histological asymmetries of primary motor cortex predict...

Histological Asymmetries of PrimaryMotor Cortex Predict Handedness in

Chimpanzees (Pan troglodytes)

CHET C. SHERWOOD,1* ELIZABETH WAHL,2 JOSEPH M. ERWIN,3,4

PATRICK R. HOF,5,6AND WILLIAM D. HOPKINS7,8

1Department of Anthropology, The George Washington University, Washington, DC 200522Department of Anthropology, Kent State University, Kent, Ohio 44242

3Department of Psychology, McDaniel College, Westminster, Maryland 211574Foundation for Comparative and Conservation Biology, Needmore, Pennsylvania 17238

5Department of Neuroscience, Mount Sinai School of Medicine, New York,New York 10029

6New York Consortium in Evolutionary Primatology, New York, New York7Department of Psychology, Agnes Scott College, Decatur, Georgia 30030

8Division of Psychobiology, Yerkes National Primate Research Center, Atlanta, Georgia 30322

ABSTRACTLike humans, chimpanzees display robust and consistent hand preferences during the

performance of certain tasks. Although correlations have been demonstrated between grossanatomic measures of primary motor cortex asymmetry and handedness in captive chimpan-zees, the relationship between histological architecture and behavioral lateralization has notyet been investigated. Therefore, we examined interhemispheric asymmetry of several dif-ferent microstructural characteristics of the primary motor cortex in the region of handrepresentation from 18 chimpanzees tested on a coordinated bimanual task before death. Atthe population level our data showed leftward bias for higher layer II/III neuron density. Ofnote, however, there was no population-level asymmetry in the areal fraction of Nissl-stainedcell bodies, a finding that differs from previous studies of this cortical region in humans.Nonetheless, we found that asymmetry in the density of layer II/III parvalbumin-immunoreactive interneurons was the best predictor of individual hand preference. Theseresults suggest that histological asymmetries are related to handedness in chimpanzees,while overall patterns of asymmetry at the population level might differ from humans. J.Comp. Neurol. 503:525–537, 2007. © 2007 Wiley-Liss, Inc.

Indexing terms: primary motor cortex; handedness; interneuron; parvalbumin; brain evolution

Once thought to distinguish humans from other ani-mals, behavioral lateralization and neuroanatomicalasymmetries have now been shown to be commonplaceamong other species (Rogers and Andrew, 2002; Halpernet al., 2005). In particular, great apes display many of thesame cerebral hemispheric biases that are also present inhumans, such as population-level asymmetry in the sur-face area of the planum temporale (Gannon et al., 1998;Hopkins et al., 1998), the length of the Sylvian fissure(Yeni-Komshian and Benson, 1976; Hopkins et al., 2000),and the sulcal anatomy of the inferior frontal cortex (Hop-kins and Cantalupo, 2004). Furthermore, humanlike lat-eralization in chimpanzees (Pan troglodytes) for preferen-tial hand use has been documented across a range ofbehaviors (Hopkins, 2006). Despite the accumulation of

data concerning such directional biases in humans andnonhumans alike, however, the functional relationship

Grant sponsor: National Institutes of Health (NIH); Grant numbers:NS42867, NS36605; Grant sponsor: National Science Foundation (NSF);Grant numbers: BCS-0515484, BCS-0549117; Grant sponsor: Wenner-Gren Foundation; Grant sponsor: James S. McDonnell Foundation; Grantnumber: 22002078.

*Correspondence to: Chet C. Sherwood, Department of Anthropology,The George Washington University, 2110 G Street, NW, Washington, DC20052. E-mail: [email protected]

Received 8 November 2006; Revised 6 March 2007; Accepted 28 March2007

DOI 10.1002/cne.21399Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 503:525–537 (2007)

© 2007 WILEY-LISS, INC.

between neuroanatomical asymmetries and behaviorallateralization is still not clearly understood.

With regard to humans, the expression of handednessshows a complex pattern of association with structuralinterhemispheric asymmetries of the primary motor cor-tex (Brodmann’s area 4) and language-related cortical ar-eas (Shapleske et al., 1999; Toga and Thompson, 2003;Sun and Walsh, 2006). Although voxel-based morphome-try of in vivo magnetic resonance images (MRI) from hu-mans has not revealed an association between brain anat-omy and handedness (Good et al., 2001), this technique isnot sensitive to subtleties of sulcal contours. In contrast,more focused examinations of asymmetry in the region ofhand representation in the primary motor cortex havedemonstrated a relationship (Hammond, 2002). In partic-ular, right-handed individuals have a deeper central sul-cus in the area of hand representation of the left hemi-sphere, whereas mixed- and left-hand dominantindividuals do not exhibit significant asymmetries of thecentral sulcus (Foundas et al., 1998; Amunts et al., 2000).This central sulcus asymmetry appears to correlate withhandedness only in males (Amunts et al., 2000). Similarly,morphological asymmetries of the central sulcus are alsoassociated with hand preference on a coordinated biman-ual task in chimpanzees (Hopkins and Cantalupo, 2004;Dadda et al., 2006) and New World capuchin monkeys(Phillips and Sherwood, 2005). Taken together, these find-ings suggest that structural asymmetries of the primarymotor cortex reflect hemispheric specialization for control-ling skilled actions of the dominant hand in primates(Hammond, 2002).

The implicit assumption behind such studies of centralsulcus asymmetry is that the observed effects are a con-sequence of underlying hemispheric differences in the his-tological composition or overall volume of this region ofprimary motor cortex. While some authors have suggestedthat gross anatomical asymmetries are due mainly tointerhemispheric differences in total neuron numbers, butnot packing density (Galaburda et al., 1990; Rosen et al.,1993), there is actually very little empirical evidence tovalidate this claim in the human brain and no evidence innonhuman primate brains.

Furthermore, the direct correspondence between micro-structural asymmetries and hand preferences also re-mains uninvestigated. Currently available data on histo-logical asymmetries in human primary motor cortexderive from studies of postmortem brain samples of un-known handedness (Amunts et al., 1996, 1997). Thesestudies have demonstrated population-level asymmetriesin the proportion of space occupied by neuropil in the siteof hand representation as measured by the gray levelindex (GLI) method. Because the overwhelming majorityof humans (�90%) are right-handed, it is assumed thatthese population-level cortical asymmetries relate to handpreferences.

One aim of the present study was to assess whetherchimpanzees show population-level microstructural asym-metries in the primary motor cortex. A second aim was toexamine the relationship between histological asymme-tries of the primary motor cortex and task-specific hand-edness in chimpanzees. Lastly, because macrostructuralasymmetries in the primary motor cortex hand area havebeen previously documented in chimpanzees, another goalof this study was to examine whether variation in asym-metries in gross morphology correlates with microstruc-

tural asymmetries measured from the same region. Ourresearch design takes advantage of the substantial body ofdata on lateralized hand use in captive chimpanzees thathas been amassed over more than a decade (Hopkins etal., 2004). Over time, several of the chimpanzee subjectsthat had been previously behaviorally characterized forhand preferences have died of natural causes. We col-lected brains postmortem from several of these chimpan-zees, providing a unique opportunity to study brain-behavioral correlations. Based on previous studies linkinghandedness with morphological asymmetries in the pre-central gyrus (Hopkins and Cantalupo, 2004; Dadda et al.,2006), we hypothesized that histological asymmetries ofthe primary motor cortex would predict individual differ-ences in hand preferences. To examine this possibility, wemeasured asymmetry in a panel of histological variablesin postmortem brains and determined their contributionto explaining variation in handedness as measured beforedeath on an experimental bimanual coordination task.

MATERIALS AND METHODS

Subjects

Eighteen chimpanzee subjects were used in this study,including 8 females (mean age at death � 36 years, SD �12.7, range � 13–48) and 10 males (mean age at death �26 years, SD � 10.7, range � 10–41). Six of the chimpan-zee subjects were wild-caught before 1973 and lived incaptivity since that time. The remaining 12 chimpanzeeswere born in captivity. All 18 subjects lived in socialgroups ranging from 2 to 13 individuals at Yerkes Na-tional Primate Research Center in Atlanta, Georgia. Allsubjects died from natural causes and were not part of anyresearch protocol that may have contributed to theirdeath.

