Handedness, Motor Skills and Maturation of theCorticospinal Tract in the Adolescent Brain

Pierre-Yves Herve,1 Gabriel Leonard,2 Michel Perron,3 Bruce Pike,2 Alain Pitiot,1

Louis Richer,4 Suzanne Veillette,3 Zdenka Pausova,1,5 and Tomas Paus1,2*

1Brain and Body Centre, University of Nottingham, Nottingham, United Kingdom2Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada

3Groupe ECOBES, CEGEP Jonquiere, Jonquiere, Quebec, Canada4Department of Psychology, University of Quebec in Chicoutimi, Chicoutimi, Quebec, Canada5Centre de recherche, Centre hospitalier de l’Universite de Montreal, Montreal, Quebec, Canada

Abstract: With anatomical magnetic resonance imaging, the signal intensity of the corticospinal tract (CST)at the level of the internal capsule is often paradoxically similar to that of grey matter. As shown previouslyin histological studies, this is likely due to the presence of very large axons. We measured the apparentgrey-matter density (aGMd) of the putative CST (pCST) in a large cohort of adolescents (n 5 409, aged 12–18 years). We tested the following hypotheses: (1) The aGMd in the pCST shows a hemispheric asymmetrythat is, in turn, related to hand preference; (2) the maturation of the CST during adolescence differs betweenboth sexes, due to the influence of testosterone; (3) variations in aGMd in the pCST reflect inter-individualdifferences in manual skills. We confirmed the first two predictions. Thus, we found a strong left > righthemispheric asymmetry in aGMd that was, on average, less marked in the 40 left-handed subjects. ApparentGMd in the pCST increased with age in adolescent males but not females, and this was particularly relatedto rising plasma levels of testosterone in male adolescents. This finding is compatible with the idea that tes-tosterone influences axonal calibre rather than myelination. The third prediction, namely that of a relation-ship between age-related changes in manual skills and maturation of the pCST, was not confirmed. We con-clude that the leftward asymmetry of the pCST may reflect an early established asymmetry in the number oflarge corticomotoneuronal fibres in the pCST.Hum Brain Mapp 30:3151–3162, 2009. VVC 2009Wiley-Liss, Inc.

Key words: pyramidal tracts; internal capsule; sex differences; functional laterality; testosterone; axons;myelin sheath; magnetic resonance imaging; neuroanatomy; postnatal development

INTRODUCTION

During the last century, numerous histological studieshave shown that, in humans, the corticospinal tract (CST)contains between 500,000 and 1,100,000 fibres of differentsizes, myelinated or not, descending from motor, premotoror sensory cortices. Importantly, the CST includes a smallpopulation of heavily myelinated, large-diameter fibres[from 7.4 lm to 22 lm, Nyberg-Hansen and Rinvik, 1962;Terao et al., 1994]. These fibres are thought to originatefrom the giant pyramidal ‘‘Betz cells’’ of Brodman’s area 4(BA4), and likely correspond to the fast, direct corticomo-

Contract grant sponsors: Canadian Institutes of Health Research,Heart and Stroke Foundation of Quebec, Canadian Foundation forInnovation.

*Correspondence to: Tomas Paus, Brain & Body Centre, Universityof Nottingham, University Park, Nottingham, NG7 2RD, UK. E-mail: tomas.paus@nottingham.ac.uk or Pierre-Yves Herve, Brain &Body Centre, University of Nottingham, University Park, Notting-ham, NG7 2RD, United Kingdom. Tel: +44(0)115 84 67552, E-mail:pierre-yves.herve@nottingham.ac.uk.

Received for publication 5 November 2008; Accepted 18 December2008

DOI: 10.1002/hbm.20734Published online 23 February 2009 in Wiley InterScience (www.interscience.wiley.com).

VVC 2009 Wiley-Liss, Inc.

r Human Brain Mapping 30:3151–3162 (2009) r

toneuronal functional component of the CST involved inmotor control [Lemon, 1993; Porter, 1985].It has been established that, depending on the MR imag-

ing sequence, the CST can be more or less apparent on T1-weighted images. A previous neuroradiological and histo-logical work demonstrated that focal differences in T2, PDor T1-weighted (T1W) intensities occur in the posteriorthird of the internal capsule, most likely due to the distinc-tive microstructure of the CST [Yagishita et al., 1994]. Onsilver-stained brain slices, the presence of the CST in theposterior third of the internal capsule [Kretschmann, 1988]was associated with a pale area containing widely spaced,large axons with thick myelin-sheaths. The location of thispale area matched that of a T1W hypo-intense patch previ-ously reported by others [Mirowitz et al., 1989]. Kitajimaextended this finding by showing that the CST and thefibers of the external sagittal lamina [see Catani et al.,2003], shared similar histological and magnetic-resonance(MR) properties [Kitajima et al., 1996]. Accordingly, bothtracts tend to appear darker than the surrounding whitematter on T1W images.An interesting consequence of this T1W hypo-intensity

