BACKGROUND
the cerebral cortex is among the most complicated structures in the brain of mammals, and comprises a large but highly variable fraction of the total brain volume.
the volume of the human cortex varies by as much as 60% among normal adults.
much of this variation is genetic  <cit>  and has been linked with measures of intelligence  <cit> , as well as with differences in susceptibility to disorders including developmental dyslexia  <cit> , anxiety-related personality traits  <cit> , and schizophrenia  <cit> .
dissecting those genetic variants that modulate normal variation of the cerebral cortex could have an impact on our understanding of cortical development, normal function, and the etiology of several pervasive diseases.
in the present study we use bxd recombinant inbred  strains of mice to investigate the genetic basis of normal variation in the size of the neocortex.
this genetic reference population, which is both genetically diverse and phenotypically well-characterized, provides an experimental system to test relations and interactions between normal variation among a potentially unlimited variety of traits  <cit> .
thus, the scientific community has been able use these mice to systematically accumulate a vast amount of morphometric, behavioral, and physiologic data for each strain.
this in turn enables scientists with different interests and expertise to test and verify entire systems of traits, their covariance, and their genetic causes  <cit> .
recently, beatty and laughlin  <cit>  identified a qtl for neocortical volume on chromosome   <dig> using images of bxd cases taken from the mouse brain library .
dong et al.
<cit>  also used the same bxd reference population – and many of the same cases and images from the mbl – and found suggestive qtls for gray matter volume on chrs  <dig>   <dig>   <dig>  and  <dig>  but not on chr  <dig>  these studies were constrained by the relatively small number of strains  and cases.
the recent addition of many new bxd strains and cases to the mbl  <cit>  has significantly increased the utility of this ri set and improved both the power and precision of qtl mapping.
in this experiment, we used unbiased stereology to estimate the neocortical volume in both right and left hemispheres in  <dig> bxd ri lines, and both parental strains.
we mapped qtls modulating neocortical volume to intervals on chrs  <dig> and  <dig>  we exploited a variety of bioinformatic resources, as well as our own neonatal and adult databases of gene expression in the neocortex, to identify potential candidate genes within these qtls.
RESULTS
neocortical volume is highly variable
all estimates of volume are fully corrected for case-by-case differences in shrinkage and should be considered close to the original size of these regions in well-fixed tissue.
bilateral neocortical volume of individual mice has a remarkably wide range – from  <dig>  to  <dig>  mm <dig> , and is normally distributed .
strain averages extend from a low of  <dig>  ±  <dig>  mm <dig> in bxd <dig> to a high of  <dig>  ±  <dig>  mm <dig> in bxd <dig> .
the right and left hemisphere neocortical volumes of individual mice ranged from  <dig> – <dig>  mm <dig> and from  <dig> – <dig>  mm <dig>  with means of  <dig>  ±  <dig>  mm <dig> and  <dig>  ±  <dig>  mm <dig>  respectively.
there is no significant left/right asymmetry in the neocortex
we computed an asymmetry coefficient δasymm by subtracting the volume of the left from right hemisphere and dividing by one half of the total volume.
to determine whether there were overall asymmetric biases irrespective of direction, we calculated an absolute asymmetry coefficient  by taking the absolute value of δasymm.
we computed the distribution of both δasymm and δabs for the neocortex .
anova with strain as the independent measure and δasymm and δabs as the dependent measures revealed no significant effects of strain .
these results suggest that there is no significant left/right neocortical asymmetry in this population.
neocortical volume is heritable, but asymmetry is not
in order to determine the heritability of neocortical volume, we computed an anova with strain as the independent measure and bilateral volume as the dependent measure.
we found a significant effect of strain .
the strain main effect accounts for 51% of the variance, which provides a reasonable upper bound on the fraction of variance that might be explained by additive and epistatic interactions.
there were no significant main effects for δasymm or δabs .
these results strongly support the notion that there are significant differences between strains on the volumetric measures, but that strain differences in asymmetry are not apparent.
we also computed heritability  using inbred strain data and the adjustment method of hegmann and possidente  <cit> .
neocortical volume is a moderately heritable trait .
the broad sense estimate of heritability, which takes into account the large sample sizes of genetically identical members of ri strains, yielded an h <dig> of  <dig> .
the heritability factor for δasymm and δabs was  <dig>  and  <dig> , respectively.
this low heritability strongly suggests that neither of these traits could be profitably mapped.