Behavioral measurements

Hand preference was considered for a task measuringcoordinated bimanual actions, referred to as the tube task(Hopkins, 1995). Handedness data from these subjectshave been previously reported (Hopkins, 1995; Hopkins etal., 2004). For this task, peanut butter is smeared on theinside edges of polyvinylchloride tubes �15 cm in lengthand 2.5 cm in diameter. Each time the subjects reachedinto the tube with their finger, extracted peanut butter,and brought it to their mouth the hand used was recorded.We used measurements of hand preference on this taskbecause it is stable across the lifespan and the strength ofhandedness elicited in chimpanzees by the tube task issignificantly higher than for other actions, such as biman-ual feeding or simple reaching (Hopkins, 2007). Althoughthe number of responses obtained from each subject dif-fered for this task, a minimum of 30 responses were ob-tained for each individual.

Binomial z-scores were calculated for each subject onthe basis of the frequency of left- and right-hand use.Subjects with z-scores greater than 1.95 or less than�1.95 were classified as right- and left-handed, respec-tively. Subjects with z-scores between �1.95 and 1.95were classified as ambidextrous. In addition, a handed-ness index (HI) was derived for each subject by subtract-ing the number of right-handed responses from the num-ber of left-handed responses and dividing by the totalnumber of responses: HI � (R � L) / (R � L). Positive

The Journal of Comparative Neurology. DOI 10.1002/cne

526 C.C. SHERWOOD ET AL.

values reflect right-hand preference and negative valuesrepresent left-hand preference. The absolute value of theHI corresponds to the consistency of directional hand pref-erence. Individuals that performed neuroanatomical mea-surements were blind to the HI score of the subjects.

MRI and measurement

Brains from each subject were obtained after death andwere immersion-fixed in 10% formalin. In most cases theprecise postmortem interval was not recorded at YerkesNational Primate Research Center; however, it was nevergreater than 14 hours. All of the brains were scannedpostmortem using a T2-weighted protocol with a 1.5 Tmagnet. Images were collected in the transverse planeusing a gradient echo protocol (pulse repetition � 22.0 s,echo time � 78.0 ms, number of signals averaged � 8–12,and a 256 � 192 matrix reconstructed to 256 � 256). The“knob” area of the precentral gyrus that corresponds to thelocation of hand representation was identified in serial1-mm slices in the axial plane following procedures previ-ously used in human and ape brains (Yousry et al., 1997;Hopkins and Pilcher, 2001). Quantification of the knobregion was obtained in the axial plane because this is themost common approach used in human studies (Ham-mond, 2002) and it is difficult to reliably quantify thisregion from other planes in chimpanzees. Nonetheless, aprevious study that quantified the hand knob in humanbrains from the sagittal plane found that these measuresof asymmetry correlated with the subjects’ handedness(Foundas et al., 1998). Because these results are largelyconsistent with the findings reported for measures fromthe axial plane, we reasoned that the axial measurementof the knob was sufficiently representative of the primarymotor cortex hand area of chimpanzees.

Morphological measurements of the hand knob wereperformed using ANALYZE software (ANALYZE, Lenexa,KS). The horizontal epsilon or inverted omega that pro-jected into the postcentral gyrus was traced on each image(Fig. 1). The dorsal and ventral edges of the knob served asthe markers for defining the boundaries of the area. Foreach slice and hemisphere, an area measurement of theregion was calculated by use of a mouse-driven pointerthat traced the region of interest. The total of area mea-surements from all slices in which the knob was presentwere summed and used to derive a volumetric measure foreach hemisphere (ranging from 5 to 13 slices in the sam-ple).

Tissue preparation andimmunohistochemistry

The region of hand representation in primary motorcortex was dissected from each hemisphere as a block �4cm thick containing the pre- and postcentral gyri. To es-timate the location of hand representation, the hand knoblandmark was viewed on horizontal MRI scans of eachbrain (Hopkins and Pilcher, 2001) and the correspondingdorsoventral level was noted on the lateral surface. Inaddition, prior to dissection the central sulcus was spreadopen to reveal the middle genu, which is anatomicallyequivalent to the hand knob seen in axial MRI sections inhuman brains (Yousry et al., 1997). The position of thehand knob landmark generally accords with previous elec-trophysiological studies of motor maps in this species(Grunbaum and Sherrington, 1903; Leyton and Sher-rington, 1917; Hines, 1940; Dusser de Barenne et al.,

1941; Bailey et al., 1950). Moreover, a recent functionalimaging study on grasping using positron emission tomog-raphy (PET) in five chimpanzees found significant activa-tion in the knob region in the hemisphere contralateral tothe hand used (Hopkins et al., 2006).

After dissection, tissue blocks were cryoprotected byimmersion in buffered sucrose solutions up to 30%, frozenon dry ice, and sectioned at 40 �m with a sliding mic-rotome perpendicular to the axis of the central sulcus.Every 10th section (400 �m apart) was stained for Nisslsubstance with a solution of 0.5% cresyl violet. For eachindividual, sections from both hemispheres were Nissl-stained together in order to ensure comparable stainingconditions for subsequent analyses. Sections not used forimmediate staining were cryoprotected in a storage solu-tion consisting of glycerol, ethylene glycol, dH2O, andphosphate buffer (3:3:3:1 volume/volume) and archived at�20°C.

Immunohistochemistry was performed for each antigenon adjacent 1:20 series of sections. Free-floating sectionswere stained with mouse monoclonal antibodies to a non-phosphorylated epitope in neurofilament H (SMI-32 anti-

Fig. 1. The region of the hand knob landmark as seen in four axialMRI sections of a chimpanzee brain from dorsal to ventral (A–D). Bluelines indicate the tracing of the central sulcus (CS). The horizontalepsilon or inverted omega that projects into the precentral gyrus wastraced on each image, shown here in yellow. The dorsal and ventraledges of the knob defined the boundaries of the area measured.


527HISTOLOGY AND HANDEDNESS IN CHIMPANZEES

body; Covance International, Netherlands; dilution1:2,000) and parvalbumin (Swant, Belinzona, Switzer-land, Cat. no. 235; dilution 1:10,000). The SMI-32 mousemonoclonal IgG1 antibody was raised against the non-phosphorylated epitope of neurofilament H isolated fromhomogenized hypothalami of Fischer 344 rats. On conven-tional immunoblots, SMI-32 visualizes two bands (200and 180 kDa), which merge into a single neurofilament Hline on two-dimensional blots (Sternberger and Stern-berger, 1983). The antibody reacts with a nonphosphory-lated epitope from 200-kD neurofilament heavy chain ofmost mammalian species. This protein is expressed in cellbodies, dendrites, and some thick axons within a subset ofneurons that are mostly pyramidal cells (Campbell andMorrison, 1989; Hof and Morrison, 1995). Other cells andtissues are unreactive and the antibody does not recognizethe phosphorylated 200-kD neurofilament heavy chain.The mouse monoclonal IgG1 PV antibody was raisedagainst PV from carp muscle. It has been shown to bindwith high affinity to the tertiary structure of PV frommultiple species including macaque monkeys and hu-mans, with binding eliminated by addition of exogenousPV (Celio et al., 1988). No crossreactivity with othercalcium-binding proteins was noted in radioimmunoassayand immunoblotting assays (Celio et al., 1988). In primatebrain tissue the pattern of staining with this antibody isconsistent with that previously established for other PVantibodies (Conde et al., 1994).

