditions and

Fig. 2. Compoundmuscleactionpotentials (CMAPs) evokedby tsMSS.A:Non-rectifiedwaveformaverages (n ¼ 10, elicited every 5 s) of CMAPs recorded from the right (R) and left (L) tibialisanterior (TA) and rectus femoris (RF) muscles in subject 4 following tsMSS. B: Non-rectifiedwaveform averages (n ¼ 10, elicited every 5 s) of CMAPs recorded from the R TA, soleus (SOL),peroneus longus (PL), and RF muscles in subject 7 following tsMSS. Note the different shape ofthese compoundaction potentials acrossmuscles.Timeat 0.02 s on thex-axis corresponds to theonset of tsMSSandassociatedspikeof thestimulusartifact.

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during conditioning stimulation. In Figure 6B, theamplitude of the conditioned soleus H-reflex from allsubjects and C-T intervals tested is shown as a per-centage of the control H-reflex. The soleus H-reflexwas significantly depressed at the C-T interval of50 ms, reaching an overall amplitude of74.9 � 8.8% of the control H-reflex (P < 0.05;Fig. 6B). At the remaining C-T intervals (20, �20,�50) no statistically significant differences betweenthe conditioned and control H-reflex were found(P > 0.05). The soleus H-reflex depression coincidedwith stable M-waves (F3,23 ¼ 0.048, P ¼ 0.986) thatranged from 3.6 � 1% to 4.3 � 1.2% of the maximalM-wave across the C-T intervals tested (Fig. 6C).

DISCUSSION

This study showed for the first time thatCMAPs evoked by magnetic stimulation of the thor-acolumbar enlargement are not susceptible to low-frequency depression, excitation of group I afferentsfrom mixed peripheral nerves in the leg affect theiramplitude in a non-somatotopical neural organizationpattern, and magnetic stimulation over the thoraco-lumbar spine significantly decreases soleus H-reflexexcitability.

The shape of the CMAPs evoked by magneticstimulation of the spine was significantly different

across muscles. The CMAPs recorded from the anklemuscles had mostly a triphasic shape, while thoserecorded from the R/L RF muscles had a polyphasicshape (Fig. 2). The first component was present atlatencies ranging from 5 to 11 ms, while the secondcomponent appeared at latencies ranging from 16 to21 ms. Similar polyphasic phases have been reportedfor the quadriceps femoris muscle following magnet-ic stimulation of the spine [Ugawa et al., 1989], thesoleus muscle when the stimulating electrode movedcaudally from T11 to S1 [Maertens de Noordhoutet al., 1988], and the biceps and deltoid muscles[Chokroverty et al., 1991]. In the latter case, whenthe magnetic coil’s position moved, the shape and/orlatency of the CMAPs were not affected [Chokrov-erty et al., 1991]. A similar polyphasic shapehas also been reported in rats when stimulatingelectrodes were implanted in the L2 epidural space[Gerasimanko et al., 2006]. The first component ofthe CMAP may represent a direct response (i.e.,M-wave) while late components may be due toreflex-mediated action potentials. If the first compo-nent resembles an M-wave, then one would expectthat it is not influenced by synaptic events that affectmotoneuronal depolarization. Indeed, the amplitudeof the first component of the RF CMAP was notaffected by magnetic stimulation at low stimulationfrequencies in this study, consistent with the lack of

Fig. 3. Latency of CMAPs evoked by tsMSS. A,B: Non-rectified waveform averages of soleus(SOL) CMAPs following tsMSS, soleusH-reflex, andmaximalM-waverecorded from two subjects.The latency for each response and the difference between SOL CMAP and soleus H-reflexlatenciesis identified.C:Average overallaverage latency (inms) of the CMAPsrecorded fromkneeandanklemuscles; the early component of the R/Lrectus femoris (RF)muscle depicted in Figure1wasexcluded.