mapping neocortical volume
we mapped bilateral neocortical volume and detected two loci  with closely matched likelihood ratio statistics  linkage scores but with opposite allelic effects .
the first locus on chr  <dig> peaks between  <dig> and  <dig> mb .
this region corresponds to human chr  <dig> at ≈  <dig> mb .
the second locus on chr  <dig> peaks between  <dig> and  <dig> mb .
in order to map variation related to volume of neocortex rather than possible confounding covariates, we performed multiple regression to remove all covariance associated with differences in age, sex, plane of section, strain epoch , and non-neocortical brain weight .
there were significant effects of non-neocortical brain weight  and epoch .
effects of sex , plane of section , or age  were modest and did not reach statistical significance.
we regressed neocortical volume for each subject against age, sex, plane of section, epoch, and non-neocortical brain weight and calculated residuals.
the use of all variables, even those that do not reach an alpha level of .
<dig>  is warranted and removes that variable as a potential confound in mapping.
these residuals were used to compute adjusted strain means.
when we re-mapped the variation in adjusted neocortical volume, we detected the same two loci as the original values , but with a modest increase in the strength of linkage .
the d <dig> allele has a positive effect of about  <dig> mm <dig> per allele at the chr  <dig> locus, whereas the b <dig> allele has a similar positive effect at the chr  <dig> locus .
we did not detect any epistatic interactions that could account for adjusted neocortical volume.
composite interval mapping of neocortical volume did not reveal any significant or consistent secondary loci for this trait.
candidate gene analysis
the chr  <dig> and chr  <dig> loci can be subdivided into large blocks that have common haplotypes in b <dig>  d <dig>  and bxd strains.
these blocks are essentially identical by descent  and have low densities of single nucleotide polymorphisms .
such regions are less likely to contain polymorphisms that modulate the volume of forebrain derivatives.
in contrast, several large blocks have dissimilar haplotypes and much higher densities of snps .
genes within these regions have a higher prior probability of containing functional polymorphisms.
chr  <dig> candidate analysis
we combined data on snp density with lrs values to rank positional candidate genes.
of ≈  <dig> genes in the chr  <dig> interval ,  <dig> were located in regions that were highly polymorphic .
members of this subset are good positional candidates, irrespective of any role that they may have in brain development or adult forebrain structure .
an additional  <dig> candidates were located in regions with low snp densities but had either missense mutations or snps in their promoters or utrs.
the restrictive criteria for inclusion were increased by requiring that strong candidates also have moderate to high expression in the neocortex at some stage of development.
to apply this filter, we extracted data on expression of all of these genes in the neocortex from the adult and p <dig> databases.
this subset of  <dig> genes was ranked using data on missense snps in these genes and by evidence of local regulatory variation that controls their own expression – so-called cis-qtls.
those genes with snps in illumina microarray probes that produced high cis-qtls were excluded from the analysis.
fifteen genes met these criteria: slit <dig>  odz <dig>  gabrg <dig>  gabra <dig>  gabrb <dig>  atp10b, pttg <dig>  slu <dig>  ttc <dig>  ublcp <dig>  ebf <dig>  adam <dig>  cyfip <dig>  thg1l, and itk .
chr  <dig> candidate analysis
the chr  <dig> interval identified here was previously associated with striatal volume, and the identification of candidate genes , was identical to those recently described  <cit> .
a subset of  <dig> genes met all the criteria: htra <dig>  tia <dig>  mxd <dig>  anxa <dig>  aak <dig>  nfu <dig>  nup <dig>  hdac <dig>  fbln <dig>  and slc25a <dig> .
gene expression correlations with neocortical volume
we asked which subset of the positional candidates at both qtls also had differences in expression across bxd strains that co-varied well with our trait.
we produced a correlation matrix based on the subset of  <dig> strains for which we have data of all types.
this matrix was then thresholded at a pearson r = | <dig> | and plotted as a graph.
there were  <dig> genes – gabra <dig>  gabrb <dig>  pttg <dig>  slu <dig>  ublcp <dig>  ebf <dig>  and thg1l – that co-varied with neocortical volume on chr  <dig> .
we further found confirmatory evidence from the allen brain atlas and gensat that these genes are expressed either in the adult brain, the developing brain, or both.
in marked contrast, none of the chr  <dig> gene expression profiles co-vary with neocortical volume.
trait correlation analysis
one advantage of employing the bxd ri set is the ability to test for covariation with other phenotypes that have also been studied in this population.
we correlated neocortical volume  with the bxd published phenotypes database of gn, which contains a large number of behavioral, anatomic, and physiologic phenotypes gathered from bxd ri sets.
alpha levels were adjusted after permuting neocortical volume and adjusted neocortical volume  <dig> times each, determining the top  <dig> correlations with the bxd published phenotypes database for each permutation, and determining the computed alpha level of the top 5% of all correlations.
from this analysis, it was determined that computed correlations by gn with p < .