Prior to immunostaining, sections were rinsed thor-oughly in phosphate-buffered saline (PBS) and pretreatedfor antigen retrieval by incubation in 10 mM sodium ci-trate buffer (pH 3.5) at 37°C in an oven for 30 minutes. Forthe SMI-32 antibody, antigen retrieval used the samebuffer with pH 8.5 at 90°C in a water bath to achieveimproved staining. Sections were then immersed in a so-lution of 0.75% hydrogen peroxide in 75% methanol toeliminate endogenous peroxidase activity. After rinsingagain, sections were incubated in the primary antibody ina diluent containing PBS with 2% normal horse serum

and 0.3% Triton X-100 for �48 hours on a rotating table at4°C. After rinsing in PBS, sections were incubated in thesecondary antibody (biotinylated antimouse IgG, VectorLaboratories, Burlingame, CA; dilution 1:200) and pro-cessed with the avidin-biotin-peroxidase method using aVectastain ABC kit (Vector Laboratories). Immunoreac-tivity was revealed using 3,3�-diaminobenzidine (DAB).Sections were counterstained with cresyl violet to visual-ize nonimmunoreactive neurons and cytoarchitecturalboundaries. Specificity of the reaction was confirmed byprocessing negative control sections as described, but ex-cluding the primary antibody. No immunostaining wasobserved in control sections. Each set of sections from eachhemisphere were stained together to control for interex-periment variation. In this way, because lateral asymme-try was of interest, differences in fixation protocols, dura-tion of fixation, storage conditions, and postmortem delaywithin each individual case would affect both hemispheresequally. Examples of immunohistochemical staining re-sults are shown in Figure 2.

Histological identification of the region ofinterest

We identified the region of interest for all subsequentquantitative measurements as primary motor cortex(Brodmann’s area 4) based on previous descriptions of thecytoarchitecture of this area in chimpanzees (Bailey et al.,1950; Sherwood et al., 2003, 2004b, 2006). In brief, theprimary motor cortex is distinguished by giant Betz cellsin the lower portion of layer V, low overall cell density,large average cellular sizes, a poorly defined layer IV, anda diffuse border between layer VI and the subjacent whitematter. The border between area 4 and area 3a, whichusually occurs close to the fundus of the central sulcus, isrecognized by the development of a well-defined granularlayer IV. Although the border between area 4 and premo-tor cortex (area 6) may occur along the convexity of theprecentral gyrus, we restricted our analyses to the portionof area 4 on the anterior bank of the central sulcus (Fig. 3).

Fig. 2. Examples of histological staining in chimpanzee primary motor cortex (Brodmann’s area 4).Staining for Nissl substance with cresyl violet (A), nonphosphorylated neurofilament protein with cresylviolet counterstain (B), and parvalbumin with cresyl violet counterstain (C) are shown. Scale bar �50 �m.



Measurement of relative layer thickness

Using a portion of the precentral gyrus where corticallayers were most easily distinguishable and not obscuredby tangential sectioning, three sections spaced 400 �mapart were selected to measure the relative thickness ofcortical layers. Measurement sites were positioned to fallalong a part of the anterior bank of the central sulcus thatwas not folded at the crown of a gyrus or within the depthof a fundus. At each measurement site a reference contourwas drawn from the pial surface to the white matterinterface following the radial orientation of cortical mini-columns at low magnification (4�) using a computerizedstereology and morphometry system consisting of a ZeissAxioplan 2 photomicroscope equipped with a Ludl XYmotorized stage, Heidenhain z-axis encoder, an OptronicsMicroFire color videocamera, a Dell PC workstation, andStereoInvestigator software v. 6 (MBF Bioscience, Willis-ton, VT). The length of each cortical layer was then mea-sured along the radial guideline. We segmented the pri-mary motor cortex along the most obvious layerboundaries: I, II/III, and V/VI (Fig. 3). In general, theselaminar subdivisions correspond to functional differencesin connectivity patterns. Molecular layer I containsmainly apical dendrites and horizontally oriented axons.Neurons in supragranular cortical layers II/III are in-volved in corticocortical integrative processes and haveaxonal projections to other ipsi- and contralateral corticalareas. Infragranular layers V/VI are comprised of neuronsparticipating in corticofugal systems projecting to the spi-nal cord, brainstem, striatum, and thalamus.

The border between layers III and V was identified bythe presence of a poorly developed, yet detectable, innergranular layer IV. In each section measurements wereperformed at two locations spaced 2 mm apart. At eachmeasurement site the fraction of the total cortical thick-

ness occupied by the width of each laminar segment wascalculated as the relative layer thickness. An averagerelative layer thickness for each hemisphere was obtainedfrom these measurements.

Areal fraction of Nissl-stained tissue

We quantified the areal fraction (AF) of tissue com-prised of Nissl-stained cell bodies of neurons, glia, andendothelial cells in layers II/III and V/VI from high-resolution images. The AF represents the proportion ofstained cellular profiles that project onto a two-dimensional measuring plane. Using the same threeNissl-stained sections used for analysis of relative layerthickness, digital images were collected using fractionatorsampling as implemented by the StereoInvestigator sys-tem. First, contours were drawn around layers II/III andV/VI at low magnification. Then a fractionator samplingdesign (grid spacing of 600 � 800 �m for layer II/III; 800 �800 �m for layer V/VI) was used to obtain a series of 8-bitgrayscale image frames in a systematic random fashionwith a 20� (0.50 N.A.) Plan-Neofluar objective lens. Priorto collection of image frames in each section, the exposureof the digital camera was standardized to an averagetarget intensity of 70%. Images covered 440 � 587 �m andwere 1,600 � 1,200 pixels in size, yielding a resolution of0.37 pixels per �m. Image frame acquisition was moni-tored during fractionator sampling and all images that felloutside of the laminar region of interest boundaries wereomitted from further processing. On average, 24.3 � 4.5(mean � SD) image frames representing each laminarregion of interest were collected for AF analysis. To mea-sure the AF, images were processed in ImageJ software v.1.32j with background subtraction using a rolling ballalgorithm (Sternberger, 1983), converted to binary by anautomated threshold routine based on Rider and Calvard

Fig. 3. The part of primary motor cortex (Brodmann’s area 4) thatwas sampled for quantitative measurements. Only the intrasulcalportion of the precentral gyrus was analyzed (A). Arrowheads illus-trate the typical borders used in defining the region of interest. Ahigher magnification view of the cytoarchitecture from the segment of

primary motor cortex indicated by the dashed line box in A is shownin B. The layers boundaries that were defined for various quantitativemeasurements are shown in B. PoG, postcentral gyrus; PrG, precen-tral gyrus; wm, white matter. Scale bar � 500 �m in B.



(1978), and dilation and erosion were applied to fill smallholes representing light staining of cellular nuclei (Fig. 4).After converting the image to binary, the percentage of themeasuring frame area occupied by pixels representingstained elements was calculated. The AF value for eachhemisphere is the section-weighted mean of AF measure-ments calculated from all frames.

Stereologic analyses

Quantification of numerical densities of cells and theirvolumes within layer II/III was performed using the Ste-reoInvestigator stereology system. We focused on layerII/III for these analyses because many prior studies con-cerning histological asymmetry in the human neocortexhave identified significant hemispheric differences specif-ically in the superficial cortical layers (Hayes and Lewis,1995, 1996; Buxhoeveden et al., 2001; Hutsler, 2003; Gar-cia et al., 2004), suggesting that the functional role ofthese layers may be particularly relevant for hemisphericspecialization.

Strict stereological quantification of total cell numbersis not feasible within the restricted region of hand repre-sentation of primary motor cortex because distinct bound-aries cannot be established to separate this area from themotor representation of adjacent body parts. Nonetheless,asymmetries of cell-type-specific numerical densities canprovide a useful reflection of hemispheric specialization.In this way, our data characterize asymmetries in thecellular composition per unit of tissue. Densities of neu-rons and glial cells were estimated from sections stainedfor Nissl substance. The density of PV-immunoreactive(ir) interneurons was also estimated. Cell densities inlayer II/III were obtained using the optical disector withfractionator sampling following methods described in aprevious study (Sherwood et al., 2007). The same threesections used for AF analysis, or those in adjacent series,were quantified. After outlining the boundaries of layerII/III at low magnification, a set of optical disector frames(30 � 30 �m for neurons and glia; 90 � 90 �m for PV-irinterneurons) were placed in a systematic random fashionto yield �30 frames per section. Disector analysis wasperformed under Koehler illumination using a 63� objec-

tive (Zeiss Plan-Apochromat, N.A. 1.4). The thickness ofoptical disectors was set to 6 �m to allow for a minimum2-�m guard zone on either side of the section after z-axiscollapse from histological processing. Cellular densities(Nv) were derived from these stereologic counts and cor-rected for shrinkage from histological processing by thenumber-weighted mean section thickness. On average,the coefficient of error (Schmitz and Hof, 2000) of mea-surements was 0.07 � 0.03 for neurons, 0.08 � 0.03 forglial cells, and 0.11 � 0.02 for PV-ir interneurons. Thesecoefficients of error are somewhat larger than is commonin stereologic studies because we restricted our counts tothe part of the precentral gyrus where cortical layers weremost easy to define. This amount of measurement errorwould be expected to reduce the probability of rejectingthe null hypothesis in statistical tests; however, findingsof statistical significance in spite of this error can beconsidered reliable.

