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he Role of the Corpus Callosum in Transcranialagnetic Stimulation Induced Interhemispheric Signal

ropagationristotle N. Voineskos, Faranak Farzan, Mera S. Barr, Nancy J. Lobaugh, Benoit H. Mulsant, Robert Chen,aul B. Fitzgerald, and Zafiris J. Daskalakis

ackground: The corpus callosum, the main interhemispheric connection in the brain, may serve to preserve functional asymmetryetween homologous cortical regions.

ethods: To test this hypothesis, 30 healthy adult subjects underwent combined transcranial magnetic stimulation (TMS)– electroen-ephalography procedures. Nineteen of these subjects also completed diffusion tensor imaging and tractography procedures. We exam-

ned the relationship between microstructural integrity of subdivisions of the corpus callosum with TMS-induced interhemispheric signalropagation.

esults: We found a significant inverse relationship between microstructural integrity of callosal motor fibers with TMS-induced interhemi-pheric signal propagation from left to right motor cortex. We also found a significant inverse relationship between microstructural integrityf genu fibers of the corpus callosum and TMS-induced interhemispheric signal propagation from left to right dorsolateral prefrontal cortex

DLPFC). We then demonstrated neuroanatomic specificity of these relationships.

onclusions: Taken together, our findings suggest that TMS-induced interhemispheric signal propagation is transcallosally mediated andeuroanatomically specific and support a role for the corpus callosum in preservation of functional asymmetry between homologousortical regions. Delineation of the relationship between corpus callosum microstructure and interhemispheric signal propagation in

europsychiatric disorders, such as schizophrenia, may reveal novel mechanisms of pathophysiology.

ey Words: Corpus callosum, diffusion tensor imaging (DTI), EEG,nterhemispheric, tractography, transcranial magnetic stimulationTMS)

he biological axiom that “structure determines function” hasproved difficult to demonstrate when studying the brains ofhealthy humans in vivo. In particular, our capability of mea-

uring the functional significance of white matter tracts is limited.arious functional roles of the largest and most studied white mat-

er tract in the brain, the corpus callosum, have been primarilynferred from lesion, surgical transection, and agenesis studies (1).n some instances, such as in interhemispheric transfer of visualnformation, facilitation of communication between hemispheres isssential for optimal function, verified by lesion studies of the sple-ium of the corpus callosum (2). However, handedness and linguis-

ic function are examples of operations that are highly lateralizednd require hemispheric specialization. Furthermore, performancen higher demand cognitive tasks of executive function can beighly lateralized, with functional imaging demonstrating asym-

rom the Centre for Addiction and Mental Health (ANV, FF, MSB, BHM, ZJD),Department of Psychiatry, University of Toronto, Toronto, Ontario; Cog-nitive Neurology (NJL), Sunnybrook Health Sciences Centre, Depart-ment of Medicine, University of Toronto, Toronto; Division of Neurology(RC), Toronto Western Research Institute, University of Toronto, Toronto,Ontario, Canada; and Alfred Psychiatry Research Centre (PBF), The Alfredand Monash University School of Psychology, Psychiatry and Psycholog-ical Medicine, Commercial Rd. Melbourne, Victoria, Australia.

uthors ANV and FF contributed equally to this article.ddress Correspondence to: Z. Jeff Daskalakis, M.D., Ph.D., FRCP, Associate

Professor of Psychiatry and Director of Brain Stimulation Treatment andResearch Program, University of Toronto, Centre for Addiction and Men-tal Health, 250 College Street, Room 713, Toronto, Ontario, CanadaM5T1R8; E-mail: Jeff_Daskalakis@camh.net.

eceived Mar 22, 2010; revised Jun 23, 2010; accepted Jun 24, 2010.

006-3223/$36.00oi:10.1016/j.biopsych.2010.06.021

metry of frontal cortical activation (3,4). Therefore, one importantfunction of neurons projecting through the corpus callosum thatconnect motor and particularly frontal cortical regions may be toensure that excessive “crosstalk” does not occur between hemi-spheres (5) during asymmetrical or highly lateralized tasks.

