Proc. Natl. Acad. Sci. USAVol. 90, pp. 3246-3250, April 1993Neurobiology

Direction of handedness linked to hereditary asymmetry of asensory system

(mouse genetics/vibrissae/barrels/brain lateralization/paw preference)

PASCAL BARNIOUD* AND HENDRIK VAN DER LoostInstitute of Anatomy, University of Lausanne, rue du Bugnon 9, 1005 Lausanne, Switzerland

Communicated by Richard F. Thompson, December 28, 1992

ABSTRACT Studies on the role of heredity in the trans-mission of handedness in nonhuman mammals have, so far, ledto the isolation of mouse strains that differed in the lateralizedversus ambidextrous use of the forepaw in a food-retrieval task(strength of paw preference). Here we report that left versusright use of the forepaw (direction of paw preference) isassociated with a genetically expressed structural asymmetry ofa sensory system, the whisker-to-barrel pathway. Mice thatexpress whisker pad asymmetry of a direction that correspondswith the asymmetry for which they were bred demonstrate anopposite shift in the distribution of handedness: a right or leftdominance of the whisker pad predicts a high proportion ofleft-handers or right-handers, respectively. Is an altered braincircuit-that is, a consequence of the asymmetry ofthe whiskerpad-associated with a change in the circuitry that governshandedness? Or, alternatively, are there two gene sets respon-sible for the phenomena that we report-one that causes"whiskeredness" and another that causes handedness?

Many explanations have been proposed to account for humanhandedness-an expression of brain asymmetry-rangingfrom purely cultural to purely genetic factors (1). Becausehuman family studies do not readily lead to a better under-standing of hereditary mechanisms and the brain substrate ofhandedness is not known, it is difficult to arrive at a uniqueexplanation. Such difficulties can be circumvented by usinganimals because handedness is not confined to our species(2). Mice exhibit a durable paw preference (3). Coflins (4)reported that preference for the right or the left paw is notsubject to genetic selection: he was unable to breed right- orleft-pawed mice over several generations by mating animalsshowing a right or a left paw preference. However, thisauthor succeeded in breeding ambidextrous as opposed tolateralized (left- or right-handed) mice by mating animals thatshow ambidextrousness or show strong lateralization, result-ing in lines that he named "LO" and "HI." Collins con-cluded that strength, but not direction, of lateralization maybe genetically defined. We here propose a genetically deter-mined asymmetry of a sensory system that may indeed beassociated with the direction of paw preference ("handed-ness") in mice.A contribution of potential interest for animal studies on

left-right asymmetry has been the breeding of mice for amorphologic trait that in certain lines of animals may occurdominantly on one side of the body or on the other. Startingwith an outbred population of ICR mice that showed mildvariations of the nearly stereotyped pattern of whiskers ontheir muzzles (5), Van der Loos et al. (6) selectively bred micefor distinct whisker patterns. Such patterns of whiskers onthe whisker pad are homeomorphic with those of the barrels(multineuronal units) in the barrel field of the primary soma-

tosensory cortex (7, 8). Moreover, we have adduced argu-ments for the notion that it is the pattern of vibrissae that isresponsible for the creation of the pattern of barrels in thebrain (9).

Twenty-three mouse strains were created, each character-ized by a different distribution of whiskers over the muzzle.Three of these were used in the present study: the strainNOR, bred for the standard, bilaterally symmetric pattern;the strain O/AP, bred for supernumerary whiskers (SWs) ina particular location on the right side of the face and corre-sponding supernumerary barrels in the left barrel field; andthe strain AP/O, bred for SWs at the same location on the leftside and corresponding supernumerary barrels in the rightbarrel field (Fig. 1). These strains allowed us to test whetherdirection and strength of handedness are associated with thedirection ofan observable left-right asymmetry ofa neuronalsystem, obtained after breeding for >40 generations of mice.To possibly discriminate between genotypic and phenotypicfactors influencing handedness, we not only tested mice thatexpressed the patterns for which they were bred but alsotested bilaterally symmetric and inversed left-right asym-metric animals from the O/AP and AP/O strains.

Part of this work has appeared in abstract form (10).