Cellular volumes of neurons immunostained for non-phosphorylated neurofilament protein (NPNFP) and PVwere estimated using the nucleator with a vertical design(Gundersen, 1988). Neurons were selected for volumemeasurement systematic randomly by applying opticalfractionator sampling in two sections. In this way, thedistribution of cell volumes obtained comprises an unbi-ased representation of the total population. The verticalaxis of the probe was a line running superior-to-inferior tothe pial surface. The centroids of neurons within the in-clusion boundaries of optical disectors were marked andtwo transect lines from randomly selected directions werecentered at the marker and superimposed over the neu-ron. The intersection of each line with the outer surface ofthe neuronal soma was marked and cellular volume wasmeasured based on the nucleator principle. Because it wasnot feasible to perform isotropic-uniform-random section-ing in these rare behaviorally characterized chimpanzeematerials, our mean cellular volume estimates contain adegree of bias due to a preferred sectioning orientation.However, due to normal variations in the orientation ofthe tissue in our preparations, not all cells were cut alongthe same axis, thereby generating a degree of randomnessin the sample. Furthermore, coronal and sagittal sections

Fig. 4. Image conversion method for measurement of areal fraction (AF). An image frame of Nissl-staining in layer II/III is shown (A). Images were processed with background subtraction and conversionto binary to measure the proportion of spaced occupied by stained cells (B). Scale bar � 50 �m.



have been shown to yield comparable results to isotropic-uniform-random sections using this probe (Schmitz et al.,1999). In each hemisphere for each individual, 73.1 � 13.6cell soma volumes were sampled for NPNFP-ir neuronsand 43.6 � 16.6 for PV-ir interneurons. Mean cell volumewas calculated for each hemisphere.

Data analysis

Lateral asymmetries in the various anatomical mea-surements were quantified by calculating an asymmetryquotient (AQ) using the formula: AQ � (R � L) / ((R � L) �0.5). Positive AQ values signify right hemisphere domi-nance, negative values signify left hemisphere dominance,and zero denotes symmetry. The absolute value of the AQindicates the degree of asymmetry. Table 1 provides AQvalues for all neuroanatomical variables in each chimpan-zee subject. Mann–Whitney U-tests did not reveal signif-icant differences between sexes for HI score or any neu-roanatomical AQ measurement; therefore, sexes werepooled in subsequent analyses. Furthermore, becausenone of the behavioral or neuroanatomical measurementsshowed a correlation with age, it was also not consideredin the analysis. Nonparametric Spearman rank order cor-relations were used to examine associations with handed-ness because coordinated bimanual HI data was not nor-mally distributed. We applied a sequential Bonferroniadjustment on a per hypothesis basis to adjust for mul-tiple comparisons in correlation and t-test analyses. How-ever, because type II error is increased by this method, wereport statistical significance at � � 0.1.

Forward stepwise multiple regression analysis wasused to examine whether the combination of various ana-tomical AQ variables could predict variation in HI score.The forward stepwise approach sequentially selects themost highly correlated independent variable, removes theassociated variance in the dependent, then enters furtherindependents into the model which most correlate withthe remaining variance in the dependent until selection ofan additional independent does not increase r2 by a sig-nificant amount (P 0.05). The final model includes areduced number of predictor variables which collectively

make the strongest, uncorrelated contributions to explain-ing variation in the dependent. Assumptions of multipleregression analysis were checked and each independentpredictor variable was normally distributed as deter-mined by Shapiro–Wilk’s W-tests. Furthermore, aftermultiple regressions, plots of studentized residuals versusunstandardized predicted values did not show nonlinear-ity or heteroscedasticity. Finally, binomial logistic regres-sion was used to explore relationships between anatomicalpredictors and categorical handedness classification. Foranalyses that did not involve Bonferroni correction, sta-tistical significance is reported at � 0.05 (two-tailed).

Photomicrography

Photomicrographs were obtained using an OptronicsMicroFire digital camera mounted on a Zeiss Axioplan 2microscope. Brightness and contrast of images were ad-justed using Adobe Photoshop 6.0 software (San Jose, CA).Adobe Illustrator 8.0 was used for assembling and label-ing figures.

RESULTS

Population-level asymmetry

In the current sample of 18 chimpanzees, there wassignificant rightward dominance of hand preference forthe coordinated bimanual task (mean HI � 0.30, SD �0.58; single-sample t-test: t17 � 2.24, P � 0.04). Based onz scores, 14 chimpanzees were classified as right-handed,3 were left-handed, and 1 was ambidextrous (4 were clas-sified as nonright-handed). In addition, this group of chim-panzees displayed a significantly larger hand knob in theleft hemisphere (mean AQ � �0.19, SD � 0.37; single-sample t-test: t17 � �2.15, P � 0.05). In a previous studyof a larger cohort of captive chimpanzees, population-levelright-hand dominance for the coordinated bimanual taskwas also demonstrated, although the hand knob region ofthe primary motor cortex did not show significant asym-metry (Hopkins and Cantalupo, 2004).

Next, single-sample t-tests were performed on the AQ ofeach histological variable to test for a significant deviation

TABLE 1. Individual Subject Data

YerkesCode Sex

Age atDeath HI HC

HandKnob

NeuronDensityin Layer

II/III

GliaDensityin Layer

II/III

AF inLayerII/III

AF inLayerV/VI

RelativeThicknessof Layer I

RelativeThicknessof Layer

II/III

RelativeThicknessof Layer

V/VI

PV-irInterneuronDensity inLayer II/III

PV-irInterneuronMean CellVolume inLayer II/III

NPNFP-ir Mean

CellVolumein Layer

II/III

C0630 F 13 0.51 R �0.06 0.01 0.00 0.03 0.03 �0.06 �0.06 0.05 �0.06 �0.07 �0.17C0576 F 20 0.34 R 0.22 �0.16 �0.03 0.05 0.05 �0.04 0.04 0.10 0.25 �0.14 �0.08C0342 F 35 0.36 R �0.28 �0.41 �0.41 0.01 0.12 �0.05 �0.05 0.04 �0.38 �0.29 �0.24C0320 F 39 �0.69 L �0.06 �0.78 �0.01 �0.21 �0.21 �0.07 0.08 �0.06 0.24 �0.35 �0.14C0406 F 41 0.91 R �0.76 0.17 0.16 �0.11 �0.09 0.31 0.08 �0.12 �0.21 0.14 0.41C0336 F 44 �0.16 A �0.44 0.04 0.22 0.19 0.20 �0.07 0.04 �0.03 0.07 �0.01 �0.28C0408 F 45 1.00 R �0.29 0.06 �0.16 �0.16 �0.03 �0.09 �0.02 0.03 �0.13 �0.04 �0.06C0242 F 48 0.68 R �0.13 �0.88 �0.50 0.01 0.08 �0.21 �0.06 0.10 �0.53 0.04 �0.02C0645 M 10 0.45 R �0.09 �0.03 0.00 �0.06 �0.04 �0.04 �0.01 0.02 �0.63 0.04 0.57C0573 M 15 0.83 R �0.51 �0.15 0.02 �0.08 �0.20 �0.09 �0.16 0.09 0.29 �0.02 �0.30C0507 M 17 0.35 R �0.78 �0.10 0.09 0.22 0.19 0.04 0.05 �0.06 �0.24 0.14 0.12C0491 M 18 0.34 R �0.08 �0.21 �0.29 0.02 0.00 �0.06 �0.04 0.05 �0.41 0.23 �0.13C0429 M 24 0.50 R �0.06 0.17 �0.05 0.03 �0.02 0.26 0.15 �0.15 �0.30 �0.63 �0.40C0423 M 25 0.36 R 0.09 �0.22 �0.05 �0.06 �0.12 0.20 0.01 �0.03 0.14 �0.02 0.40C0369 M 32 0.77 R �0.59 �0.40 �0.22 0.00 0.04 0.13 0.17 �0.05 �0.27 0.12 0.01C0301 M 35 �0.71 L 0.81 �0.18 �0.34 �0.33 �0.29 0.41 0.16 0.14 0.62 �0.47 0.69C0273 M 40 0.65 R �0.23 �0.21 �0.09 �0.13 �0.13 �0.40 �0.17 0.17 0.16 0.03 0.29C0367 M 41 �1.00 L �0.15 �0.37 �0.02 �0.04 �0.06 0.08 �0.04 0.03 0.20 0.10 0.83