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changes observed in the SOL CMAP first componentfollowing voluntary background contraction of thesoleus muscle and Achilles tendon vibration [Maertensde Noordhout et al., 1988].

One of the behaviors that cause a motor re-sponse to be considered reflexly mediated is the ad-aptation of its amplitude to different stimulationfrequencies. For example, the soleus H-reflex isdepressed at low stimulation frequencies [Knikou,2008; Jessop et al., 2012]. This H-reflex depressionis induced by repetitive activation of Ia afferents thatoccurs in the synapse between Ia afferents and amotoneurons, without concomitant changes in the

membrane potential of a motoneurons ascribed to apresynaptic inhibitory mechanism [Hultborn et al.,1996] known as homosynaptic depression. In thisstudy, the amplitude of the second component ofthe CMAP was not decreased at low stimulation fre-quencies for all muscles (Fig. 4). It should be notedthat homosynaptic or post-activation depression isnot a widespread phenomenon in the spinal cord,depends on the type of group I afferents and targetneurons, and affects group I excitation that is mediat-ed through the g loop [Lamy et al., 2005]. Followinga double stimuli at an interstimulus interval of50 ms, the amplitude of the second component of theCMAP evoked by tsESS was significantly reduced[Courtine et al., 2007; Dy et al., 2010]. This effect,however, is not consistent with short-latency mono-synaptic reflexes since the soleus H-reflex is facilitat-ed when double stimuli are delivered at interstimulusintervals that range from 25 to 100 ms [Katz et al.,1977; Crayton and Rued, 1980], which are likely due

A

C

E

G

D

F

B

Fig. 4. CMAPsevokedat different interstimulusintervals.Averageoverall amplitude (fromall subjects) of the CMAPs recorded bilat-erally from the RF (A,B), SOL (C,D),TA (E,F), and RPL (G) musclesat the interstimulus intervals of1, 3, 5, and 8 s are presented as apercentage of the mean amplitude of the associated CMAPevoked every10 s. Error bars represent the SEM. No statisticallysignificant differences in the amplitude of the CMAPs recordedfrom all muscles were found across the interstimulus intervalstested.

Fig. 5. Interaction of CMAPs evoked by tsMSS with afferentvolleys. Average overall amplitude of the CMAPs recordedfrom the R/L RF, SOL,TA, and R PL muscles conditioned by (A)posterior tibial and (B) commonperonealnerve stimulation at theconditioning-test intervalof 50 ms.Asterisksindicate statisticallysignificant differences of the conditioned CMAPs to the controlCMAPvalues.Errorbarsindicate theSEM.

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to oscillatory properties of the nervous system. Incontrast, the F-wave is depressed at an interstimulusinterval of 80 ms due to the refractoriness of motoraxons by the conditioning afferent volley [Mastagliaand Carroll, 1985]. The aforementioned support thenotion that CMAPs evoked by electric or magneticstimulation of the spine are not due to the excitationof spinal alpha motoneurons and interneurons but tothe activation of nerve roots [Maertens de Noordhoutet al., 1988]. This is consistent with the shorter laten-cy of the SOL CMAPs compared to the soleusH-reflex latency we observed here (Fig. 3).

At this point, we should consider whether thesecond component of the CMAP is an F-wave.F-waves are produced at high stimulation intensitiesof a mixed peripheral nerve and are recurrent dis-charges of antidromically activated motoneurons[Magladery and McDougal, 1950]. F-waves are simi-lar when the recurrent discharges occur in the samemotor unit, vary significantly in amplitude, latency,shape and duration among different motor units, andthe same motor units have to discharge in both directmotor responses and F-waves. Vibration of the Achil-les tendon decreases the amplitudes of the late

component of the SOL CMAP [Maertens de Noordh-out et al., 1988; Dy et al., 2010], F-wave [Shahaniand Young, 1976], and H-reflex [Abbruzzesse et al.,1997], suggesting that post-synaptic changes in alphamotoneurons is apparent in all three types ofresponses. Collision experiments conducted to ad-dress whether transcutaneous stimulation of the spineproduced an M-wave and an antidromic F-waveshowed that F-waves could be recorded upon con-comitant supramaximal peripheral nerve and spinalstimulation, with an F-wave latency 2.2 ms longerthan the CMAP’s evoked by electric and/or magneticstimulation of the spine [Mills and Murray, 1986;Maertens de Noordhout et al., 1988; Ugawa et al.,1989].