Recent advances in neuroimaging now permit sophisticatedvisualization and measurement of corpus callosum microstructure.Using DTI, microstructure of white matter fibers can be measuredby indexing fractional anisotropy (FA) (6). FA reflects the degree towhich diffusion of water molecules is restricted by microstructuralelements such as cell bodies, axons, myelin, and other constituentsof cytoskeleton (7). DTI tractography permits anatomically plausi-ble visualization of white matter tracts in the brain (8) and has led toneuroanatomic advances (9) and reliable quantification (10) of seg-mentation of the corpus callosum.

Transcranial magnetic stimulation (TMS) can be used to studypotential transcallosal processes noninvasively (11), which mayprovide insights into the mechanisms leading to hemispheric spe-cialization and corpus callosum function. TMS can be combinedwith electroencephalography (EEG) to measure changes in cortical-evoked activity, including signal propagation to the contralateralhemisphere, which should be primarily mediated by the corpuscallosum. A major advantage of TMS-EEG is that cortical activity canbe measured in motor cortex and dorsolateral prefrontal cortex(DLPFC) (12). TMS-EEG indexing from the DLPFC is more broadlyrelevant to human cognition and neuropsychiatric disorders (13).

The purpose of this study, therefore, was to examine the rela-tionship between microstructural integrity of callosal fibers withTMS-induced interhemispheric signal propagation (ISP; the ratio ofright to left cortical evoked activity) between the homologous cor-tical regions that these fibers connect. Consistent with a role for thecorpus callosum in maintaining laterality and hemispheric special-ization in motor and frontal regions, we hypothesized that 1) micro-

structural integrity of callosal motor fibers would be inversely cor-

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elated with TMS-induced ISP between left and right motor cortex,) microstructural integrity of callosal frontal fibers would be in-ersely correlated with TMS-induced ISP between left and rightLPFC, and 3) these relationships would demonstrate neuroana-

omic specificity.

ethods and Materials

articipantsThirty right-handed healthy subjects (mean age � 34.0 � 7.9,

ange � 20 – 48 years; 18 males, 12 females) completed TMS-EEGrocedures, of whom 19 completed DTI (mean age � 35.7 � 6.5ears, range � 23– 47 years; 14 males, five females). All subjectsere recruited via advertisements or self-referral, and psychopa-

hology was ruled out through the Personality Assessmentcreener (Psychological Assessment Resources, Lutz, Florida). Ex-lusion criteria included a self-reported medical illness or a historyf drug or alcohol abuse. Handedness was confirmed using theldfield Handedness Inventory (14). The protocol was approved by

he Research Ethics Board of the Centre for Addiction and Mentalealth, and all subjects gave their written informed consent inccordance with the declaration of Helsinki.

ranscranial Magnetic StimulationSingle monophasic TMS pulses were administered to the left

otor cortex and left DLPFC using a 7-cm figure-of-eight coil and aagstim 200 stimulator (Magstim Company, Carmarthenshire,ales). In motor cortex, the TMS coil was placed at the optimal

osition for eliciting motor evoked potentials (MEPs) from the rightbductor pollicis brevis (APB) muscle, which typically correspondedo a region between FC3 and C3 electrodes on the 10-20 EEG system15). Localization of the DLPFC was achieved through neuronaviga-ion techniques using the MINIBIRD system (Ascension Technolo-ies, Milton, Vermont) and MRIcro/reg software using a T1-weightRI scan obtained for each subject with seven fiducial markers in

lace. Stimulation was directed at the junction of the middle andnterior one third of the middle frontal gyrus [Talairach Coordinatesx, y, z) � (–50, 30, 36)] corresponding with posterior regions ofrodmann area 9, which overlap with the superior section of Brod-ann area 46 as previously described (16). The optimal positionas marked on the EEG cap to ensure identical placement of the

oil, and the handle of the coil pointed backward, perpendicular tohe presumed direction of the central sulcus, approximately 45° tohe midsagittal line.