Neuroanatomical data are reported as asymmetry quotients (AQs).F, female; M, male; HI, coordinated bimanual handedness index; HC, handedness classification; R, right; L, left; A, ambidextrous; AF, areal fraction of Nissl-stained tissue.



from symmetry, i.e., a reference constant of zero (Table 2).Figure 5 shows mean AQs for all neuroanatomical mea-surements. Of all the histological measures, only layerII/III neuron density showed a significant population-levelbias (mean AQ � �0.20, SD � 0.29, t17 � �3.01, P �0.008, corrected P � 0.08). The left hemisphere tended tocontain a higher density of neurons than the right.

Test of correlation between externalmorphology and histology

A series of Spearman rank order correlations were usedto examine the relationship between asymmetry in theexternal morphology of the primary motor cortex and itsunderlying microstructural organization. Only the corre-lation between the hand knob AQ and layer II/III PV-ircell volume AQ was significant (r � �0.61, n � 18, P �0.008, corrected P � 0.08).

Prediction of the direction and strength ofhand preference

We calculated a Spearman correlation matrix to exam-ine the bivariate relationships between each neuroana-tomical AQ value and coordinated bimanual HI sepa-rately. The hand knob AQ (r � �0.50, P � 0.04, n � 18)showed an association with the HI score; however, it wasnot significant after adjusting for multiple comparisons.No other correlations were significant.

Next we performed forward stepwise multiple regres-sion analysis to evaluate whether a linear combination ofindependent variables could predict HI scores on the co-ordinated bimanual task. The AQ values for all neuroana-tomical measures were entered as independent variables.This analysis yielded a model that explained a significantproportion of variance in handedness (adjusted r2 � 0.51,P � 0.02). In this analysis, the standard partial regressioncoefficient (�) represents a measure of the unique contri-bution of each independent variable. It is the averageamount the dependent variable changes when the inde-pendent varies by one standard deviation and other inde-pendent variables are held constant. Listed in order of thestrength of the standard partial regression coefficient, thepredictors that contributed to this model included layerII/III PV-ir interneuron density AQ (� � �0.62, t11 ��2.84, P � 0.02), hand knob AQ (� � �0.45, t11 � �2.09,P � 0.06), relative thickness of layer V/VI AQ (� � 0.44,t11 � 2.15, P � 0.05), AF in layer II/III AQ (� � �0.40,t11 � �1.73, P � 0.11), layer II/III neuron density AQ (� �

0.39, t11 � 2.21, P � 0.05), and layer II/III NPNFP-ir meancell volume AQ (� � �0.37, t11 � �1.98, P � 0.07). Biva-riate plots of these predictor variables and coordinatedbimanual HI score are shown in Figure 6.

Test of differences between right-handedand nonright-handed chimpanzees

Visual inspection of Figure 6 makes it clear that datafrom the nonright-handed subjects (i.e., left-handed andambidextrous) exerted strong leverage on the regressionanalysis. These results prompted us to also test for differ-ences among subjects that were categorized dichoto-mously as either right- or nonright-handed. First, we useda series of Mann–Whitney U-tests to examine whetherneuroanatomical AQ values differed between handednessgroups. Of all the comparisons, only layer II/III PV-irinterneuron density displayed a difference between hand-edness categories (U4,14 � 8, z � 2.12, P � 0.03); however,it was not significant after adjusting for multiple com-parisons. We also used binomial logistic regression anal-ysis to examine whether the combination of neuroana-tomical AQ values could differentiate individuals thatwere categorized dichotomously as either right-handed ornonright-handed. The logistic regression approached sig-nificance (�2 � 18.13, df � 11, P � 0.08).

Fig. 5. The population-level mean asymmetry quotient (AQ) forneuroanatomical measures in the sample of 18 chimpanzees. PositiveAQs represent rightward bias and negative AQs represent leftwardbias. Bars indicate mean, error bars indicate � standard deviation.

TABLE 2. Descriptive Statistics of Handedness Index (HI) and AsymmetryQuotient (AQ) Values and Results of One-Sample T-tests Using a Reference

Constant of Zero

MeanAQ SD t P

Coordinated bimanual HI 0.30 0.58 2.24 0.04Hand knob �0.19 0.37 �2.15 0.05Neuron density in layer II/III �0.20 0.29 �3.01 0.01Glia density in layer II/III �0.09 0.19 �2.05 0.06AF in layer II/III �0.03 0.13 �1.12 0.28AF in layer V/VI �0.03 0.13 �0.85 0.40Relative thickness of layer I 0.02 0.19 0.34 0.74Relative thickness of layer II/III 0.01 0.10 0.42 0.68Relative thickness of layer V/VI 0.02 0.09 0.85 0.41PV-ir interneuron density in layer II/III �0.07 0.33 �0.85 0.40PV-ir interneuron mean cell volume in layer II/III �0.07 0.23 �1.22 0.24NPNFP-ir mean cell volume in layer II/III 0.08 0.36 0.97 0.35

P values are uncorrected.



Fig. 6. Bivariate plots of predictors of coordinated bimanual handedness index from multiple regres-sion analysis, presented in the order of the strength of the relationship (A–D). Open circles indicateright-handed subjects; closed circles indicate nonright-handed subjects.



DISCUSSION

Our findings indicate that the region of hand represen-tation of the primary motor cortex of chimpanzees exhibitspopulation-level asymmetry of histological architecture,although in a manner that differs from humans. Further-more, our data reveal an association between individualhand preferences and microstructural asymmetries of thiscortical area. This study is of special significance becauseit represents one of the first characterizations of histolog-ical asymmetry within any region of the chimpanzee neo-cortex and therefore provides an essential comparativecontext for understanding the evolutionary history of cor-tical asymmetries in humans.

Methodological considerations

In discussing our results, it is important to keep in mindthat the AF value represents the Nissl-stained cellularvolume fraction, which is not the same as the numericaldensity of the constituent cells. The AF is based on boththe number of cells and their sizes. Furthermore, the AF iscalculated from all Nissl-stained cellular profiles andhence represents the contribution of different classes ofneurons, various types of glia, and endothelial tissue. Inthe present study, we used AF for two reasons. First, theAF is inversely proportional to the amount of neuropilspace available for synapses, dendrites, and axons. There-fore, it provides a useful indirect measure of the fraction oftissue involved in interconnectivity among neurons. Sec-ond, for the purpose of analyzing hemispheric asymmetryin histological architecture, our measurement of AF wasdesigned to be comparable to the GLI as reported in pre-vious studies of human cortex (Amunts et al., 1996, 1997,1999, 2003). The GLI technique is used for multidimen-sional characterization of cytoarchitecture and definitionof cortical area boundaries. In plotting vertical changes incell volume densities, the GLI is measured from manysmall fields within the image frame across a series oftransects that run from the pial surface to the whitematter interface. Our method differs in that we used frac-tionator sampling to collect image frames and extractedan AF measurement from the entire image. Nonetheless,one application of the GLI method in prior studies ofasymmetry of human neocortex has been to calculate av-erage layer-wise or whole cortex GLI values. As discussedbelow, this particular application of the GLI method,which provides data that are similar to our technique formeasuring AF, has consistently found asymmetries in hu-mans.