of the putative CST (pCST), probably due to the largeaxons, is that the pCST tends to be iso-intense with greymatter and may be misclassified as such by automated tis-sue-classification algorithms [Levy-Cooperman et al., 2008].In other words, depending on the strength of this effect,some hypointense white-matter structures can paradoxi-cally appear as grey matter, and this can have implicationsfor analyses based on local tissue-densities in a stereotaxicspace [i.e. voxel based morphometry (VBM); Wright et al.,1995]: we noticed that this phenomenon caused the pCSTto become evident on the average stereotaxic grey-mattermap of a large cohort of adolescent siblings aged from 12to 18 years from the Saguenay Youth Study (SYS, T1Wdataset, Fig. 1). We were able to segment the average leftand right pCST and then to measure the individual‘‘apparent grey-matter densities’’ (aGMd) in these two vol-umes of interest in a sample of 409 adolescents (see Fig. 2).We took advantage of this phenomenon and tested three

hypotheses:Firstly, we predicted that the aGMd in the pCST shows

a hemispheric asymmetry that is, in turn, related to handpreference. Several reports of a leftward asymmetry of theCST exist [Dubois et al., 2008; Galaburda et al., 1978;Nathan et al., 1990; Rademacher et al., 2001; Westerhausenet al., 2007; but see White et al., 1997], and it may be that

the very adaptable structure and function of the CST[Lemon and Griffiths, 2005] would reflect the bias towardsright-hand preference found in 90% of the human popula-tion. An association with handedness, however, has so farremained elusive [Westerhausen et al., 2007]. We hypothe-sized that the CST should display a leftward asymmetry inright-handers, and a rightward asymmetry in left-handers.Secondly, we hypothesized that the maturation of the

CST during adolescence differs between both sexes, due tothe influence of testosterone. This prediction is based onthe previous observations of a faster growth of white mat-ter in males during childhood and adolescence [De Belliset al., 2001; Lenroot et al., 2007] and our recent findingthat age-related increase in the overall volume of whitematter during male adolescence is mediated, in part, bytestosterone [Perrin et al., 2008]. Based on the discrepancybetween age-related changes in volume and those in mag-netization-transfer ratio, an indirect index of myelination,we speculated that this global increase is related to anincrease in axonal calibre [Perrin et al. 2008]. Given theknown relationships between T1W intensity on one handand, on the other hand, axonal calibre and/or axon densityat the level of the CST [Yagishita et al., 1994], we predictedthat the aGMd will increase with age in males but not infemales and that this sex-specific increase is explained bythe rise of testosterone.Thirdly, we predicted that variations in aGMd in the

pCST reflect inter-individual differences in manual skills.It is not known whether, within the human species, inter-individual variations in manual skills relate to the mor-phology of the CST.

MATERIALS AND METHODS

Participants

Analyses were conducted on a set of 409 healthy FrenchCanadian adolescents (age ranging between 12 and 18years, 204 males, 205 females) from the Saguenay YouthStudy dataset [SYS, detailed in Pausova et al., 2007]. Inthis sample, there were 40 self-reported left-handers (22females). Self-reported handedness was recorded by a psy-chometrician before the performance of motor tasks; thisinformation was used to determine the ordering of the useof left and right hand when testing manual skills. Handed-ness was also documented using an 18-item questionnaire,with scores ranging from 18 to 90 for extreme right-hand-

Figure 2.

A: Slice of the average grey matter map (z 5 10 mm) showing

the left putative corticospinal tract at the level of the internal

capsule, with the outline of the automatically determined region

of interest for that slice in red. B: Watershed transform of the

same slice, with isocontours of the probabilistic map of the cor-

ticospinal tract from Rademacher et al. [2001]. A warmer colour

reflects a lower rank of the corresponding watershed basin

(smaller seed or peak value). This transform of the image

allowed the segmentation of the pCST by adding the 30 highest

pixels to the seed value within the pCST patch (in red). C: Final

volumes of interest, computed over 26 slices, and rendered over

the average grey matter density map.

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Figure 1.

Left: The corticospinal tract (outlined in blue)

as it appears on the grey-matter average of

409 non-linearly normalised brains (MNI coor-

dinates: x 5 23 mm, y 5 215 mm, z 5 7

mm). Right: superimposition with the mye-

loarchitectonic maps from Rademacher et al.

[2001].

Figure 2.

ers and extreme left-handers respectively [based on Cro-vitz and Zener, 1962] (Table I). We used the questionnaireto verify that the self-reported handedness was consistentwith the ‘‘writing hand’’ item from this questionnaire. Inthis report, handedness was based on the hand used forwriting, as reported by the subject during neuropsycholog-ical testing and in the Crovitz & Zener questionnaire. Wechose a simple binary classification based on the handused for writing, a much practised and extremely lateral-ized task that requires an early and extensive learning[Perelle and Ehrman, 2005]. This definition has been usedin large populations [Peters et al., 2006], and provides anunambiguous operational criterion of handedness [Perelleand Ehrman, 2005]. No handedness information was avail-able for three participants; they were not included in therelevant analyses. There was no difference in terms of ageor IQ (WISC III) between the four sex and handednessgroups (Table I).