MATERIAL AND METHODSWhereas the NOR strain had been "pure" [i.e., virtually allanimals showed the standard whisker pattern from the eighthgeneration onward (6)], the patterns of the O/AP and AP/Ostrains took longer to consolidate. As breeding proceededbeyond the 10th generation, we continued the best lines bybrother-sister mating of animals with the desired phenotypeand by occasional back-crossing of offspring to parents. Atgeneration 30 we considered we were dealing with inbredstrains. Over the period of the present experiment, the NORstrain had maintained its standard pattern, whereas the twoasymmetric strains still contained symmetric animals andthose showing inversed left-right asymmetry.Table 1 summarizes the whisker patterns found in the mice

from the asymmetric strains used in our experiment. Thestrains O/AP and AP/O mice that express whisker padasymmetry of the direction corresponding with the patternfor which they were bred were termed O/AP(+) and AP/O(+), respectively; those that express whisker pad asymme-try of inversed direction were termed O/AP(-) and AP/O(-), respectively; and those that express whisker padsymmetry were termed O/AP(s) and AP/O(s), respectively.All animals tested were females. [We have studied femalemice because they have been shown to be slightly more

Abbreviations: SW, supernumerary whisker; RPE, number of rightpaw entries; LPE, number of left paw entries; PPE, number ofpreferred paw entries.*Present address: C.R.V., Rhone-Poulenc-Rorer, 13, quai JulesGuesde, 94403 Vitry s. Seine, France.tTo whom reprint requests should be addressed.

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FIG. 1. (Upper) Left whisker pad of an AP/O(+) mouse. NoteSWs A5 and A'3,4 (arrows). (Lower) Representation of the corre-sponding barrel field in the right cortex of the same mouse. Notesupernumerary barrels A5 and A'3,4 (arrows). The barrel field isoriented such that topologic equivalence is most easily appreciated.In both illustrations, rows A of vibrissal follicles and of barrels areat the top, and rows E are at the bottom. a, ,B, 'y, and 8 are the caudal,"straddling" barrels, which correspond with the four caudal, tallest"straddling" whiskers. R, rostral; D, dorsal.

lateralized, in terms of strength of handedness, than are malemice (refs. 4, 11, and nonpublished personal observation). Asthe overall sex ratio of all seven groups of animals tested wasthe same--1:1-we have no a priori reason to assume thatsex is a factor that would significantly influence our results.]The 93 strain NOR subjects came from 27 litters of the 43rdto 51st generations, the 184 strain O/AP subjects came from65 litters of the 31st to 45th generations, and the 1% strainAP/O subjects came from 88 litters of the 31st to 44thgenerations. In neither strain did we find a shift in testperformance in subsequent generations. The vibrissal pat-terns were evaluated with the aid of a dissecting microscope,under identical conditions for all animals, usually within 24hr after birth. At that time, the mice, including their muzzles,are free of fur, allowing for ready identification of whiskersand, thus, of SWs.

Our interest with respect to the whisker pattern wasdirected to the following points: in strain NOR, row A hasfour vibrissae. In strains O/AP and AP/O, the SWs ofinterest are situated at the rostral end of row A and termedA5 and/or just dorsal to this row, usually in a positionbetween row A3 and A4; these latter are termed A'3,4. Thelocations ofthe supernumerary barrels in the b.,Tel field werefound to be homeomorphic to those of the correspondingSWst (Fig. 1). NOR animals that did not adhere strictly to thestandard pattern (i.e., the pattern shown in Fig. 1A minus theA5 and A'3,4 whiskers-known to be accompanied by thepattern formed by the white barrels in Fig. 1B) were rejected,as were occasional strains O/AP and AP/O mice that hadSWs other than A5 and A'3,4. (The small whiskers rostral torows C7, D8, and E9, which in the adult are submerged incommon fur and represented in the cortex by small circularbarrels, were not taken into account.)Handedness was determined by using the paw-preference

test designed by Collins (4). The mice were food-deprived 12to 24 hr before testing. They were then put into a testingcubicle in which some crushed conventional food was avail-able in a narrow tube. The sequence, in which the right or theleft paw was used to reach the food crumbs, was scored; allanimals were tested five times, once weekly. A test sessionwas terminated after 50 reaches for food and scored for eachmouse. The first session took -30 min; subsequent sessionstook less time. Two parameters define handedness: directionand strength. The number of right paw entries (RPE) or thenumber of left paw entries (LPE) measures the direction ofhandedness, whereas the number of preferred paw entries(PPE) serves as a measure of the strength of handedness:PPE = ILPE - RPE|/(LPE + RPE) x 100%6 [= 2 x 15 -2RPEI% because LPE + RPE = 50]. The direction andstrength ofhandedness were compared in the seven groups ofmice in every test session with the parametric two-wayanalysis of variance (the interindividual factor is group, andthe intraindividual factor is session). Using the Kolmogorov-Smirnov test for normality of the 35 statistical matrices(seven experimental groups by five behavioral sessions), wefound that, for PPE, the different experimental groups weredistributed normally. With respect to RPE-the parameterwe chose to express the direction of handedness-we foundthat 29 of the 35 matrices were normally distributed. We,therefore, used a parametric two-way analysis of variancetest on the RPE and PPE scores and, in addition, a Friedmantwo-way analysis obtained by ranking RPE within blocks(sessions), followed by a main-effects analysis of variance onthese ranks (12). Because both tests gave similar results, wepresent only the parametric analysis. The means were com-pared two-by-two using the Newman-Keuls test.