Population-level effects and comparison tohumans

Population-level asymmetries of histological organiza-tion have been described for areas of the human cerebralcortex that are known to be functionally lateralized suchas the primary motor cortex, Broca’s area, and the planumtemporale (Hayes and Lewis, 1995; Amunts et al., 1996,1999; Anderson et al., 1999; Buxhoeveden et al., 2001;Hutsler, 2003; Uylings et al., 2006). Interpretation of therelationship between functional lateralization and ana-tomical asymmetry is complicated in these postmortemstudies, however, because information is generally notavailable for subjects regarding hemispheric dominancefor language and handedness. Nonetheless, conclusionsabout such an association are often drawn under the as-

sumption that any robust anatomical asymmetries relateto the high incidence of right-handedness and left hemi-sphere language dominance in humans (Toga and Thomp-son, 2003).

In this context, the most direct comparison of our re-sults with findings from postmortem human brain studiesconcerns population-level asymmetry. To make our anal-yses comparable to existing human data, we measuredrelative layer thicknesses, neuron density, AF, andNPNFP-ir pyramidal cell volumes. Additional measure-ments of glia density, PV-ir interneuron density, andPV-ir cell volumes were performed to extend beyond cur-rently documented asymmetries in the human cerebralcortex. To our knowledge, the sample size of behaviorallycharacterized chimpanzee brains that were available forthe current study (n � 18) exceeds the sample size fromnearly all studies of adult humans, with the exception ofone study of pyramidal cell size asymmetry in area 45(Hayes and Lewis, 1995). It is also important to note thatright-handedness occurs in a smaller proportion of captivechimpanzees than in humans. Approximately two-thirdsof all captive chimpanzees examined are right-handed onthe bimanual coordination task (Hopkins, 2006). Of thechimpanzees in the current study, 78% (14 out of 18) wereclassified as right-handed. Thus, it might be expected thatany population-level tendencies for histological asymme-tries will be more difficult to detect in chimpanzees than inhumans based on small sample sizes. Nonetheless, wefound that neuron density in layer II/III was significantlyhigher in the left hemisphere primary motor cortex acrossthis sample of chimpanzees. Neuron densities have not yetbeen examined for asymmetries in the primary motorcortex of humans using stereologic methods. However,asymmetries of neuronal numerical density in other cor-tical areas of humans have been reported. In normal hu-mans, neuron density distributions do not show asymme-tries within the planum temporale (Anderson et al., 1999)or Broca’s area (areas 44 and 45) (Garcia et al., 2004;Uylings et al., 2006), whereas dorsolateral prefrontal cor-tex (area 9) has greater overall neuron densities in the lefthemisphere (Cullen et al., 2006). This pattern of results inhumans raises the question of whether neuron densityasymmetries relates to functional lateralization.

Besides neuron density, we did not find evidence ofpopulation-level asymmetry of any other histological fea-ture, including AF. In fact, the AF AQ values in layersII/III and V/VI were among the least asymmetric of any ofthe variables analyzed (Table 1, Fig. 5). These resultscontrast with findings from humans, where significantlylower GLI (i.e., a greater amount of neuropil space) wasfound in the left hemisphere primary motor cortex in asample of 12 adult individuals of unknown handedness(Amunts et al., 1996). A later study by Amunts et al.(1997) showed that neuropil asymmetry of human pri-mary motor cortex develops within the infragranular cor-tical layers in childhood, whereas the subsequent appear-ance of neuropil asymmetry does not occur insupragranular layers until adulthood. Furthermore, anal-yses of Broca’s area (areas 44 and 45) (Amunts et al.,1999), the cortex of the planum temporale (area Tpt)(Anderson et al., 1999; Buxhoeveden et al., 2001), as wellas areas V1, V2, and V5/MT� (Amunts et al., 2007) inhumans have all reported left dominance of the neuropilvolume fraction. Interestingly, neuropil asymmetry hasnot been found in area Tpt of chimpanzees or rhesus



macaques (Buxhoeveden et al., 2001). Although cautionshould be exercised when interpreting results that fail toshow asymmetry in nonhuman species based on smallsample sizes, it is notable that several different corticalregions in humans appear to exhibit relatively greaterneuropil space in the left hemisphere at the populationlevel, whereas no such asymmetry has yet been detectedin chimpanzees or other primates.

Taken together with these previous studies, our find-ings suggest that the human cortical phenotype differsfrom chimpanzees in showing a fundamental structuralasymmetry in the space occupied by neuropil versus cellsomata. Such divergence in the microstructural architec-ture of homotopic cortical areas might be, at least in part,determined by initial asymmetries in neuronal connectiv-ity occurring early in development and guided by a com-bination of intrinsic and extrinsic signals (Stephan et al.,2007). Indeed, several genes that show differential expres-sion between the cerebral hemispheres during early fetaldevelopment of humans also exhibit elevated rates ofcoding-sequence and regulatory evolution in humans com-pared to chimpanzees (Sun et al., 2006). This evidencesuggests that some of the genes associated with brainasymmetry in humans have been the target of naturalselection, since chimpanzees and humans diverged from acommon ancestor 6–8 million years ago. Of the genes thatare both upregulated and show higher levels of expressionin the left hemisphere of human fetal cortex, those com-prising the upper 10% of fold-differences between speciesinclude genes involved in functions such as protein assem-bly, intracellular transport, and transcription (PIN1),c-fos signaling pathways (CROC4), enzymatic regulationof glycolysis (PFKP), and assembly of the cytoskeleton(CKAP1).

Histological relationships with handedness

Gross anatomical asymmetries of the region of handrepresentation of the primary motor cortex have beenshown to correlate with hand preferences in humans,chimpanzees, and capuchin monkeys (Amunts et al., 2000;Hopkins and Cantalupo, 2004; Phillips and Sherwood,2005; Dadda et al., 2006), suggesting that handedness isfacilitated by a greater total mass of neural tissue devotedto controlling the dominant hand. This hypothesis is fur-ther supported by evidence from magnetoencephalogra-phy in humans (Volkmann et al., 1998) and intracorticalmicrostimulation in New World squirrel monkeys (Nudoet al., 1992) indicating that the size of forelimb movementrepresentation is significantly increased in the primarymotor cortex opposite to the preferred hand. However,beyond these large-scale interhemispheric biases in thecortical tissue dedicated to hand movement representa-tion, do changes to the internal histological wiring of pri-mary motor cortex also subserve the expression of hand-edness? As discussed previously, current data frompostmortem human brain studies cannot address thisquestion directly.

Our results demonstrate that variation in the directionand magnitude of hand preference in chimpanzees can bepredicted by asymmetries of certain histological featuresof the primary motor cortex. It is unlikely that the neuro-anatomical asymmetries observed among the nonright-handed chimpanzees in our sample can be explained byother variables aside from hand preference, such as age orsex (see Table 1). The four nonright-handed chimpanzees

were equally balanced for sex and their age did not sig-nificantly differ from the right-handed subsample (Mann–Whitney U8,10 � 11, z � �1.3, P � 0.21).

Among the various statistical approaches that we usedto examine these associations, asymmetries in layer II/IIIPV-ir interneuron density and the knob showed the stron-gest relationship with hand preference. Thus, asymme-tries at both macro- and microstructural levels may beinvolved in organizing lateralized deployment of thehands. Interestingly, PV-ir interneuron density AQ madea stronger unique contribution than knob AQ in explain-ing handedness on the coordinated bimanual task in thefinal reduced multiple regression model. Our multiple re-gression analysis also identified several other variablesthat made a unique contribution to explaining hand pref-erences. However, none of these variables showed a sig-nificant relationship with handedness in simple bivariatecorrelations or comparisons between handedness catego-ries, indicating that there is a complex interaction amongthese histological features that may account for lateral-ized behavior better than any single one of these variablesin isolation.