In the motor cortex, TMS was delivered in three conditions:ctive suprathreshold, active subthreshold, and sham. Active referso the conditions in which TMS is held tangential to the scalp. In theLPFC, TMS was delivered in two conditions: active suprathresholdnd sham. For each condition, a total of 100 stimuli were deliveredith an interstimulus interval of 5 sec. The order of all conditionsas randomly counterbalanced between subjects to avoid order

ffects. The TMS intensity was determined at the beginning of eachxperiment. For each subject, resting motor threshold was defineds the minimum stimulus intensity that elicits a MEP of more than0 �V in 5 of 10 trials (17). This corresponded to a mean (� SD) of2.5% � 9.8% in 30 subjects who received TMS to the left motorortex and DLPFC and to 52.1% � 6.6% in 19 subjects who under-ent DTI procedures. The suprathreshold intensity was set to elicit

n average MEP of 1 mV peak-to-peak amplitude upon delivery of0 pulses over the motor cortex; this corresponded to 66.1% �2.8% in 30 subjects and to 67.6% � 10.0% in the 19 subjects whonderwent DTI procedures. To control for the effect of TMS click-

nduced auditory activation and TMS-induced cutaneous sensation

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on cortical evoked potentials, sham stimulation was administeredusing the same parameters in 26 subjects in the motor cortex andDLPFC with the coil angled at 90° from the scalp resting on one wingof the coil (18). Finally, in a subset of 14 subjects, subthresholdstimulation was delivered at 80% of resting motor threshold (RMT),which corresponded to 35.7% � 8.9% of stimulator output.

Electromyogram RecordingElectromyogram was captured by placing two disposable disk

electrodes over the right APB in a tendon-belly arrangement. MEPswere filtered (bandpass 2–5 kHz), digitized at 5 kHz, and collectedthrough commercially available software (Signal; Cambridge Elec-tronics Design, Cambridge, United Kingdom).

EEG RecordingTo evaluate TMS-induced cortical evoked potentials, EEG was

acquired through a 64-channel Synamps2 (Compumedics, Char-lotte, North Carolina) EEG system using a 64-channel EEG cap. Fouradditional electrodes were placed on the outer side of each eye andabove and below the left eye to monitor eye movement artifacts. Allelectrodes were referenced to an electrode placed posterior to theCz electrode. EEG signals were recorded with filters at DC to 100 Hzat 20 kHz sampling rate, which was shown to minimize the TMSrelated artifacts (12).

EEG AnalysisThe EEG recordings were first processed off-line by commer-

cially available software, Neuroscan (Compumedics, Charlotte,North Carolina). The EEG data were down-sampled to 1-kHz sam-pling frequency, and segmented with respect to the TMS pulse suchthat each epoch included 1000-msec prestimulus baseline and1000 msec poststimulus activity (Figure S1 in Supplement 1). Ep-ochs were baseline corrected with respect to the TMS-free pre-stimulus interval. The baseline-corrected poststimulus intervalsthat were not contaminated by TMS artifact were extracted anddigitally filtered by using a zero-phase shift 1- to 100-Hz bandpassfilter (48 dB/Oct). At this stage, epochs were manually reviewed,and trials (an average of 5.6% � .4% of trials) contaminated withmuscle activity or movement were excluded from further analysis.Finally, the average signals at each recording site were computedand were fed into an automated eye-blink correction algorithm(19). The eye-blink corrected average EEG waveforms were thenimported into MATLAB (MathWorks, Natick, Massachusetts), andfurther analyses were carried by EEGLAB toolbox (20).

TMS-Induced ISPTo quantify the propagation of TMS-induced cortical evoked

activity from left to right cortex for each subject, the average TMS-induced cortical evoked potentials following TMS pulse were band-pass filtered (1–50 Hz), and TMS-induced ISP was calculatedthrough Equation 1. To evaluate TMS-induced ISP in the motorcortex, C3 (left hemisphere) and C4 (right hemisphere) electrodeswere used, which are the electrodes closest to the optimal site ofAPB activation through TMS (21). In the DLPFC, the recording elec-trodes of interest were AF3 (left hemisphere) and AF4 (right hemi-sphere), which optimally represent the overlap of Brodmann’s areas9 and 46 of the DLPFC. In the left hemisphere, the area under therectified curve was obtained between 50 and 150 msec poststimu-lus (Figure S2 in Supplement 1). The onset (50 msec) was chosenbecause it represents the earliest artifact free data that was reliablyrecorded post-stimulus. The offset (150 msec) was chosen becauseit represents the mean duration of the probable gamma-aminobu-tyric acid–B receptor mediated inhibitory neurotransmission that is

recorded neurophysiologically (22) and is related to callosal inhibi-

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ory mechanisms (11). An average interhemispheric transfer time of0 msec was chosen to account for the time it takes for the signal toropagate from the site of the stimulation to the opposite hemi-phere (23). Thus, in the right hemisphere, the area under the recti-ed curve was obtained between 60 and 160 msec poststimulus.