<dig> were significant at an adjusted alpha level of .
<dig>
both neocortical and adjusted neocortical volume correlated highly with a large number of morphological traits, including volume of dorsal thalamus, dorsal striatum, lateral geniculate nucleus, hippocampus, basolateral amygdala, fimbria, trigeminal tract, nucleus accumbens, and corpus callosum.
there were no significant correlations with non-morphological traits.
discussion
we systematically explored sources of genetic variation in the size of the neocortex.
using unbiased stereological estimations of this trait in the bxd ri genetic reference population, we found that neocortical volume is highly heritable and variable.
there was a  <dig> -fold difference between the neocortical volume of the smallest and largest individuals in this study, and a  <dig> -fold difference when strain means were compared.
the heritability of this trait is consistent with that reported for similar neuroanatomic traits including olfactory bulb, hippocampus, striatum, neocortex, and thalamus  <cit> .
even with a large sample size and a powerful paired t-test design, we did not find any significant asymmetry between the left and right hemispheres of mice.
neocortical volume was associated with qtls on chrs  <dig> and  <dig>  large microarray data sets were used to efficiently nominate small numbers of viable candidate genes, one or more of which may modulate the volume of the neocortex, and these results supported candidates on the chr  <dig> interval as modulating neocortical volume.
there is a lack of anatomic asymmetry in the neocortex
because previous research has found evidence of volumetric asymmetry in the rodent brain  <cit> , we attempted to assess this variable in the bxd ri set.
we found that there was little evidence of asymmetry in the neocortex.
this was true both for measures that assessed directional  asymmetry and those that assessed magnitude .
these results are consistent with previous reports that failed to reveal neocortical asymmetries in the mouse brain  <cit> .
the results reported here suggest that there are no population level anatomic asymmetries in the neocortex, but do not discount the possibility that anatomic asymmetries at the individual level may correlate with functional asymmetries.
qtls for neocortical volume
using images from the mbl, beatty and laughlin  <cit>  reported significant qtls for neocortical volume on chr  <dig> and for non-neocortical brain volume on chr  <dig>  a more recent study, which again used images from the mbl  <cit> , did not find any statistically significant qtls for gray matter volume, although there were suggestive qtls on chrs  <dig>   <dig>   <dig>  and  <dig>
we find some support in this study for the chr  <dig> qtl reported by beatty and laughlin  <cit> .
although the chr  <dig> interval identified by beatty and laughlin is more proximal than that described in the current study, the intervals do overlap.
in some ways, this was not surprising given that both beatty and laughlin and we analyzed brains from the mbl.
yet there were a number of methodological differences that could have worked against this.
our estimations of cerebral volume were performed using unbiased stereology on histologic tissue that was corrected for section thickness, whereas beatty and laughlin used planimetric techniques on images from the mbl to estimate volume.
despite these differences in methodology, there was a highly significant correlation  between the neocortical volume estimates of beatty and laughlin and our own for the  <dig> strains common to both studies, and the correlation between the regressed cerebral volume estimates – the variable used to map these traits – is also quite high .
this was encouraging given that we regressed out the effects of brain weight , age, plane of section, epoch, and sex, whereas beatty and laughlin regressed out brain weight and logarithm of age only.
there were differences, however, between our results and those published previously.
none of the early data sets detect a qtl on chr  <dig>  and we find no support for any of the qtls identified by dong et al.