Fine Motor Skills

In the Grooved Pegboard task, participants wererequired to fit key-shaped pegs into similarly shaped holeson a 4-by-4 inches board, beginning at the left side of theboard with the right hand and at the right side with theleft hand. Participants were instructed to complete the taskas rapidly as possible. Two trials were performed witheach hand, starting with the dominant hand (self-report).The order was RLLR for right-handers, LRRL for left-handers. The average completion times were used asscores.In the repetitive tapping task [Leonard et al., 1988], the

subjects had to hit a target (metal plate) located on eitherthe left or the right of a board, as fast as possible, with astylus for a period of 15 s. One trial was performed witheach hand, starting with the dominant hand. We recordedthe number of taps.

Bioavailable Testosterone

Fasting blood samples were taken in the morning(between 8 and 9 am) and analysed via radioimmunoassay(Testosterone RIA DSL-4000, Diagnostic Systems Labora-tory, Inc., TX) to measure levels of testosterone (nmol/l)and sex-hormone-binding-globulin (SHBG). The level ofbioavailable testosterone (nmol/l) was then calculated

[Sodergard et al., 1982]. Testosterone levels were not differ-ent between the two handedness groups but, as expected,were very different between males and females (Table I).

Magnetic Resonance Imaging

For each participant, a high-resolution T1W image of thebrain was acquired on a Phillips 1.0-T scanner using thefollowing parameters: 3D RF-spoiled gradient echo scanwith 160 slices, 1-mm isotropic resolution, TR 5 25ms, TE5 5 ms and flip angle 5 308.

Image Processing

T1-weighted images were first non-uniformity corrected[Sled et al., 1998] and intensity normalized. They werethen non-linearly registered to the SYS333 study-specifictemplate, an average of 333 subjects of the SYS dataset reg-istered to the MNI305 template. This step was achievedusing the Image Registration Tool Kit software package[Rueckert et al., 1999], with a sampling distance of 5 mm.The registered brain images were then classified [Zij-denbos et al., 2002] into grey, white matter and cerebro-spinal fluid (GM, WM, CSF images).The average of the 409 grey matter images was then

slightly smoothed using a 2 3 2 3 1 mm FWHM Gaussianfilter. The pCST was clearly apparent on this mean imageas a patch of apparent grey-matter in the posterior part ofthe internal capsule, a location consistent with previousreports [Yagishita et al., 1994]. In order to segment thepCST patch, a 2-D watershed transform [Vincent andSoille, 1991] was applied to 26 consecutive axial slices cov-ering the internal capsule from z 5 24 to z 5 21 mm (seeFig. 2). We used the 8-neighbour definition of connectivity,and pixels with grey-matter probabilities below a thresh-old of 0.15 were excluded from the transform. In eachhemisphere, the watershed basin corresponding to thepCST was first identified on the most ventral slice (z 524). The criterion for this was the highest average proba-bility of being included in the CST as defined by the mye-lin-based stereotaxic probability map [Rademacher et al.,2001]. On subsequent slices, the watershed whose peakcoordinates were closest to those of the pCST watershedbasin of the previous slice (Euclidean distance) wasselected. In each hemisphere and each slice, we then

TABLE I. Demographics

Sex handedness

Females Males

Left-handers Right-handers Left-handers Right-handers

Age (months) 188.3 6 25.5, n 5 22 183.1 6 23.2, n 5 181 179.4 6 23.4, n 5 18 180.7 6 22.4, n 5 185Bioavailable testosterone (nmol/l) 0.5 6 0.38, n 5 22 0.5 6 0.37, n 5 181 10.4 6 06.6, n 5 18 8.7 6 05.4, n 5 181IQ 105.8 6 11.5, n 5 22 104.7 6 12.1, n 5 181 103.6 6 13.9, n 5 18 105.4 6 12.8, n 5 185Handedness scores 74.0 6 11.2, n 5 22 27.2 6 07.1, n 5 181 60.8 6 17.6, n 5 18 26.1 6 06.2, n 5 185

Means 6 SD for age, bioavailable testosterone levels, IQ (WISC III), and an 18 item handedness scale, ranging 18–90 (extreme right-handers to extreme left-handers, 54 being the center).

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applied a 2-D region-growing method to add sequentially30 neighbouring pixels with the highest probability ofbeing classified as grey matter within the pCST. The num-ber of 30 pixels gave the most reliable results. This rater-independent strategy allowed for the creation of masks ofsimilar volume in the left and right hemispheres, thusmaking the hemispheric comparisons unbiased [Hagmannet al., 2006]. The region was not allowed to grow into anyadjacent watersheds (thalamus or putamen/globus pal-lidus); it remained free, however, to extend into the inter-nal capsule. By combining the regions drawn on all slices,we obtained two volumes of interest of 806 and 804 voxelson the left and right side respectively (two voxels lackedat z 5 6 mm on the right because the watershed was too

constraining). These masks were used to measure the aver-age aGMd in the pCST region in each individual grey-mat-ter map. Both the masks and the grey-matter maps wereprojected back to native space, and a 2-mm 3D Gaussianblur was applied to the GM maps.