ISP � �Area under rectified curve �Right Cortex�Area under rectifed curve �Left Cortex� � � 100

(1)

To rule out signal degradation as a potential contributing factor,e computed the signal propagated ipsilaterally over the sameistance from the stimulation site as that between left and rightortex (Results in Supplement 1). To control for effects of TMSlick-induced auditory activation and TMS-induced cutaneous sen-ation, we subtracted the cortical evoked potentials following shamtimulation from active stimulation in motor cortex and DLPFC.inally, the reliability of TMS-EEG procedures was evaluated in 14

ndividuals who returned for repetition of TMS-EEG procedures.

TIImage Acquisition. DTI images were acquired using an eight-

hannel head coil on a 1.5-Tesla GE Echospeed system (Generallectric Medical Systems, Milwaukee, Wisconsin), which permitsaximum gradient amplitudes of 40 mT/m. A single shot spin echo-

lanar sequence was used with diffusion gradients applied in 23oncollinear directions and b � 1000s/mm2. Two b � 0 imagesere obtained. Whole-brain coverage (no gap) was acquiredblique to the axial plane. Slice thickness was 2.6 mm, and voxelsere isotropic. The field of view was 330 mm, and the size of the

cquisition matrix was 128 � 128 mm, with echo time � 85.5 msecnd repetition time � 15,000 msec. To improve the signal-to-noiseatio, the entire sequence was repeated three times. Inversion re-overy prepped spoiled gradient recall and fast spin echo T2-eighted images were also acquired in the event of need for regis-

ration and to ensure anatomic accuracy.Tractography. The DTI data were transferred to a workstation

nd a diffusion-weighted image was created for each of the threeepetitions. The three DTI repetitions were coregistered to the first� 0 image in the first repetition using FSL (http://www.fmrib.ox.

c.uk) to produce a new averaged image, with gradients reori-nted. Registration corrects eddy current distortions and subjectotion, important artifacts that can affect the data, and averag-

ng improves the signal-to-noise ratio. A brain “mask” was thenenerated.

More detailed descriptions of the tractography approach and clus-ering segmentation algorithm have been described (24). In brief,oints were seeded throughout each voxel of the brain, followed byhole-brain deterministic streamline tractography (Runge-Kutta or-er two tractography with a fixed step size of .5 mm). The three thresh-ld parameters for tractography were Tseed, Tstop, and Tlength, Tseed, and

stop and are anisotropy thresholds based on the linear anisotropyeasure CL (25), where CL � (�1 � �2)/�1. The goal of the anisotropy

thresholds is to limit tractography to the white matter. Parameterschosen for this study were Tseed � .3, Tstop � .15, and Tlength � 20 mm.Tractography and creation of white-matter fiber tracts were per-formed using 3D Slicer (open source software, http://www.slicer.org)and MATLAB. For the clustering step, pairwise fiber trajectory similaritywas quantified by first computing a pairwise fiber distance. The meanclosest point distance was employed and directed distances betweenfibers “A” and “B” was converted to a symmetrical pairwise fiber dis-tance. Each distance was then converted to an affinity measure suit-able for spectral clustering via a Gaussian kernel (Wij)� e^ (–d(2)

ij/�2), a

method that is employed in the clustering literature (26). The role of �(�� 60 mm used in this study) is to define the size scale of the problemby setting the distance over which fibers can be considered similar. Aspectral embedding of fibers was then created based on the top 15eigenvectors of the fiber affinity matrix (24) to calculate the most im-portant shape similarity information for each fiber, using a k-way nor-malized cuts clustering algorithm (27).

After the whole-brain cluster model was produced, the operator(ANV) combined the clusters that correspond to the genu of thecorpus callosum, motor fibers of the corpus callosum, and left andright corticospinal tract fibers (Figure 1) as previously described(10), for which high reliability was shown. MATLAB was used tocalculate FA (mean FA was calculated along the selected tracts). Theoperator was blind to any TMS-EEG data. Correct selection of tractswas further verified by superimposing clusters on both the FA andT1 images (28).