<cit> .
the most obvious difference among these studies is the inclusion of the new bxd strains  <cit> .
qtl detection and map precision improves with number of strains, whereas increasing the number of subjects within a strain has only marginal effects  <cit> .
we have increased the number of strains by over 50%  in this experiment, and it is likely that our ability to detect qtls that modulate a smaller percent of the variance of neocortical volume is improved.
positional candidate genes
before considering positional candidate genes, it may be useful to review factors that are likely to impact normal variation in brain volume.
cerebral cortical volume can be modified by a variety of developmental factors, as well as those that appear during adulthood.
thus, genes modulating cell proliferation, differentiation, and/or death may contribute to the variation in cerebral cortical volume reported here.
in adulthood, cerebral cortical volume can be modified by neurodegenerative changes, some of which may be under genetic control.
in the current experiment, age was not a significant predictor of cerebral cortical volume, and none of the mice would be qualified as senescent.
when considering potential candidate genes, we therefore concentrated our efforts on those genes known to modulate factors in brain development.
we have recently reported a qtl modulating striatal volume on the same interval on chr  <dig> as that in the current report  <cit> .
a detailed analysis of the genes lying within that chromosome  identified a number of potential candidate genes.
analysis of gene expression strain distribution profiles revealed that a number of these genes correlated well with striatal volume.
in contrast, none correlated with cerebral cortical volume in the current study.
in a complementary manner, none of the chr  <dig> candidates reported here correlated with striatal volume.
there are a number of genes in the chr  <dig> interval whose functions suggest that they could play a role in modulating neocortical volume.
slit <dig> is a well-characterized gene that is part of the slit family of ligands for the robo receptors  <cit> , and which are important for neuronal migration, axon outgrowth, and dendritic development .
moreover, it has recently been demonstrated that this gene is upregulated in the caudal ganglionic eminence, which gives rise to neocortical gabaergic neurons  <cit> .
this is particularly intriguing given the inclusion of three gaba receptor subtype genes  in the qtl interval.
odz <dig> is expressed in the mouse brain quite early during development  <cit> , although its specific role is not yet known.
cyfip <dig> has been shown to be an interactor with fmrp , which has been implicated in fragile × syndrome, and is highly expressed in neurons throughout the brain  <cit> .
of the candidate genes on chr  <dig>  we found seven whose expression levels were highly correlated with neocortical volume.
all are strong cis-qtls; that is, a sequence variant in or very close to the gene itself controls its expression .
many of these genes have been documented to have roles during brain development.
gaba receptors  are obvious candidates given their roles in brain development in general and neocortical functioning in specific .
pttg <dig> is upregulated during development of the mouse telencephalon  <cit> .
likewise, ublcp <dig> is upregulated in fast growing or tumor tissues perhaps through its activity as a regulator of the phosphorylation state of rna polymerase ii  <cit> .
the transcription factor ebf <dig> plays a pivotal role in ensuring that a set of thalamic axons reaches the neocortex as well as in the development of striatal neurons  <cit> .
though the last two transcripts, slu <dig> and thg1l, have not yet been implicated in neural growth, their molecular functions suggest a developmental role.
the homolog of slu <dig> is well characterized in s. cerevisae as essential during the second catalytic step in the pre-mrna splicing process  <cit> .
additionally, thg1l is important for trna priming for protein translation  <cit> .
future directions
mechanisms that control numbers of neurons and glial cells assigned during development to the neocortex are crucial in understand human behavior, human evolution, and the basis of individual differences.
this study can be considered an essential step in a more refined regional genetic anatomy of the several dozen distinct cytoarchitectonic regions that together make up the entire neocortex.
the cellular demographics of the neocortex are a strong determinant of functional repertoire.
this is most obvious when comparisons are made at a coarse taxonomic level comparing, for example, the massive auditory cortex of echo-locating bats and the complex, highly evolved whisker fields of mice and rats, and the expanded prefrontal cortical regions of humans.
but even within a species, pronounced differences in cytoarchitecture are associated with function  <cit> .
now that we have a baseline, we can begin the more painstaking genetic studies of discrete regions of the mouse neocortex and determine if there are qtls with smaller but regionally intense effects on, for example, barrel cortex, primary visual cortex, or temporal regions.
a number of research strategies that can further refine the qtl interval are available.
higher density maps of the b <dig> and d <dig> parental strains will allow a more refined screen for sequence variants.
knockouts of the candidate genes are another potentially productive avenue of investigation.
although none of the chr  <dig> candidates have been knocked out, pttg <dig>  ebf <dig>  ublcp <dig>  slu <dig>  thg1l, and gabrb <dig> have been targeted by the knockout mouse project .
additionally, there are a number of forward genetic methods that can be employed to confirm our results.
the interval identified here using the bxd ri line could be confirmed in other recombinant inbred lines, including the axb, bxa, and lxs.
the collaborative cross – an 8-strain cross to generate hundreds of ri lines, each with more recombinations than existing ri strains – will enhance precision of qtl mapping  <cit> .
recombinant inbred intercross  schemes – whereby selective generation of f <dig> progeny from recombinant inbred parents allows for the examination of known heterozygous and homozygous intervals – extends the number of genomes that can be phenotyped by n/ <dig>  where n is the number of original recombinant inbred strains .
the rix lines have known genomes and quantifiable phenotypes and, therefore, will contribute to mapping traits to their genetic determinants  <cit> .