Based on these results we propose that CMAPsevoked by stimulation of the spine at the thoracolum-bar or cervicothoracic enlargement are not F-wavesbut are due to excitation of nerve root fibers and an-terior root fibers that are excited 2–4 cm distal to themotor neuron cell bodies [Mills and Murray, 1986].Peristimulus time histograms of single motor unitsfollowing stimulation of the spine with an implantedelectrode in the epidural space concluded that the

Fig. 6. Effects tsMSS of the spine on the soleus H-reflex. A: Non-rectified waveform averages(n ¼ 20) of the soleus H-reflex in four subjects under control conditions (dashed lines) andfollowing tsMSSat the conditioning-test intervalof 50 ms.Note that the soleusH-reflexdepressionoccurredwith stable M-waves.B: Average overall amplitude of the soleus H-reflex conditioned bytsMSSas a percentage of the control H-reflex with respect to the conditioning-test interval tested.Asterisk indicates statistically significant differences between the conditioned and control soleusH-reflex. C: Average overall amplitude of the soleus M-wave as a percentage of the maximalM-wave. M-waves were constant during reflex conditioning recordings. Error bars indicate theSEM.

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short latency that increased the firing probability ofmotoneurons arises from the antidromic activation ofIa afferents [Hunter and Ashby, 1994]. This is consis-tent with the proposed excitation site, that is, distalto the anterior horn cells, based on differencesbetween indirect (spinal stimulation) and direct(F-wave) latencies, and computed tomography scanmeasurements of the distance between the dura andintervertebral foramina [Ugawa et al., 1989; Chok-roverty et al., 1991; Epstein et al., 1991]. This meansthat the nerve roots are excited near their exit fromthe spinal column or near the emergence of the axonsfrom the anterior horn cells [Cros et al., 1990], inde-pendent of the type of stimulation (magnetic or elec-tric) [Mills and Murray, 1986]. This stimulation siteis further supported by results obtained through arecent simulation study, which showed that actionpotentials are initiated along the posterior and anteri-or root fibers, exciting the fibers at the spinal cordentry or at their exit from the spinal canal [Landen-bauer et al., 2010]. The excitation site appears not toinfluence the latency of the CMAPs because whenthe magnetic coil moved caudally or horizontallyfrom the midline [Ugawa et al., 1989; Chokrovertyet al., 1991] or when stimulation intensities were in-creased [Ugawa et al., 1989] the latencies remainedconstant.

In this study, we found that tsMSS significantlydecreases the soleus H-reflex amplitude at a C-T in-terval of 50 ms (Fig. 6B). The soleus H-reflex de-pression might have been driven by the potentiationof Ia afferent hyperpolarization through the antidrom-ic excitation of group Ia afferents, or to the collisionbetween orthodromic and antidromic afferent volleysevoked by posterior tibial nerve and spine stimula-tion, respectively. Because magnetic stimulation doesnot itself stimulate neural or skin tissue but the mag-netic field acts as a vehicle to induce an ion flow(or electric current) [Barker, 1991], soleus H-reflexdepression mediated by cutaneous afferents canbe excluded [Knikou, 2007]. The excitation of fast-conducting group I afferents from the leg hadcomplex effects on the CMAPs’ amplitude (Fig. 5).Because both peripheral nerves were stimulated ator above the M-wave threshold, we cannot excludethe possibility that Golgi tendon organ afferents (Ib)were excited following transcutaneous spine orperipheral nerve stimulation, resulting in complexinteractions.