Statistical Analysis

Hypothesis 1: We assessed the effects of hemisphere, sexand handedness on aGMd values using a repeated-meas-ures type II ANOVA (with hemisphere as a within-subjectfactor, sex and handedness as between-subjects factors).Hypothesis 2: In order to assess the effects of sex and age

on aGMd in the pCST and their interaction, we computedlinear models that included sex and age as explanatory vari-ables of the left or right aGMd and of the asymmetry index.To complement these analyses, the effects of testosteronewere also tested by linear regression separately in malesand females, with and without age correction.Hypothesis 3: Both the unimanual performance on peg-

board and tapping and the asymmetry between the left andright hands were analyzed in the same way as the aGMddata (repeated measures type II ANOVA, sex and handed-ness as between-subjects factors and performing hand aswithin-subjects factor). Subjects with scores distant by morethan 3 standard deviations from the mean on the left or righthand or the asymmetry index were rejected from the analy-sis. We then correlated aGMd values with the hand skill vari-ables in males and females separately, with and without agecorrection. When used, asymmetry indices were computedwith the formula (L 2 R)/(L 1 R) for the pCST, and (R 2L)/(L 1 R) for the manual performance variables.Statistics were computed using R (http://www.r-project.

org, ‘‘car’’ package), and power calculations with the Gpower software.

RESULTS

Measurements of aGMd in pCST

On 25 consecutive 1-mm-thick axial slices of a grey-mat-ter probability map obtained in the population of 409 ado-

Figure 3.

Seed pixel profiles for the two volumes of interest. The coordi-

nates are given in the two first graphs (absolute x and y coordi-

nate versus z level), the seed density in the last graph. The left

hemisphere appears in closed circles, the right in open circles.

The apparent grey matter density is higher on the left, and also

increases dorso-ventrally.

TABLE II. Descriptive statistics

Sex handedness

Females Males

Left-handers Right-handers Left-handers Right-handers

Side L R L R L R L RaGMd 0.31 6 0.18 0.27 6 0.16 0.31 6 0.15 0.26 6 0.15 0.37 6 0.17 0.38 6 0.15 0.41 6 0.17 0.36 6 0.16n 22 181 18 185Pegboard (s) 12.2 6 1.4 12.4 6 1.7 13.1 6 2.1 11.5 6 1.6 12.9 6 1.7 14.0 6 2.2 13.8 6 2.1 12.5 6 1.6n 21 181 18 182Tapping 98.0 6 8.1 93.1 6 8.6 87.3 6 9.4 101.2 6 10.7 99.9 6 11.6 99.3 6 13.4 93.4 6 11.9 107.6 6 12.0n 21 176 18 174

Descriptive statistics (mean 6 SD) for the apparent grey matter density of the putative corticospinal tract, pegboard task (execution timein s) and tapping task (number of taps in 15 s). The data are sorted by handedness, sex and side (hemisphere or hand). The sample sizes(n) fluctuate due to missing values and outlier exclusion (3 standard deviations criterion).

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lescents (see Fig. 2), we were able to identify automaticallythe aGMd peaks corresponding to the left and right pCST(seed pixels). The aGMd of the seed pixels was higher in theleft hemisphere at all levels except in the lowest, denser, sli-ces (see Fig. 3). This effect was also present when consider-ing the individual aGMd densities averaged over the entireleft and right pCST region (Table II). The left and rightaGMd values were highly correlated (r 5 0.86).

Hypothesis 1: Hemispheric Asymmetry of the

pCST, and Effects of Sex and Handedness

In the repeated measures ANOVA, with handednessand sex as between-subject factors and hemisphere aswithin-subject factor, the three-way interaction was not