CONCLUSIONS
this study is the first to use the expanded set of bxd strains to map neocortical volume.
with the enhanced precision garnered from the addition of over  <dig> strains, we found that neocortical volume is a heritable trait with large inter-strain variability.
by quantifying cerebral cortical volume for both the right and left hemispheres, we were able to conclude that asymmetry is most likely not genetically modulated, at least in this group of inbred mice.
we identified two qtls, one on chr  <dig> and another on chr  <dig>  that are likely to contain gene variants that modulate neocortical volume.
from correlational analysis to gene expression data, we identified a number of candidates on the chr  <dig> interval that may modulate this trait.
future research exploiting this expanded bxd genetic reference population will allow us to further dissect other important neuroanatomic phenotypes that may contribute to variation in regional volume, such as proliferation, death, and number and packing density of neurons.

 the cerebral cortex is among the most complicated structures in the brain of mammals, and comprises a large but highly variable fraction of the total brain volume. the volume of the human cortex varies by as much as 60% among normal adults. much of this variation is genetic and has been linked with measures of intelligence, as well as with differences in susceptibility to disorders including developmental dyslexia, anxiety-related personality traits, and schizophrenia. dissecting those genetic variants that modulate normal variation of the cerebral cortex could have an impact on our understanding of cortical development, normal function, and the etiology of several pervasive diseases. in the present study we use bxd recombinant inbred strains of mice to investigate the genetic basis of normal variation in the size of the neocortex. this genetic reference population, which is both genetically diverse and phenotypically well-characterized, provides an experimental system to test relations and interactions between normal variation among a potentially unlimited variety of traits. thus, the scientific community has been able use these mice to systematically accumulate a vast amount of morphometric, behavioral, and physiologic data for each strain. this in turn enables scientists with different interests and expertise to test and verify entire systems of traits, their covariance, and their genetic causes. recently, beatty and laughlin identified a qtl for neocortical volume on chromosome using images of bxd cases taken from the mouse brain library. dong et al. also used the same bxd reference population – and many of the same cases and images from the mbl – and found suggestive qtls for gray matter volume on chrs and but not on chr these studies were constrained by the relatively small number of strains and cases. the recent addition of many new bxd strains and cases to the mbl has significantly increased the utility of this ri set and improved both the power and precision of qtl mapping. in this experiment, we used unbiased stereology to estimate the neocortical volume in both right and left hemispheres in bxd ri lines, and both parental strains. we mapped qtls modulating neocortical volume to intervals on chrs and we exploited a variety of bioinformatic resources, as well as our own neonatal and adult databases of gene expression in the neocortex, to identify potential candidate genes within these qtls.  neocortical volume is highly variable all estimates of volume are fully corrected for case-by-case differences in shrinkage and should be considered close to the original size of these regions in well-fixed tissue. bilateral neocortical volume of individual mice has a remarkably wide range – from to mm, and is normally distributed. strain averages extend from a low of ± mm in bxd to a high of ± mm in bxd. the right and left hemisphere neocortical volumes of individual mice ranged from – mm and from – mm with means of ± mm and ± mm respectively. there is no significant left/right asymmetry in the neocortex we computed an asymmetry coefficient δasymm by subtracting the volume of the left from right hemisphere and dividing by one half of the total volume. to determine whether there were overall asymmetric biases irrespective of direction, we calculated an absolute asymmetry coefficient by taking the absolute value of δasymm. we computed the distribution of both δasymm and δabs for the neocortex. anova with strain as the independent measure and δasymm and δabs as the dependent measures revealed no significant effects of strain. these results suggest that there is no significant left/right neocortical asymmetry in this population. neocortical volume is heritable, but asymmetry is not in order to determine the heritability of neocortical volume, we computed an anova with strain as the independent measure and bilateral volume as the dependent measure. we found a significant effect of strain. the strain main effect accounts for 51% of the variance, which provides a reasonable upper bound on the fraction of variance that might be explained by additive and epistatic interactions. there were no significant main effects for δasymm or δabs. these results strongly support the notion that there are significant differences between strains on the volumetric measures, but that strain differences in asymmetry are not apparent. we also computed heritability using inbred strain data and the adjustment method of hegmann and possidente. neocortical volume is a moderately heritable trait. the broad sense estimate of heritability, which takes into account the large sample sizes of genetically identical members of ri strains, yielded an h