Some biophysical properties of magnetic stimu-lation that affect currents, which in turn initiateaction potentials, need to be considered. For exam-ple, cortical current densities of magnetic stimulationover the scalp are determined by the stimulus

waveform, the stimulating coil, and the relative coil-to-tissue distribution, while the magnitude of the cur-rent density is related to the conductivity, heterogene-ity, and anisotropy of the tissue [Miranda et al.,2003; Wagner et al., 2004, 2009]. Phantom modelrecordings, imaging studies, deep electrode record-ings, and electromagnetic models have shown thatthe focality of magnetic stimulation delivered with afigure-of-eight coil at 90–100% of the maximumstimulator output is approximately 25 mm2, the areaof stimulation is 100–200 mm2, and the strength ofthe magnetic field decreases 25 mm below the coilsurface [Barker, 1991; Jalinous, 1991; Wagner et al.,2009]. Further, a simulation model that incorporatedspecific dimensions of anatomical structures of thevertebral column including skin, fat, and cerebrospi-nal fluid showed that transcutaneous stimulationcould generate action potentials in neural tissue witha depth of 5 cm [Ladenbauer et al., 2010]. If we as-sume that similar principles apply when the magneticcoil was placed on the Th 10, it is apparent that elec-trical signals produced by the magnetic coil coveredat least four spinal processes and reached the spinalcord. It is clear that dosimetric evaluation of thestimulation currents as well as the induced electricalfield within the tissues warrant further investigation.Such findings will contribute to the development oftreatment protocols based on an in-depth understand-ing of the underlying physiological and biophysicalmechanisms that will result in effective neuromuscu-lar stimulation.

Limitations of this study are that CMAPs fromthe leg muscles were recorded at a specific stimulatoroutput and the input–output curve was not deter-mined, and neural characteristics of the CMAPs werenot examined in detail with the current flowing in aclockwise direction. A third limitation is the inabilityto effectively assess the exact excitation site withinthe spine. This can be addressed in future studiesincorporating invasive recordings in animals as wellas mathematical modeling based on the physiologicalfindings.

Clinical Application of Findings

Transcutaneous stimulation of the spine is amodality used widely to treat chronic neuropathic orischemic pain [Linderoth and Foreman, 1999]. Fur-ther, invasive continuous dorsal column stimulationin two people with multiple sclerosis improved theirmotor, reflex, and bladder functions [Illis et al.,1976]. A recent study showed that continuous epidu-ral stimulation enabled a person with complete para-plegia to achieve full-weight bearing and locomotor-like EMG activity [Harkema et al., 2011]. These

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results were obtained with the stimulation adminis-tered invasively, thus limiting the possibility of theirapplication to a larger number of patients. This studyshowed that tsMSS depressed spinal reflex excitabil-ity and therefore could potentially be utilized inupper motor neuron lesions to normalize reflexhyperexcitability. Nonetheless, the effects of tsMSSon spinal cord segmental reflex circuits in neuro-logical disorders remains to be determined, as wellas the parameters of stimulation, such as duration,frequency, and amplitude. Furthermore, becauseCMAPs induced by tsMSS bypass alpha motoneur-ons, their latency may diagnose abnormal spine-to-muscle conduction times in both limbs in centraland/or peripheral nervous system disorders, as wellas restore the spinal conduction time after a rehabili-tation protocol.

CONCLUSION

CMAPs evoked by magnetic stimulation of thespine represent composite excitatory potentials ofmotor nerve fibers excited orthodromically and dif-ferent types of afferents excited antidromically. It isclear that in order to outline the neurophysiologicalproperties of these CMAPs in detail, single motorunit studies and methods exploiting inhibitory andfacilitatory neural events are needed. These canprovide information about spinal conduction timethat does not depend on the excitability of alphamotoneurons in central or peripheral nervous systemdisorders.

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