Statistical AnalysisTwo-tailed paired t tests were used to examine the difference

between left- and right-sided cortical evoked potentials for bothmotor cortex and DLPFC during the active and sham condition andalso to examine whether cortical evoked activity was different be-tween the active and sham conditions. TMS-induced ISP at sub-threshold activation was similarly examined.

Pearson correlation coefficients were used to assess the rela-tionship between 1) TMS-induced ISP in motor cortex with micro-structural integrity of callosal motor fibers, genu fibers, and cortico-spinal tract and 2) TMS-induced ISP in DLPFC with microstructuralintegrity of genu fibers and callosal motor fibers. For tests con-ducted on both motor and frontal regions, a Bonferroni correctedalpha � .025 was applied. All statistical analyses were performedusing SPSS 15.0 (SPSS, Chicago, Illinois).

Results

TMS-EEGActive suprathreshold stimulation of the left motor cortex re-

sulted in significant activation of the right motor cortex comparedwith sham (active: 297.1 � 183.3 �v.msec, sham: 106.1 � 51.7�v.msec; t � 5.7, df � 25, p � .001; Figure 2A). In the active condition,activation of the right motor cortex was significantly lower comparedwith the activity in left motor cortex (right: 291.31 � 175.54 �v.msec,left: 797.1 � 536.4 �v.msec; t � 6.0, df � 29, p � .001; Figure 2B). The

Figure 1. Diffusion tensor imaging tractography of white-matter tracts. Illustration of the genu (in red) of the corpuscallosum (left panel); callosal motor fibers (in red) of thecorpus callosum (middle panel), and the left corticospinaltract (right panel).

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ean TMS-induced ISP, measured through Equation 1, was 46.7% �7.0%.

Active suprathreshold stimulation of the left DLPFC resulted inignificant activation of the right DLPFC compared with sham stim-lation (active: 578.0 � 412.4 �v.msec, sham: 210.6 � 92.4 �v.msec;� 4.9, df � 25, p � .001; Figure 2C). In the active condition,

ctivation of the right DLPFC was significantly lower compared withhe activity in left DLPFC (right: 586.2 � 406.2 �v.msec, left: 951.2 �91.8 �v.msec; t � 4.9, df � 29, p � .001; Figure 2D). The meanMS-induced ISP was 65.7% � 35.8%.

Topographic plots (Figure S3 in Supplement 1) illustrate theMS-induced cortical activation in motor cortex and DLPFC.

At subthreshold stimulation (i.e., 80% RMT), both left and rightotor cortices were significantly activated compared to sham (ac-

ive left: 210.7 � 116.2 �v.msec, sham left: 111.1 � 76.1 �v.msec;� 3.2, df � 11, p � 0.008; active right: 173.8 � 87.8 �v.msec, shamight: 120.7 � 62.1 �v.msec; t � 2.8, df � 11, p � .02). Activation ofight motor cortex was not significantly different from corticalvoked potentials in the left motor cortex (left: 231.1 � 132.9v.msec, right: 180.8 � 87.8 �v.msec; t � 1.4, df � 13, p � .18; ISP:7.0% � 61.1%). Importantly, in these same subjects, suprathresh-ld stimulation (corresponding to 66.4% � 11.8% of stimulatorutput) resulted in significantly reduced activity of contralateralotor cortex compared with the stimulation site (left: 748.6 � 376.7v.msec, right: 318.6 � 200 �v.msec; t � 4.9, df � 13, p � .001; ISP:8.6% � 23.0%). Finally, subthreshold ISP was significantly greaterhan suprathreshold ISP (t � 2.5, df � 13, p � .029).

When measured over equal distances, intrahemispheric signalropagation was significantly greater than ISP (Results in Supple-ent 1). In addition, any potential confounding effect of TMS click-

nduced auditory activation was ruled out (Results in Supplement), and our findings demonstrated high reliability (Results in Sup-lement 1).

MS-EEG and DTIIn the 19 subjects who completed TMS-EEG and DTI procedures,

e observed similar TMS-induced ISP in both motor cortex and

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DLPFC, compared with the entire sample of 30 subjects (data notshown).