Direction as well as strength of handedness was recordedat the end of every behavioral session in 475 mice: thenumbers of animals from the NOR, O/AP(+), O/AP(s),O/AP(-), AP/O(+), AP/O(s), and AP/O(-) groups were 93,56, 66, 64, 72, 56, and 68, respectively.

1To confirm this point, 10 mice of each group were prepared forhistological analysis. Animals of groups NOR, O/AP(+), andAP/O(+) were killed at the end of the experiment, under sodiumpentobarbital anesthesia (Nembutal, Abbott, 60 mg/kg i.p.), bytranscardial perfusion with 10% neutralized formalin in a 0.9%o NaClsolution. Upon perfusion, the skull was opened, and the head waspostfixed overnight in fresh fixative of the same composition. Thebrain and the muzzle were then removed, stored in fresh fixative,and 2 days before cutting in a cryostat, immersed in 30%6 sucrose.The brains were cut at 40 um tangential to the pial surface over thebarrel field, and the sections were mounted and stained with a Nisslmethod (cresyl violet). The muzzles were cut tangential to the skinsurface, also at 40 Am, and then mounted and stained with amodified Lillie method for myelin.

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Table 1. Numbers of O/AP and AP/O mice tested for SWs in different configurations

GroupSW pattern O/AP(+) AP/O(-) AP/O(+) O/AP(-) O/AP(s) AP/O(s)

-/A5 18 30A5/- 24 20-/- 32 45-/A'3,4 16 12A'3,4/- 26 8A5/A5 16 6-/A5 A'3,4 5 17A5 A'3,4/- 16 8A'3,4/A'3,4 2 1A5/A5 A'3,4 17 6A5 A'3,4/A5 6 28A'3,4/A5 A'3,4 0 3A5 A'3,4/A'3,4 0 0A5 A'3,4/A5 A'3,4 16 4

The letters and numbers in the SW pattern (phenotype) mark the presence and location ofone or moreSWs on the left/right side of the muzzle; a dash signifies absence of SWs. Animals whose phenotypecorresponded with the whisker pattern for which they were bred are named O/AP(+) and AP/O(+);those whose phenotype was in an opposite direction are named O/AP(-) and AP/O(-); and those witha bilaterally symmetric distribution are named O/AP(s) and AP/O(s). Note that identical phenotypesoccur in groups O/AP(+) and AP/O(-); and, in an inverse direction, identical phenotypes occur ingroups AP/O(+) and O/AP(-).

RESULTS AND DISCUSSIONAnalyzing the direction of handedness, we demonstrated thatthe RPE differed between groups [F(6, 474) = 4.18, P =0.0004] (Fig. 2). The O/AP(+) mice had a preference for theleft paw, and the AP/O(+) had a preference for the right one;the other groups had no preference. The differences in direc-tion of handedness between O/AP(+), AP/O(+), and theother five groups were apparent at the second test session andbecame marked in the subsequent sessions [interaction strainx session factors: F(24, 1900) = 3.22, P = 0.004]. At the firstsession, no difference for the direction of handedness wasobserved between groups; at the second session (occurring 1week later) O/AP(+) and AP/O(+) groups differed from oneanother but did not differ significantly from the five othergroups; at the third, fourth, and fifth sessions, the oppositedirections of handedness demonstrated by the O/AP(+) andAP/O(+) mice differed from the five other groups.To test whether the phenotype was the all-determining

factor associating the direction of handedness with whiskerasymmetry, we analyzed the paw-preference in animals that

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expressed an asymmetry contrary to that for which theselective breeding was performed [groups O/AP(-) andAP/O(-)]. Interestingly, these animals did not demonstratebias for handedness (Fig. 2). This result would seem toindicate that the genotype is an important factor leading to thebehavioral shift seen in the O/AP(+) and AP/O(+) groups.

Fig. 3 illustrates the RPE distributions of the seven groupsanalyzed. Because the RPEs were stable from the third to thefifth test sessions, mice were distributed among five classesby the means of the RPEs during these last three sessions.The limits of the middle class were defined by their statisticaldifference from 25. The number of observations per mousebeing 150, the lower and the higher means of RPE, statisti-callyjust not different from 25, are 20 and 30, respectively (x2(1 degree offreedom) = 2.64;P = 0.10). Thus, mice belongingto class 20-30 were considered as ambidextrous, those inclasses 0-9 and 10-19 were considered as sinistral, and thosein classes 31-40 and 41-50 were considered as dextral. Thehandedness distributions of all groups were compared byusing the x2 test.