significant (F1,402 5 2.46, P 5 0.117). We noticed, however,a two-way interaction between handedness andhemisphere (F1,402 5 5.85, P 5 0.016) indicating that theinter-hemispheric difference was different in the right andleft-handers irrespective of sex (mean difference in inter-hemispheric difference between right-handers and left-handers 5 0.034, Cohen’s d 5 0.40). The L > R asymmetryof the pCST was observed in right-handers but was lowerin left-handers. The hemispheric asymmetry was not sig-nificant in the left-handed group (mean inter-hemisphericdifference 5 0.013, Cohen’s d 5 0.14; one-sample t-test, n5 40, P 5 0.39). It is interesting to note that male left-handers were on average symmetric, whereas all othergroups showed a L > R asymmetry (see Fig. 4). Given thesmall number of left-handed subjects in each sex group,we explored this trend with post-hoc tests (Table III). Aone-sample t-test, assessing the presence of a hemisphericasymmetry in the male left-handed group, did not reachsignificance (Table III). In contrast, female left-handers stilldisplayed a trend for an L > R asymmetry (Table III, Fig.4); a larger sample of n 5 45 would have been needed forthis asymmetry to become significant (alpha 5 0.05 andpower 5 0.8). The L > R asymmetry was evident andhighly significant in both (large sample-size) right-handedgroups, with a ‘‘medium’’ effect size (d > 0.5, Table III).The effect of handedness on the asymmetry was significantonly in males (two-sample student’s t-test, males, P 50.009; females, P 5 0.4; Table III). Although the three-wayinteraction was not significant, the two-way interactionbetween hemisphere and handedness appears to be drivenby males (see Fig. 4). In fact, the small number of left-handedsubjects makes it difficult to detect differences in inter-hemi-spheric asymmetries between male and female left-handers(Table III). The sex-by-hemisphere interaction was not signifi-cant (F1,402 5 0.68, P 5 0.4), indicating that sex did not influ-ence the asymmetry in pCST aGMd. The sex-by-handednessinteraction was not significant (F1,402 5 0.07, P 5 0.79), indi-cating that the effect of sex was similar in both handednessgroups. The ‘‘main effect’’ of hemisphere, i.e. the L > R

Figure 4.

Boxplots of the inter-hemispheric difference (L 2 R) in the appa-

rent grey-matter density (aGMd) of the putative corticospinal

tract (pCST) at the level of the internal capsule, in the four sex

and handedness groups. The small boxes represent the means.

TABLE III. Asymmetry of the putative corticospinaltract

Test Mean 6 SD [95% CI] Cohen’s d T P valueCorr.

P valueDetectable

difference (d)

Male LHd (n 5 18) 20.013 6 0.102 [20.064 0.037] 0.13 20.56 0.58 – 0.0715 (0.70)Male RHd (n 5 185) 0.045 6 0.089 [0.032 0.058] 0.51 6.92 7 3 10211 6 3 10210 0.0185 (0.21)Female LHd (n 5 22) 0.034 6 0.079 [–0.001 0.069] 0.43 2.02 0.056 0.45 0.0495 (0.62)Female RHd (n 5 181) 0.048 6 0.080 [0.036 0.060] 0.60 8.08 9 3 10214 7 3 10213 0.0168 (0.21)RHd–LHd, males (n 5 203) 0.059 6 0.090 [0.015 0.103] 0.65 2.63 0.009 0.072 0.063 (0.70)RHd–LHd, females (n 5 203) 0.014 6 0.082 [20.050 0.021] 0.17 0.77 0.44 – 0.052 (0.64)Males–females, LHd (n 5 40) 20.047 6 0.089 [20.106 0.011] 0.53 21.65 0.11 0.88 0.080 (0.91)Males–females, RHd (n 5 186) 20.003 6 0.088 [20.020 0.015] 0.03 0.30 0.77 – 0.026 (0.29)

Post-hoc comparisons for the hemispheric asymmetries in pCST aGMd (L–R). The presence of a significant asymmetry in the four sexand handedness groups and differences between groups were assessed with one- and two-sample Student’s t-tests. Cohen’s d is a mea-sure of effect size (ratio of mean and standard deviation). Corrected P values (Bonferroni for 8 tests) are also given. Statistical powerestimates are presented in the form of the detectable difference with alpha 5 0.05 and power 5 0.8 (mean difference and Cohen’s d).

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asymmetry in the whole population, was highly significant(F1,402 5 104.87, P < 10215). The main effect of sex was alsovery significant (F1,402 5 42.30, P < 10210). The aGMd valueswere overall greater in males as compared with females in

both hemispheres. There was no significant main effect ofhandedness (F 1,402 < 0.01, P 5 0.99).

Hypothesis 2: Sex Difference in the

Developmental Course of the pCST During

Adolescence and the Influence of Testosterone

In both hemispheres, the aGMd in the pCST increasedwith age in males (P < 1024; simple regression in males left:r 5 0.35, right: r 5 0.38, Fig. 5, Table IV) but not in females(left r 5 0.06, right 5 20.01, non significant). This sex differ-ence in the effect of age on the aGMd in the pCST was statis-tically significant (linear models, interaction between age andsex, left: P 5 6 3 1023; right: P 5 1024). Thus, the observedsex effect was age dependent: the predicted means for thetwo groups at 12 years of age were not different betweenmales and females, whereas they were very different at 18years of age. The asymmetry index of the aGMd did notvary with age in either males or females.Bio-available testosterone shows a significant increase

with age during adolescence in males only (r 5 0.65, P <1024, Table IV). There was a significant relationshipbetween aGMd in the pCST and bio-available testosteronein males (left: r 5 0.36, right: r 5 0.38, both P < 1024). Fur-thermore, this relationship between testosterone andaGMd in pCST in males survived a statistical correctionfor the effects of age, albeit explaining less variance attrib-utable to testosterone above and beyond the effect of age(P 5 5 3 1023, R2 5 0.04, on both sides after removing theeffects of age on both the dependent and independentvariable, i.e. partial correlation). In the light of the slopesobserved in males (Table IV), the plasma levels of testos-terone in females (Table I) were not variable enough toallow a meaningful regression analysis. But computing the

Figure 5.