of. the heritability factor for δasymm and δabs was and, respectively. this low heritability strongly suggests that neither of these traits could be profitably mapped. mapping neocortical volume we mapped bilateral neocortical volume and detected two loci with closely matched likelihood ratio statistics linkage scores but with opposite allelic effects. the first locus on chr peaks between and mb. this region corresponds to human chr at ≈ mb. the second locus on chr peaks between and mb. in order to map variation related to volume of neocortex rather than possible confounding covariates, we performed multiple regression to remove all covariance associated with differences in age, sex, plane of section, strain epoch, and non-neocortical brain weight. there were significant effects of non-neocortical brain weight and epoch. effects of sex, plane of section, or age were modest and did not reach statistical significance. we regressed neocortical volume for each subject against age, sex, plane of section, epoch, and non-neocortical brain weight and calculated residuals. the use of all variables, even those that do not reach an alpha level of. is warranted and removes that variable as a potential confound in mapping. these residuals were used to compute adjusted strain means. when we re-mapped the variation in adjusted neocortical volume, we detected the same two loci as the original values, but with a modest increase in the strength of linkage. the d allele has a positive effect of about mm per allele at the chr locus, whereas the b allele has a similar positive effect at the chr locus. we did not detect any epistatic interactions that could account for adjusted neocortical volume. composite interval mapping of neocortical volume did not reveal any significant or consistent secondary loci for this trait. candidate gene analysis the chr and chr loci can be subdivided into large blocks that have common haplotypes in b d and bxd strains. these blocks are essentially identical by descent and have low densities of single nucleotide polymorphisms. such regions are less likely to contain polymorphisms that modulate the volume of forebrain derivatives. in contrast, several large blocks have dissimilar haplotypes and much higher densities of snps. genes within these regions have a higher prior probability of containing functional polymorphisms. chr candidate analysis we combined data on snp density with lrs values to rank positional candidate genes. of ≈ genes in the chr interval, were located in regions that were highly polymorphic. members of this subset are good positional candidates, irrespective of any role that they may have in brain development or adult forebrain structure. an additional candidates were located in regions with low snp densities but had either missense mutations or snps in their promoters or utrs. the restrictive criteria for inclusion were increased by requiring that strong candidates also have moderate to high expression in the neocortex at some stage of development. to apply this filter, we extracted data on expression of all of these genes in the neocortex from the adult and p databases. this subset of genes was ranked using data on missense snps in these genes and by evidence of local regulatory variation that controls their own expression – so-called cis-qtls. those genes with snps in illumina microarray probes that produced high cis-qtls were excluded from the analysis. fifteen genes met these criteria: slit odz gabrg gabra gabrb atp10b, pttg slu ttc ublcp ebf adam cyfip thg1l, and itk. chr candidate analysis the chr interval identified here was previously associated with striatal volume, and the identification of candidate genes, was identical to those recently described. a subset of genes met all the criteria: htra tia mxd anxa aak nfu nup hdac fbln and slc25a. gene expression correlations with neocortical volume we asked which subset of the positional candidates at both qtls also had differences in expression across bxd strains that co-varied well with our trait. we produced a correlation matrix based on the subset of strains for which we have data of all types. this matrix was then thresholded at a pearson r = | | and plotted as a graph. there were genes – gabra gabrb pttg slu ublcp ebf and thg1l – that co-varied with neocortical volume on chr. we further found confirmatory evidence from the allen brain atlas and gensat that these genes are expressed either in the adult brain, the developing brain, or both. in marked contrast, none of the chr gene expression profiles co-vary with neocortical volume. trait correlation analysis one advantage of employing the bxd ri set is the ability to test for covariation with other phenotypes that have also been studied in this population. we correlated neocortical volume with the bxd published phenotypes database of gn, which contains a large number of behavioral, anatomic, and physiologic phenotypes gathered from bxd ri sets. alpha levels were adjusted after permuting neocortical volume and adjusted neocortical volume times each, determining the top correlations with the bxd published phenotypes database for each permutation, and determining the computed alpha level of the top 5% of all correlations. from this analysis, it was determined that computed correlations by gn with p <. were significant at an adjusted alpha level of. both neocortical and adjusted neocortical volume correlated highly with a large number of morphological traits, including volume of dorsal thalamus, dorsal striatum, lateral geniculate nucleus, hippocampus, basolateral amygdala, fimbria, trigeminal tract, nucleus accumbens, and corpus callosum. there were no significant correlations with non-morphological