We found a significant inverse relationship between TMS-in-duced ISP in motor cortex, and callosal motor fiber FA (r � �.56, p �.01; Figure 3A). This correlation remained significant when the sub-ject with greater than 100% TMS-induced ISP was removed fromthe analysis (n � 18, r � �.55, p � .02). There was no relationshipbetween sham condition TMS-induced ISP and callosal motor fiberFA (n � 17, r � �.30, p � .24).

We also found a significant inverse relationship betweenTMS-induced ISP in DLPFC and genu fiber FA (r � �.58, p � .01;Figure 3B). There was no relationship between sham conditionTMS-induced ISP in DLPFC and genu fiber FA (n � 17, r � .19, p �.46). Neuroanatomic specificity of these findings was demon-strated: there was no correlation between callosal motor fiber FAand TMS-induced ISP in DLPFC (r � �.41, p � .08) or betweengenu fiber FA and TMS-induced ISP in motor cortex (r � �.11,p � .65). Furthermore, there was no correlation between TMS-induced ISP in motor cortex and left corticospinal tract FA (r ��.25, p � .3) or right corticospinal tract FA (r � �.25, p � .3).Because there may be gender differences with respect to thecorpus callosum (29), data were analyzed in men only, and sig-nificant correlations were found (Figure S4 in Supplement 1).Finally, given the potential role of the corpus callosum in main-taining laterality, correlations between LQ and ISP and betweenLQ and callosal fibers were examined, but no significant relation-ships were found (Results in Supplement 1).

Subthreshold StimulationCallosal motor fiber FA was positively correlated with TMS-

induced ISP at the subthreshold intensity (n � 7, r � .87, p � .01),whereas in the same subjects, suprathreshold TMS-induced ISPwas negatively correlated with callosal motor fiber FA (n � 7, r �.86, p � .01; Figure 4). This correlation was neuroanatomicallyspecific, because there was no correlation with genu FA (n � 7,

Figure 2. Cortical evoked potentials in ipsi- and contralat-eral hemispheres following the application of transcranialmagnetic stimulation (TMS) to the left motor cortex anddorsolateral prefrontal cortex (DLPFC). The waveformsrepresent mean rectified cortical potentials following thedelivery of a single pulse of TMS to the left motor cortex(A and B) and to the left DLPFC (C and D). In all figures, xaxes represent the time after the delivery of the TMS, andthe y axes represent the cortical evoked potentials (�v).(A, and C) These figures illustrate that application of activeTMS (solid waveforms) results in significant activation ofcontralateral hemisphere compared with sham stimula-tion (dashed waveforms) in 26 subjects who receivedboth active and sham stimulation. (B and D) Data derivedfrom 30 subjects. In these figures, application of activeTMS to the left hemisphere (solid waveforms) results incortical evoked potentials in the contralateral hemi-sphere (dashed waveforms) measured through C4 elec-trodes in the motor cortex (B), and AF4 electrode in theDLPFC (D) that are of lower amplitude than the ipsilateralresponse (solid waveform) measured through C3 andAF3, respectively.

r � .34, p � .50).

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iscussion

This study represents the first attempt to investigate directly theelationship between ISP of TMS-induced cortical evoked poten-ials and microstructural integrity of the corpus callosum. Our find-ngs provide in vivo support for the theory that the corpus callosum

ay serve to limit “excessive crosstalk” between hemisphereshen cortical regions are asymmetrically activated. First, there wassignificant inverse correlation between TMS-induced ISP in theotor cortex and FA in callosal motor fibers. Second, there was a

ignificant inverse correlation between TMS-induced ISP in theLPFC and FA of genu fibers. Third, there was no correlation be-

ween callosal motor fiber FA and TMS-induced ISP in DLPFC oretween genu fiber FA and TMS-induced ISP in motor cortex. Taken

igure 3. The relationship between microstructural integrity (measured asractional anisotropy [FA]) of callosal fibers and transcranial magnetic stim-lation (TMS)-induced interhemispheric signal propagation (ISP) in the mo-

or cortex and dorsolateral prefrontal cortex (DLPFC). Data obtained from 19ubjects that underwent diffusion tensor imaging and received suprath-eshold TMS to the left motor cortex (A) and left DLPFC (B). (A) The x axisepresents the FA of callosal motor fibers, and the y axis illustrates theMS-induced ISP from the left motor cortex to the right motor cortex ob-ained through Equation 1 (refer to Methods and Materials). The inverseelationship between these two measures suggest that subjects with higherA of callosal motor fibers have a lower TMS-induced ISP. (B) In this figure,he x axis represents the FA of genu fibers, and the y axis represents theMS-induced ISP from the left DLPFC to the contralateral DLPFC. Similar tohe motor cortex, in the DLPFC, the higher integrity of genu fibers is associ-ted with lower signal propagation from the left to right DLPFC.