Effects of sex and age (in months) on the putative corticospinal

tract (pCST) apparent grey-matter density (aGMd), for the left

and right hemispheres. Females appear in open circles and

dashed lines, males in closed circles and solid lines. The regres-

sion over the whole group is given by the dotted line.

TABLE IV. Effects of age, sex and testosterone on the pCST apparent grey matter density

Intercept Std err P value Slope Std err P value R2

Left pCST aGMd, linear model with age and sexMales 0.306 0.021 – 0.0027 0.0005 5 3 1028 0.146Males–females 20.012 0.030 NS 20.0024 0.0007 5.7 3 1024

Left pCST aGMd, linear regression with bioavailable testosterone (n 5 404)Total (n 5 404) 0.305 0.010 – 0.0112 0.0013 2 3 10215 0.145Males (n 5 200) 0.307 0.021 – 0.0111 0.0020 1027 0.131

Right pCST aGMd, linear model with age and sexMales 0.270 0.019 – 0.0027 0.0005 1028 0.171Males–females 20.006 0.028 NS 20.0027 0.0006 2.5 3 1025

Right pCST aGMd, linear regression with bioavailable testosterone (n 5 404)Total (n 5 404) 0.260 0.009 – 0.0115 0.0013 <2 3 10216 0.167Males (n 5 200) 0.272 0.019 – 0.0105 0.0018 4 3 1028 0.142

Testosterone by ageMales (n 5 200) 3.12 nmol/l 0.57 1027 0.160 nmol/l/mth 0.013 <2 3 10216 0.417

Apparent grey-matter density (aGMd) of the putative corticospinal tract (pCST) as explained by age and sex or testosterone, and rela-tionship between age and testosterone. The intercept is placed at the lowest age in the study (145 months, 12 years old). When pCSTaGMd is explained by sex and age, the first row gives the intercept and slope in males, whereas the second row gives the relative differ-ence in intercept and slope between males and females. All these models were strongly significant.

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regression in males only or in males and females togetherdid not affect the slope or intercept of the regression line(Fig. 6, Table IV) indicating that the age-dependent sex dif-ference could be mediated by the progressive increase inbio-available testosterone in males during adolescence.

Hypothesis 3: Development of Manual Skills

During Adolescence and Maturation of the pCST

In both left and right-handers, the time taken to insert allthe pegs into the board was shorter with the dominant handthan with the non-dominant (performing-hand by handed-ness interaction, F1,398 5 36.69, P 5 3 3 1029, Fig. 7, TableII). Similarly, the dominant hand was also able to hit the tar-get a greater number of times in 15 s than the non-dominanthand (F1,385 5 123.57, P < 10215). The left-handers weremore symmetric than the right-handers at this task (TableII). Over the whole sample, with a majority of right-handers,the right-hand was better (main effect of performing handfor pegboard and tapping: F1,398 5 148.48, F1,385 5 733.5,both P < 10215). Females outperformed males at the peg-board task (main effect of sex: F1,398 5 29.62, P 5 1 3 1027)while the converse was true for the repetitive tapping task(F1,385 5 35.15, P 5 7 3 1029).Completion times in the pegboard task improved signifi-

cantly with age for both hands (see Fig. 7) in females (P <

1024, left: r 5 20.40, right: r 5 20.27) but not in males (P> 0.2, left r 5 20.09, right r 5 20.006; linear models, sexby age interaction: left, P 5 0.002; right, P 5 0.01). Theasymmetry in the performance of this task with the domi-nant and non-dominant hands decreased significantly withage in females only (r 5 0.16, P 5 0.02 in females; r 5 0.06,P 5 0.38 in males but no significant slope differencebetween the two sexes). This was indeed due to the lefthand improving with age faster than the right hand. Asregards the tapping task, the number of taps improved sig-nificantly with age in both males and females, and for bothhands (simple regression, all four P values < 3 3 1023, rranging 0.2120.42). The asymmetry tended to decreaseslightly with age in males only (r 5 20.15, P 5 0.04, but nosignificant slope difference between the two sexes).Prior to the statistical correction for age, we observed

modest correlations between increasing aGMd values andimprovement in manual performance as measured withthe repetitive tapping task in males (r ranging 0.16–0.26, P< 0.05) but not in females. Similar tests conducted withthe pegboard data did not yield any significant result. Theabove correlation observed in males did not survive a sta-tistical correction for the effects of age. In keeping with theeffect of handedness observed in males, there was a slightbut significant relationship between the degree of asymme-try in tapping and the asymmetry in the aGMd of thepCST in this group (r 5 0.176, P 5 0.01); no such relation-ship was observed in the female group. This effect was notdependent on age.