traits. discussion we systematically explored sources of genetic variation in the size of the neocortex. using unbiased stereological estimations of this trait in the bxd ri genetic reference population, we found that neocortical volume is highly heritable and variable. there was a -fold difference between the neocortical volume of the smallest and largest individuals in this study, and a -fold difference when strain means were compared. the heritability of this trait is consistent with that reported for similar neuroanatomic traits including olfactory bulb, hippocampus, striatum, neocortex, and thalamus. even with a large sample size and a powerful paired t-test design, we did not find any significant asymmetry between the left and right hemispheres of mice. neocortical volume was associated with qtls on chrs and large microarray data sets were used to efficiently nominate small numbers of viable candidate genes, one or more of which may modulate the volume of the neocortex, and these results supported candidates on the chr interval as modulating neocortical volume. there is a lack of anatomic asymmetry in the neocortex because previous research has found evidence of volumetric asymmetry in the rodent brain, we attempted to assess this variable in the bxd ri set. we found that there was little evidence of asymmetry in the neocortex. this was true both for measures that assessed directional asymmetry and those that assessed magnitude. these results are consistent with previous reports that failed to reveal neocortical asymmetries in the mouse brain. the results reported here suggest that there are no population level anatomic asymmetries in the neocortex, but do not discount the possibility that anatomic asymmetries at the individual level may correlate with functional asymmetries. qtls for neocortical volume using images from the mbl, beatty and laughlin reported significant qtls for neocortical volume on chr and for non-neocortical brain volume on chr a more recent study, which again used images from the mbl, did not find any statistically significant qtls for gray matter volume, although there were suggestive qtls on chrs and we find some support in this study for the chr qtl reported by beatty and laughlin. although the chr interval identified by beatty and laughlin is more proximal than that described in the current study, the intervals do overlap. in some ways, this was not surprising given that both beatty and laughlin and we analyzed brains from the mbl. yet there were a number of methodological differences that could have worked against this. our estimations of cerebral volume were performed using unbiased stereology on histologic tissue that was corrected for section thickness, whereas beatty and laughlin used planimetric techniques on images from the mbl to estimate volume. despite these differences in methodology, there was a highly significant correlation between the neocortical volume estimates of beatty and laughlin and our own for the strains common to both studies, and the correlation between the regressed cerebral volume estimates – the variable used to map these traits – is also quite high. this was encouraging given that we regressed out the effects of brain weight, age, plane of section, epoch, and sex, whereas beatty and laughlin regressed out brain weight and logarithm of age only. there were differences, however, between our results and those published previously. none of the early data sets detect a qtl on chr and we find no support for any of the qtls identified by dong et al.. the most obvious difference among these studies is the inclusion of the new bxd strains. qtl detection and map precision improves with number of strains, whereas increasing the number of subjects within a strain has only marginal effects. we have increased the number of strains by over 50% in this experiment, and it is likely that our ability to detect qtls that modulate a smaller percent of the variance of neocortical volume is improved. positional candidate genes before considering positional candidate genes, it may be useful to review factors that are likely to impact normal variation in brain volume. cerebral cortical volume can be modified by a variety of developmental factors, as well as those that appear during adulthood. thus, genes modulating cell proliferation, differentiation, and/or death may contribute to the variation in cerebral cortical volume reported here. in adulthood, cerebral cortical volume can be modified by neurodegenerative changes, some of which may be under genetic control. in the current experiment, age was not a significant predictor of cerebral cortical volume, and none of the mice would be qualified as senescent. when considering potential candidate genes, we therefore concentrated our efforts on those genes known to modulate factors in brain development. we have recently reported a qtl modulating striatal volume on the same interval on chr as that in the current report. a detailed analysis of the genes lying within that chromosome identified a number of potential candidate genes. analysis of gene expression strain distribution profiles revealed that a number of these genes correlated well with striatal volume. in contrast, none correlated with cerebral cortical volume in the current study. in a complementary manner, none of the chr candidates reported here correlated with striatal volume. there are a number of genes in the chr interval whose functions suggest that they could play a role in modulating neocortical volume. slit is a well-characterized gene that is part of the slit family of ligands for the robo receptors, and which are important for neuronal migration, axon outgrowth, and dendritic