ogether, our findings suggest that TMS-induced ISP is transcal-

losally mediated in a neuroanatomically specific manner and isdependent on the microstructural integrity of callosal fibers thatconnect homologous cortical regions. These findings also highlighta potential inhibitory role for callosal fibers, particularly at suprath-reshold stimulation.

Our findings are consistent with evidence demonstrating thatthe corpus callosum may play either an inhibitory or excitatory(facilitatory) role (30). When we activated motor cortex at sub-threshold stimulation, the corpus callosum seemed to play a facili-tatory role. This was demonstrated by three observations. First,TMS-induced ISP following subthreshold stimulation was greaterthan during the suprathreshold condition. Second, activity in rightmotor cortex was not significantly different from left motor cortex(importantly, both were greater than sham), suggesting full or near-full ISP from left to right motor cortex. Third, for this subthresholdcondition, FA of callosal motor fibers was positively correlated withTMS-induced ISP. Therefore, our findings suggest that facilitatoryfibers may be activated during subthreshold stimulation unlikesuprathreshold stimulation, when inhibitory fibers may be primar-ily activated. Our findings support the contention that inhibitoryand facilitatory transcallosal fibers may have different thresholds ofactivation (31). It may be advantageous to activate different typesof callosal fibers depending on the extent of cortical activation. Thatis, low-intensity cortical activity may be associated with activation

Figure 4. The relationship between fractional anisotropy (FA) of callosalmotor fiber and subthreshold versus suprathreshold activation of motorcortex. Data obtained from seven subjects in whom both subthreshold (80%of resting motor threshold) stimulation of left motor cortex and DTI proce-dure were administered. The x axes represent the FA of callosal motor fiber.The y axes illustrate the TMS-induced interhemispheric signal propagation(ISP) from the left motor cortex to the right motor cortex obtained throughEquation 1 (refer to methods) following application of subthreshold (A) and

suprathreshold TMS (B) to the left motor cortex.

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f facilitatory fibers, whereas high-intensity cortical activity may bessociated with activation of inhibitory callosal fibers, perhaps toaintain hemispheric specialization.

Our findings support a previous report (32) demonstrating aorrelation between TMS motor indices of interhemispheric inhibi-ion and FA of the hand area of corpus callosum. In our study,owever, evoked activity was recorded directly over the motorortex through EEG, which may represent a more direct measure ofortical evoked activity than peripheral motor measures (12). Most

mportantly, our finding relating genu FA to TMS-induced ISP inLPFC is novel and is particularly relevant to neuropsychiatric dis-rders. Patients with callosal agenesis demonstrate higher rates ofeuropsychiatric disorders, including schizophrenia and autism (1).ecent neuropathologic findings suggest a relationship betweenisrupted cortical asymmetry and the corpus callosum in schizo-hrenia (33) that may underlie impairments in functionally asym-etrical domains of auditory processing and language in schizo-

hrenia patients. Therefore, potential disruption of the corpusallosum, and of the genu in particular, has been a topic of study inchizophrenia (34), and with the advent of DTI, measurement of theorpus callosum is now more sophisticated (9). In addition, DLPFCysfunction is arguably one of the most well-established findings inchizophrenia (35), supported by neuroimaging, postmortem, andognition studies. Both genu microstructure (36) and DLPFC struc-ure and function (37) have been independently associated witharious executive function tasks, including working memory per-ormance, a well-characterized cognitive deficit in schizophreniahat is closely tied to functional outcome (38). The relationshipetween genu structure and TMS-induced ISP from left to rightLPFC may be a mechanism that contributes to executive cognitive

unction impairments in schizophrenia, whereby normal functionalsymmetry is disrupted.