DISCUSSION

This study described the morphological variability of thepCST and its behavioural relevance in the largest sampleof healthy volunteers to date. We confirmed two out of thethree hypotheses.

Hypothesis 1: Hemispheric Asymmetry

and Handedness

We found a significant left > right hemispheric asymme-try in the pCST in both males and females right-handers,which was reduced in left-handers, particularly males.This is to our knowledge the first report of an associationbetween handedness and morphological features of theputative CST. The asymmetry of the pCST was, however,not reversed in left-handers. The fact that we did not findsuch an asymmetry reversal is not necessarily surprising.At the behavioural, functional or anatomical levels, asmaller degree of asymmetry was often described in left-handers as compared with right-handers [Amunts et al.,2000; Kim et al., 1993; Schmidt et al., 2000a; Solodkin et al.,2001]. From what we inferred of the microstructural basesof the T1W intensity of the pCST (see below), we wouldhypothesize that the handedness-related asymmetry stemsfrom an inter-hemispheric difference in the proportion oflarge CST axons. This asymmetry would thus be estab-

Figure 6.

Effect of testosterone on the putative corticospinal tract (pCST)

apparent grey-matter density (aGMd), for the left and right

hemispheres. Females appear in open circles and dashed lines,

males in closed circles and solid lines. The average regression is

given by the dotted line.

r Herve et al. r

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lished early during brain development [Dubois et al.,2008], and remain essentially unchanged until the onset ofthe aging process. This would further explain the absenceof the relationship between pCST and the improvement ofmanual skills during adolescence; the number of largefibres from the Betz cells could only decrease, leaving noroom for further improvement. Theoretically, larger axonson the dominant side would allow for faster conductiontimes [Hartline and Colman, 2007; Rushton, 1951] and/orincreased axonal transport capacities [but Inestrosa andAlvarez, 1988]; yet, an increase in the axon diameterthroughout adolescence was not shown to have any influ-ence on motor skills. If we assume that the fast, largefibres are indeed involved in fine motor control, a largernumber of such fibres on the dominant side could affordthe dominant M1 a relatively better control over the moto-neurons of the hand. This frequent left hemispheric biaswould facilitate the preferred use of the right hand duringdevelopment. Theoretically, the subjects in whom this bias

would be absent or reversed would in contrast be morelikely to become left-handed. The fact that the associationbetween pCST asymmetry and hand preference was morecomplex than we had initially hypothesized, and the possi-ble interaction with sex (evidenced by Amunts et al. [2000]at the level of the central sulcus), suggests that other fac-tors than pCST asymmetry are involved in the early deter-mination of hand preference. No relationship was foundbetween levels of bio-available testosterone and eitherhandedness or asymmetry of the pCST, suggesting thattestosterone does not play a role in the establishment ofthese particular behavioural and neuroanatomical asym-metries—as far as it can be observed during adolescence.

Hypothesis 2: Age and Sex

We observed a marked sex-specific effect of age: the twosexes had the same average aGMd in the pCST at age 12years, with an increase occurring only in males over the

Figure 7.

Evolution of manual skills with age (in months) at adolescence. First row: grooved pegboard exe-

cution time (s), second row: repetitive tapping (number of taps in 15 s). Third column: asymme-

try indices (R 2 L)/(L 1 R). Males appear in triangles and solid lines, females in open circles and

dashed lines. The average regression is given by the dotted line.

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next 6 years resulting, in turn, in a large sex difference atthe age of 18 years. Testosterone levels were differentbetween the two sexes, with this divergence emerging dur-ing puberty. Testosterone alone was just as explanative asa model involving a sex-by-age interaction.Increases in T1W signal in white matter typically accom-

pany early stages of myelination [Barkovich, 2000], whiledecreases of the signal are associated with pathologiessuch as amyotrophic lateral sclerosis [Yagishita et al., 1994]or Wallerian degeneration [increase in T2 contrast after 14weeks, Kuhn et al., 1989]. The fact that we saw an age-related increase rather than decrease in aGMd in malesargues against myelination being a relevant determinant ofthe observed changes in the pCST. According to Yagishitaet al. [1994], the diameter of the largest fibers is the majordeterminant of the T1W hypointensity of the pCST. BothYagishita et al. [1994] and Mirowitz et al. [1989] hadobserved a dramatic increase in the detection rates of thepCST after infancy, and they established a compelling par-allel between this phenomenon and Lassek’s early histo-metric data on the development of the pyramidal-axon di-ameter during the first two years of life, and then at 22and 80 years old [Lassek, 1942]. Lassek reported that whilein neonates all fibers are about 1 lm or less in size, at 8–11months a differentiation between large and thick myelin-ated axons and medium-to-small axons occurs. At 7 yearsof age, the size of the largest fiber is of 12 lm [Verhaart,1950], half of the mature size, and the pCST is still seldomdetected with MRI. After 13 years of age, the MRI detec-tion rates become very high. Mirowitz’s detection ratesshow a tendency to decrease with age throughout adult-hood, which is consistent with the age-related decline inthe axonal calibre [Mirowitz et al., 1989; Terao et al., 1994].It should be noted that in previous MR studies white-mat-ter density in the internal capsule increased with age dur-ing childhood and adolescence [Paus et al., 1999], and washigher in the left vs. right hemisphere [Herve et al., 2006].In these two studies, it is possible that the pCST was notdetected as apparent grey-matter by the tissue classifica-tion procedure because of different image contrast and/orclassification algorithms.Our results suggest that the age-dependent changes