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FIG. 2. Evolution, in succeeding test sessions, of the direction of handedness in O/AP(+) (o), O/AP(-) (v), and O/AP(s) (s) (A) and inAP/O(+) (o), AP/O(s) (m), and AP/O(-) (v) (B), as compared with NOR (A) mice. Note that the group NOR is represented in both A and B.See text for number of animals per group. Vertical line segments represent SEMs.

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FIG. 3. Distributions ofRPE score of mice of groups O/AP(+), O/AP(-), O/AP(s), NOR, AP/O(s), AP/O(-), and AP/O(+). Phenotypes(+) are symbolized by black bars; phenotypes (-) are symbolized by grey bars; phenotypes (s) are symbolized by open bars. The scores areindicated below the NOR group. For the other groups, bars representing scores are given in the same order. See text for number of animalsper group. Note that, although the AP/O(-), AP/O(s), NOR, O/AP(s), and O/AP(-) mice exhibit a symmetric distribution, the O/AP(+)distribution is shifted to the left, and the AP/O(+) distribution is shifted to the right.

In the NOR mice, left- and right-handedness were foundabout equally distributed. Collins (3, 4) reported similarfindings for mice of other strains. Also, the tendency forleft-handed reaching that we observed in the NOR strain hadbeen reported in other mouse strains (4, 13), as well as inother nonhuman mammalian species (14, 15). The percentageof ambidextrous mice (class 20-30) of the NOR strain seemslarge (29%) as compared with that found in mice of otherinbred strains (3, 4). However, the handedness distribution ofNOR mice resembles that of Collins' LO line, obtained bymating ambidextrous mice (4). Whereas the handedness ofO/AP(-), O/AP(s), AP/O(s), and AP/O(-) was distributedsimilarly to that in NOR mice, the handedness of O/AP(+)mice was shifted about one class toward the left paw, and thatof the AP/O(+) mice was shifted about one class toward theright paw [X2 (24 degrees offreedom) = 44.15, P < 0.007; Fig.3].

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With respect to the strength of handedness (defined byPPE), we did not find a difference between the sevenexperimental groups [F(6, 474) = 0.793, P = 0.58]. However,this parameter dramatically increased with succeeding ses-sions [Fig. 4; F(4, 1900) = 60.4, P < 0.00001], a finding inagreement with observations by Coffins (4). What is signifi-cant and different in the present observations is that O/AP(+)and AP/O(+) mice, in succeeding test sessions, exhibitopposite directions of handedness.

It has been reported that the strength of handedness isinfluenced by hereditary factors (4, 13, 16) and that thedirection of handedness is influenced by environmental fac-tors (17), even by teaching (18). The present study associatesthe direction of handedness with a genetically determinedasymmetry of a neuronal system. The question remainswhether the association between "whiskeredness" andhandedness is made at the level of the wiring of the brain orat the genomic level. In the former case a link would existbetween stations in the sensory pathway in question-stations that are modified as a consequence ofthe asymmetryof the whisker pad-and brain centers responsible for theexpression of handedness. In the latter case two gene setswould be linked: one set that, when penetrant, causes thedesired whiskeredness and another set that is responsible fora given direction of handedness.

We thank P. G. H. Clarke, N. Jeanpretre, R. Kraftsik, H. P. Lipp,and members of the Lausanne Barrel Club for valuable comments;T. D. H. Iuliano-Dao, M. V. Bartolome, V. De Icco, and J. Oured-nik for the evaluation of mouse whisker patterns; F. X. Dote, B.Laurent, and G. Filletta for administration and maintenance of theanimal colony; M.-C. Cruz for histology; S. Daldoss, A. Bernardi,and M. Birchen for art work; and the direction of H6tel de la Foret(Col de la Fonclaz, V.S. Switzerland) for shelter during a critical

SESSIONS phase in the preparation of the manuscript. This work was supportedby Swiss National Science Foundation Grant 31-30932.

0 1 2 3 4 51. Corballis, M. C. & Morgan, M. J. (1978) Behav. Brain Sci. 2,

261-298.2. Walker, S. F. (1980) Br. J. Psychol. 71, 329-340.3. Collins, R. L. (1968) J. Hered. 59, 9-13.

FIG. 4. Evolution, in succeeding test sessions, of the strength ofhandedness (PPE) in the same mice whose evolution of direction isshown in Fig. 2. Vertical line segments represent SEMs.

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4. Collins, R. L. (1985) in Cerebral Lateralization in NonhumanSpecies, ed. Glick, S. D. (Academic, New York), pp. 41-72.

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