There are some limitations to our findings. First, we cannot beertain that TMS-induced ISP is exclusively mediated by transcal-

osal pathways, although callosal pathways comprise the vast ma-ority of commissural white matter fibers and almost all connectionsetween homologous cortical regions. Nevertheless, there arether commissural pathways that we did not measure (e.g., anteriornd posterior commissures, fornix of hippocampus). However,hese tracts are difficult to measure with high reliability using DTI39), and they project neither to motor cortex nor to DLPFC. Second,

e only considered left to right transmission, and asymmetry inallosal transfer has previously been documented (40). Third, somef the signal difference between right and left cortex might be due

o signal degradation. However, we demonstrated that at equalistances, TMS-induced intrahemispheric signal propagation wasignificantly greater than interhemispheric signal propagation inoth motor cortex and DLPFC, suggesting that signal degradation

s unlikely to account for the demonstrated difference betweenight and left cortical activity. Fourth, we studied subthreshold stim-lation in motor cortex at 80% RMT. Although the sample size wasmall, at suprathreshold stimulation, the data were similar in thisubset of individuals to that of the overall data set. Although ourndings provide insight into potential interhemispheric facilitationt subthreshold stimulation in the motor cortex, we can only spec-late that such findings might extend to DLPFC. Fifth, althoughham stimulation may produce weaker cutaneous sensation thanctive stimulation, it is unlikely that our findings would be mediatedxclusively through somatosensory pathways. Secondary fibers inomatosensory pathways of the trigeminal nerve cross to the con-ralateral somatosensory cortex at the level of the brainstem andot at the corpus callosum (41,42), making it unlikely that the rela-

ionship between callosal FA and ISP is mediated by somatosensory-

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evoked potentials from trigeminal activation. Finally, our main findingsare limited to the motor cortex and DLPFC. Future studies areneeded to examine TMS-induced ISP between homologous corticalregions other than motor cortex and DLPFC.

In conclusion, our experiments demonstrate that TMS-inducedISP is a form of interhemispheric communication and is related tothe microstructural integrity of callosal fibers. The nature of suchcommunication (i.e., inhibitory vs. facilitatory) may depend on themagnitude of cortical activation. Future work, examining the rela-tionship between interhemispheric communication and special-ized cognitive functions, may help elucidate the mechanisms in-volved in higher order cognitive functions, which rely onhemispheric specialization for optimal performance. Furthermore,examination of corpus callosum microstructure in relation to TMS-induced ISP in disease populations may provide novel insight intothe neurobiological underpinnings of severe mental illness, such asschizophrenia.

This work was funded in part by the Canadian Institutes of HealthResearch (Grant No. P62917 to RC), Canadian Institutes of Health Re-search Doctoral Award (FF), CIHR Clinician Scientist Award (ZJD, ANV),by an operating and studentship award from the Ontario MentalHealth Foundation (ZJD and MSB, respectively), by a National Healthand Medical Research Council (NHMRC) Practitioner Fellowship (PBF),by Constance and Stephen Lieber through a National Alliance for Re-search on Schizophrenia and Depression Lieber Young Investigatoraward (ZJD, PBF), by a National Health and Medical Research CouncilPractitioner Fellowship (PBF), and by an APA (APIRE) AstraZenecaYoung Minds in Psychiatry Award (ANV). The authors gratefully ac-knowledge the assistance of all persons and volunteers whose partici-pation was essential in the successful completion of the study.

ZJD and PBF have both received external funding through Neuro-netics, Inc. ZJD has received external funding through Aspect Medical,Inc., and travel support through Pfizer, Inc. PBF has received equipmentfor research studies from MagVenture, A/S and Brainsway, Ltd. RC issupported by a Canadian Institutes of Health Research—Industry Part-nered (Medtronic) Investigator Award. BHM has received research sup-port or honoraria from AstraZeneca, Bristol-Myers Squib, Corcept, Ei-sai, Eli Lilly, Lundbeck, Forest, GlaxoSmithKline, Janssen, Pfizer, andWyeth. Dr. Mulsant owns stock of less than $10,000 in value in Akzo-Nobel, Alkermes, AstraZeneca, Celsion, Elan, Eli Lilly, Forest, GeneralElectric, and Orchestra Therapeutics. All other authors report no bio-medical financial interests or potential conflicts of interest.

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