observed in males during adolescence are mediated by an

effect of testosterone on axonal calibre. To affect axonal

calibre, it would be necessary for testosterone to influence

the number and/or properties of microtubules and/or

neurofilaments [Hoffman et al., 1987; Marszalek et al.,

1996]. This hypothesis is consistent, for example, with ex-

perimental findings of testosterone-induced upregulation

of b-tubulin expression [Butler et al., 2001; Matsumoto

et al., 1994], or androgen regulation of axonal growth in

motoneurons [for a review, see Fargo et al., 2008]. Since

the effect of testosterone during adolescence would obvi-

ously relate to the late maturation of axon calibre, this phe-

nomenon would likely affect the whole fibre spectrum of

the pCST, small or large, causing the decrease in aGMd.

Hypothesis 3: Manual Skills and pCST

The increase in performance and decrease in inter-man-ual skill asymmetry with age are consistent with the litera-ture on the development of manual skills, as were theobserved sex differences [Bryden and Roy, 2005; Roy et al.,2003; Schmidt et al., 2000a,b]. It is interesting to note, how-ever, that the difference between females and males in thepegboard task seems to emerge during adolescence while,in the tapping task, the sex difference was already presentat 12 years of age. Yet, contrary to our third hypothesis,we found no reliable correlation between manual skillsand the increase in aGMd in the pCST in males duringadolescence. Overall, we conclude that the increase inmanual performance during adolescence is more likely tobe mediated by other structures than the CST. It has beensuggested that the evolution of the manual skills asymme-try pattern could itself be related to the maturation ofinter-hemispheric interactions through the corpus cal-losum, or to an asynchronous maturation of the left andright hemispheres, the left starting earlier than the right[Roy et al., 2003].The effect of age on aGMd of the pCST seen in males

may be a local expression of the global maturation of WMduring adolescence, bearing no specific relevance to handskills. Although one would assume that such a global phe-nomenon would contribute to the establishment of cogni-tive differences seen between adult males and females[Gur et al., 1999], the functional role of this sex-relatedchange itself, if any, is unclear at present. Furthermore,whether or not it could be of importance for brain disor-ders remains to be assessed. Differential maturation ofwhite matter could be a relevant factor for the emergenceof psychopathology during adolescence [Paus et al., 2008],and also later in life [Cahill, 2006]. For instance, sex differ-ences in recovery after stroke have been documented[Stein, 2007; Sue-Min et al., 2005].

CONCLUSION

Here, we took advantage of the presence of a T1Whypo-intensity in the internal capsule likely correspondingto the CST and tested three hypotheses about the relation-ship between hemispheric asymmetry in this anatomicalphenomenon, handedness and sex-specific brain matura-tion of pCST during adolescence. The relationship betweenthe hemispheric asymmetry in aGMd in the pCST andhandedness is best explained by an early appearing (con-stitutive) difference in the number of large fibres in theCST of the ‘‘hand-dominant’’ hemisphere. The observedsexual dimorphism in the T1W hypointensity, establishedduring adolescence, appears to be strongly associated withthe progressive rise of testosterone during male adoles-cence. This latter finding parallels the larger increase inwhite matter volume seen in males over this period. Wespeculate that testosterone affects axonal cytoskeleton and,

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in turn, axonal calibre, rather than myelin-generatingoligodendrocytes.

ACKNOWLEDGMENTS

P.Y.H. is supported by a grant from the Fondation Re-cherche Medicale. We thank the following individuals fortheir contributions in designing the protocol and acquiringand analyzing the data: MR team (Dr. Michel Berube, Syl-vie Masson, Suzanne Castonguay, Julien Grandisson,Marie-Josee Morin), psychometricians (Chantale Belleau,Melanie Drolet, Catherine Harvey, Stephane Jean, HeleneSimard, Melanie Tremblay, Patrick Vachon), ECOBES team(Nadine Arbour, Julie Auclair, Marie-Eve Blackburn,Marie-Eve Bouchard, Annie Houde, Catherine Lavoie, Dr.Luc Laberge) and Julie Berube. We thank Dr. Jean Mathieufor the medical follow-up of subjects in whom we detectedmedically relevant abnormalities.

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