Layer II/III PV-ir interneurons tended to be denser inthe hemisphere opposite to the direction of hand prefer-ence on the coordinated bimanual task. Asymmetries ofPV-ir interneurons may correspond to hemispheric spe-cializations for processing of complex temporal sequences.Interneurons that express the calcium-binding protein PVare characterized by fast-spiking physiological propertiesand comprise chandelier and basket cell phenotypes(Markram et al., 2004). These interneuron types have thecapacity to exert strong inhibitory influence on pyramidalcells via synapses on the soma and axon initial segment.In the dorsolateral prefrontal cortex of macaque monkeys,for example, PV-ir interneurons are involved in maintain-ing sustained firing in neuronal ensembles during thedelay phase of working memory (Wilson et al., 1994; Raoet al., 1999). Thus, this class of interneuron can shape thetemporal pattern of activation in neuronal populations, afunction that may be especially important in the process-ing of complex sequences inherent to dexterous manualbehaviors. In addition, this finding complements neuro-physiological data from squirrel monkeys that shows thatforelimb movement maps display a higher degree of frag-mentation and spatial complexity within the side of pri-mary motor cortex opposite to the dominant hand (Nudo etal., 1992). In this context, relatively greater numbers ofPV-ir interneurons, with their horizontally directed ax-onal domains, may be important in coordinating pyrami-dal cell firing across more spatially dispersed movementrepresentations.

It is also interesting to note that this class of interneu-ron shows phylogenetic variation that might relate to spe-cies differences in motor control. PV-ir interneurons areproportionally more frequent in the orofacial representa-tion of primary motor cortex in hominids (humans andgreat apes) as compared with Old World monkeys (Sher-wood et al., 2004a), whereas visual cortex does not showthis relative increase (Sherwood et al., 2007). Becausehominids display a greater degree of dexterous motor con-trol of the orofacial muscles, these comparative data,along with the current findings, suggest that variation inthe distribution of PV-ir interneurons may comprise animportant microanatomical substrate for fine motor coor-dination in the cerebral cortex.



CONCLUSION

We examined histological asymmetry in the region ofhand representation of the primary motor cortex in chim-panzees. Our analyses revealed an association betweenhand preference and layer II/III PV-ir interneuron densityasymmetry that is distinct from the previously docu-mented relationship to gross anatomical asymmetries ofthe precentral gyrus. Whereas a correlation between mor-phological asymmetry of the precentral gyrus and hand-edness has also been demonstrated in humans, there areno data available concerning hemispheric bias in the dis-tribution of interneurons. Because of the close phyloge-netic relationship between humans and chimpanzees, thepresent findings provide important insight into the evolu-tion of brain and behavioral asymmetries within the hu-man lineage. In the future, it will be essential to deter-mine whether asymmetry of PV-ir interneuronsrepresents a common neuroanatomical substrate for handpreferences that is shared between these species due tohomology. Based on our data, it is evident that AF asym-metry does not display a strong association with handpreference in chimpanzees. These results in chimpanzeesgive cause to reevaluate the proposed functional relation-ship between AF asymmetry and handedness in humans.Instead, the population-wide bias toward greater neuropilspace in the left hemisphere of primary motor cortex andother cortical areas in humans might represent a noveltrait that has emerged recently in evolution since the splitfrom the last common ancestor shared with chimpanzees.

ACKNOWLEDGMENTS

We thank C.D. Stimpson and A.R. Garrison for techni-cal assistance, Dr. T.M. Preuss for providing brain tissue,and Dr. K.A. Phillips and two anonymous reviewers forhelpful comments on an earlier draft of th article. Some ofthe brains used in this study were loaned by the Great ApeAging Project and the Foundation for Comparative andConservation Biology.

LITERATURE CITED

Amunts K, Schlaug G, Schleicher A, Steinmetz H, Dabringhaus A, RolandPE, Zilles K. 1996. Asymmetry in the human motor cortex and hand-edness. Neuroimage 4:216–222.

Amunts K, Schmidt-Passos F, Schleicher A, Zilles K. 1997. Postnataldevelopment of interhemispheric asymmetry in the cytoarchitecture ofhuman area 4. Anat Embryol 196:393–402.

Amunts K, Schleicher A, Burgel U, Mohlberg H, Uylings HB, Zilles K.1999. Broca’s region revisited: cytoarchitecture and intersubject vari-ability. J Comp Neurol 412:319–341.

Amunts K, Jancke L, Mohlberg H, Steinmetz H, Zilles K. 2000. Interhemi-spheric asymmetry of the human motor cortex related to handednessand gender. Neuropsychologia 38:304–312.

Amunts K, Schleicher A, Ditterich A, Zilles K. 2003. Broca’s region: cyto-architectonic asymmetry and developmental changes. J Comp Neurol465:72–89.

Amunts K, Armstrong E, Malikovic A, Homke L, Mohlberg H, Schleicher A,Zilles K. 2007. Gender-specific left-right asymmetries in human visualcortex. J Neurosci 27:1356–1364.

Anderson B, Southern BD, Powers RE. 1999. Anatomic asymmetries of theposterior superior temporal lobes: a postmortem study. Neuropsychia-try Neuropsychol Behav Neurol 12:247–254.

Bailey P, von Bonin G, McCulloch WS. 1950. The isocortex of the chim-panzee. Urbana, IL: University of Illinois Press.

Buxhoeveden DP, Switala AE, Litaker M, Roy E, Casanova MF. 2001.

Lateralization of minicolumns in human planum temporale is absent innonhuman primate cortex. Brain Behav Evol 57:349–358.

Campbell MJ, Morrison JH. 1989. Monoclonal antibody to neurofilamentprotein (SMI-32) labels a subpopulation of pyramidal neurons in thehuman and monkey neocortex. J Comp Neurol 282:191–205.

Celio MR, Baier W, Scharer L, de Viragh PA, Gerday C. 1988. Monoclonalantibodies directed against the calcium binding protein parvalbumin.Cell Calcium 9:81–86.

Conde F, Lund JS, Jacobowitz DM, Baimbridge KG, Lewis DA. 1994. Localcircuit neurons immunoreactive for calretinin, calbindin D-28k or parv-albumin in monkey prefrontal cortex: distribution and morphology.J Comp Neurol 341:95–116.

Cullen TJ, Walker MA, Eastwood SL, Esiri MM, Harrison PJ, Crow TJ.2006. Anomalies of asymmetry of pyramidal cell density and structurein dorsolateral prefrontal cortex in schizophrenia. Br J Psychiatry188:26–31.

Dadda M, Cantalupo C, Hopkins WD. 2006. Further evidence of an asso-ciation between handedness and neuroanatomical asymmetries in theprimary motor cortex of chimpanzees (Pan troglodytes). Neuropsycho-logia 44:2582–2586.

Dusser de Barenne JG, Garol HW, McCulloch WS. 1941. The “motor”cortex of the chimpanzee. J Neurophysiol 4:287–304.

Foundas AL, Hong K, Leonard CM, Heilman KM. 1998. Hand preferenceand magnetic resonance imaging asymmetries of the central sulcus.Neuropsychiatry Neuropsychol Behav Neurol 11:65–71.

Galaburda AM, Rosen GD, Sherman GF. 1990. Individual variability incortical organization: its relationship to brain laterality and implica-tions to function. Neuropsychologia 28:529–546.

Gannon PJ, Holloway RL, Broadfield DC, Braun AR. 1998. Asymmetry ofchimpanzee planum temporale: humanlike pattern of Wernicke’s brainlanguage area homolog. Science 279:220–222.

Garcia RR, Montiel JF, Villalon AU, Gatica MA, Aboitiz F. 2004. AChE-rich magnopyramidal neurons have a left-right size asymmetry inBroca’s area. Brain Res 1026:313–316.

Good CD, Johnsrude I, Ashburner J, Henson RN, Friston KJ, FrackowiakRS. 2001. Cerebral asymmetry and the effects of sex and handednesson brain structure: a voxel-based morphometric analysis of 465 normaladult human brains. Neuroimage 14:685–700.

Grunbaum ASF, Sherrington CS. 1903. Observations on the physiology ofthe cerebral cortex of the anthropoid apes. Proc Royal Soc 72:152–155.

Gundersen HJ. 1988. The nucleator. J Microsc 151:3–21.Halpern ME, Gunturkun O, Hopkins WD, Rogers LJ. 2005. Lateralization

of the vertebrate brain: taking the side of model systems. J Neurosci25:10351–10357.

Hammond G. 2002. Correlates of human handedness in primary motorcortex: a review and hypothesis. Neurosci Biobehav Rev 26:285–292.

Hayes TL, Lewis DA. 1995. Anatomical specializations of the anteriormotor speech area: hemispheric differences in magnopyramidal neu-rons. Brain Lang 49:289–308.

Hayes TL, Lewis DA. 1996. Magnopyramidal neurons in the anterior motorspeech region. Dendritic features and interhemispheric comparisons.Arch Neurol 53:1277–1283.

Hines M. 1940. Movements elicited from the precentral gyrus of adultchimpanzees by stimulation with sine wave currents. J Neurophysiol3:442–466.

Hof PR, Morrison JH. 1995. Neurofilament protein defines regional pat-terns of cortical organization in the macaque monkey visual system: aquantitative immunohistochemical analysis. J Comp Neurol 352:161–186.

Hopkins WD. 1995. Hand preferences for a coordinated bimanual task in110 chimpanzees (Pan troglodytes): cross-sectional analysis. J CompPsychol 109:291–297.

Hopkins WD. 2006. Comparative and familial analysis of handedness ingreat apes. Psychol Bull 132:538–559.

Hopkins WD. 2007. Hemispheric specialization in chimpanzees: evolutionof hand and brain. In: Shackelford T, Keenan JP, Platek SM, editors.Evolutionary cognitive neuroscience. Boston: MIT Press.

Hopkins WD, Cantalupo C. 2004. Handedness in chimpanzees (Pan trog-lodytes) is associated with asymmetries of the primary motor cortex butnot with homologous language areas. Behav Neurosci 118:1176–1183.

Hopkins WD, Pilcher DL. 2001. Neuroanatomical localization of the motorhand area with magnetic resonance imaging: the left hemisphere islarger in great apes. Behav Neurosci 115:1159–1164.

Hopkins WD, Marino L, Rilling JK, MacGregor LA. 1998. Planum tempo-



rale asymmetries in great apes as revealed by magnetic resonanceimaging (MRI). NeuroReport 9:2913–2918.

Hopkins WD, Pilcher DL, MacGregor L. 2000. Sylvian fissure asymmetriesin nonhuman primates revisited: a comparative MRI study. BrainBehav Evol 56:293–299.

Hopkins WD, Wesley MJ, Izard MK, Hook M, Schapiro SJ. 2004. Chim-panzees (Pan troglodytes) are predominantly right-handed: replicationin three populations of apes. Behav Neurosci 118:659–663.

Hopkins WD, Taglialatela J, Russell J. 2006. Localizing handedness in thechimpanzee brain: a combined MRI and PET study. Am J Primatol68:120.

Hutsler JJ. 2003. The specialized structure of human language cortex:pyramidal cell size asymmetries within auditory and language-associated regions of the temporal lobes. Brain Lang 86:226–242.

Leyton ASF, Sherrington CS. 1917. Observations on the excitable cortex ofthe chimpanzee, orang-utan, and gorilla. Q J Exp Psychol 11:137–222.

Markram H, Toledo-Rodriguez M, Wang Y, Gupta A, Silberberg G, Wu C.2004. Interneurons of the neocortical inhibitory system. Nat Rev Neu-rosci 5:793–807.

Nudo RJ, Jenkins WM, Merzenich MM, Prejean T, Grenda R. 1992. Neu-rophysiological correlates of hand preference in primary motor cortex ofadult squirrel monkeys. J Neurosci 12:2918–2947.

Phillips KA, Sherwood CC. 2005. Primary motor cortex asymmetry iscorrelated with handedness in capuchin monkeys (Cebus apella). Be-hav Neurosci 119:1701–1704.

Rao SG, Williams GV, Goldman-Rakic PS. 1999. Isodirectional tuning ofadjacent interneurons and pyramidal cells during working memory:evidence for microcolumnar organization in PFC. J Neurophysiol 81:1903–1916.

Rider TW, Calvard S. 1978. Picture thresholding using an iterative selec-tion method. IEEE Trans Syst Man Cybernet 8:630–632.

Rogers LJ, Andrew RJ. 2002. Comparative vertebrate lateralization. Cam-bridge, UK: Cambridge University Press.

Rosen GD, Sherman GF, Galaburda AM. 1993. Neuronal subtypes andanatomic asymmetry: changes in neuronal number and cell-packingdensity. Neuroscience 56:833–839.

Schmitz C, Hof PR. 2000. Recommendations for straightforward and rig-orous methods of counting neurons based on a computer simulationapproach. J Chem Neuroanat 20:93–114.

Schmitz C, Schuster D, Niessen P, Korr H. 1999. No difference betweenestimated mean nuclear volumes of various types of neurons in themouse brain obtained on either isotropic uniform random sections orconventional frontal or sagittal sections. J Neurosci Methods 88:71–82.

Shapleske J, Rossell SL, Woodruff PW, David AS. 1999. The planumtemporale: a systematic, quantitative review of its structural, func-tional and clinical significance. Brain Res Rev 29:26–49.

Sherwood CC, Lee PH, Rivara C-B, Holloway RL, Gilissen EPE, SimmonsRMT, Hakeem A, Allman JM, Erwin JM, Hof PR. 2003. Evolution of

specialized pyramidal neurons in primate visual and motor cortex.Brain Behav Evol 61:28–44.

Sherwood CC, Holloway RL, Erwin JM, Hof PR. 2004a. Cortical orofacialmotor representation in Old World monkeys, great apes, and humans.II. Stereologic analysis of chemoarchitecture. Brain Behav Evol 63:82–106.

Sherwood CC, Holloway RL, Erwin JM, Schleicher A, Zilles K, Hof PR.2004b. Cortical orofacial motor representation in Old World monkeys,great apes, and humans. I. Quantitative analysis of cytoarchitecture.Brain Behav Evol 63:61–81.

Sherwood CC, Stimpson CD, Raghanti MA, Wildman DE, Uddin M, Gross-man LI, Goodman M, Redmond JC, Bonar CJ, Erwin JM, Hof PR. 2006.Evolution of increased glia-neuron ratios in the human frontal cortex.Proc Natl Acad Sci U S A 103:13606–11311.

Sherwood CC, Raghanti MA, Stimpson CD, Bonar CJ, de Sousa AA, PreussTM, Hof PR. 2007. Scaling of inhibitory interneurons in areas V1 andV2 of anthropoid primates as revealed by calcium-binding proteinimmunohistochemistry. Brain Behav Evol 69:176–195.

Stephan KE, Fink GR, Marshall JC. 2007. Mechanisms of hemisphericspecialization: Insights from analyses of connectivity. Neuropsycholo-gia 45:209–228.

Sternberger SR. 1983. Biomedical image processing. IEEE Comput Soc6:22–34.

Sternberger LA, Sternberger NH. 1983. Monoclonal antibodies distinguishphosphorylated and nonphosphorylated forms of neurofilaments insitu. Proc Natl Acad Sci U S A 80:6126–6130.

Sun T, Walsh CA. 2006. Molecular approaches to brain asymmetry andhandedness. Nat Rev Neurosci 7:655–662.

Sun T, Collura RV, Ruvolo M, Walsh CA. 2006. Genomic and evolutionaryanalyses of asymmetrically expressed genes in human fetal left andright cerebral cortex. Cereb Cortex 16(Suppl 1):i18–25.

Toga AW, Thompson PM. 2003. Mapping brain asymmetry. Nat Rev Neu-rosci 4:37–48.

Uylings HB, Jacobsen AM, Zilles K, Amunts K. 2006. Left-right asymmetryin volume and number of neurons in adult Broca’s area. Cortex 42:652–658.

Volkmann J, Schnitzler A, Witte OW, Freund H. 1998. Handedness andasymmetry of hand representation in human motor cortex. J Neuro-physiol 79:2149–2154.

Wilson FA, O’Scalaidhe SP, Goldman-Rakic PS. 1994. Functional syner-gism between putative gamma-aminobutyrate-containing neurons andpyramidal neurons in prefrontal cortex. Proc Natl Acad Sci U S A91:4009–4013.

Yeni-Komshian GH, Benson DA. 1976. Anatomical study of cerebral asym-metry in the temporal lobe of humans, chimpanzees, and rhesus mon-keys. Science 192:387–389.

Yousry TA, Schmid UD, Alkadhi H, Schmidt D, Peraud A, Buettner A,Winkler P. 1997. Localization of the motor hand area to a knob on theprecentral gyrus. A new landmark. Brain 120:141–157.



histological asymmetries of primary motor cortex predict...

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