development. moreover, it has recently been demonstrated that this gene is upregulated in the caudal ganglionic eminence, which gives rise to neocortical gabaergic neurons. this is particularly intriguing given the inclusion of three gaba receptor subtype genes in the qtl interval. odz is expressed in the mouse brain quite early during development, although its specific role is not yet known. cyfip has been shown to be an interactor with fmrp, which has been implicated in fragile × syndrome, and is highly expressed in neurons throughout the brain. of the candidate genes on chr we found seven whose expression levels were highly correlated with neocortical volume. all are strong cis-qtls; that is, a sequence variant in or very close to the gene itself controls its expression. many of these genes have been documented to have roles during brain development. gaba receptors are obvious candidates given their roles in brain development in general and neocortical functioning in specific. pttg is upregulated during development of the mouse telencephalon. likewise, ublcp is upregulated in fast growing or tumor tissues perhaps through its activity as a regulator of the phosphorylation state of rna polymerase ii. the transcription factor ebf plays a pivotal role in ensuring that a set of thalamic axons reaches the neocortex as well as in the development of striatal neurons. though the last two transcripts, slu and thg1l, have not yet been implicated in neural growth, their molecular functions suggest a developmental role. the homolog of slu is well characterized in s. cerevisae as essential during the second catalytic step in the pre-mrna splicing process. additionally, thg1l is important for trna priming for protein translation. future directions mechanisms that control numbers of neurons and glial cells assigned during development to the neocortex are crucial in understand human behavior, human evolution, and the basis of individual differences. this study can be considered an essential step in a more refined regional genetic anatomy of the several dozen distinct cytoarchitectonic regions that together make up the entire neocortex. the cellular demographics of the neocortex are a strong determinant of functional repertoire. this is most obvious when comparisons are made at a coarse taxonomic level comparing, for example, the massive auditory cortex of echo-locating bats and the complex, highly evolved whisker fields of mice and rats, and the expanded prefrontal cortical regions of humans. but even within a species, pronounced differences in cytoarchitecture are associated with function. now that we have a baseline, we can begin the more painstaking genetic studies of discrete regions of the mouse neocortex and determine if there are qtls with smaller but regionally intense effects on, for example, barrel cortex, primary visual cortex, or temporal regions. a number of research strategies that can further refine the qtl interval are available. higher density maps of the b and d parental strains will allow a more refined screen for sequence variants. knockouts of the candidate genes are another potentially productive avenue of investigation. although none of the chr candidates have been knocked out, pttg ebf ublcp slu thg1l, and gabrb have been targeted by the knockout mouse project. additionally, there are a number of forward genetic methods that can be employed to confirm our results. the interval identified here using the bxd ri line could be confirmed in other recombinant inbred lines, including the axb, bxa, and lxs. the collaborative cross – an 8-strain cross to generate hundreds of ri lines, each with more recombinations than existing ri strains – will enhance precision of qtl mapping. recombinant inbred intercross schemes – whereby selective generation of f progeny from recombinant inbred parents allows for the examination of known heterozygous and homozygous intervals – extends the number of genomes that can be phenotyped by n/ where n is the number of original recombinant inbred strains. the rix lines have known genomes and quantifiable phenotypes and, therefore, will contribute to mapping traits to their genetic determinants.  this study is the first to use the expanded set of bxd strains to map neocortical volume. with the enhanced precision garnered from the addition of over strains, we found that neocortical volume is a heritable trait with large inter-strain variability. by quantifying cerebral cortical volume for both the right and left hemispheres, we were able to conclude that asymmetry is most likely not genetically modulated, at least in this group of inbred mice. we identified two qtls, one on chr and another on chr that are likely to contain gene variants that modulate neocortical volume. from correlational analysis to gene expression data, we identified a number of candidates on the chr interval that may modulate this trait. future research exploiting this expanded bxd genetic reference population will allow us to further dissect other important neuroanatomic phenotypes that may contribute to variation in regional volume, such as proliferation, death, and number and packing density of neurons. 
the discovery and characterization of these genes and their variants can contribute to an understanding of individual differences in brain development, behavior, and disease susceptibility. this study is the first to use the expanded set of bxd strains to map neocortical volume, and we found that normal variation of this trait is, at least in part, genetically modulated. these results provide a baseline from which to assess the genetic contribution to regional variation in neocortical volume, as well as other neuroanatomic phenotypes that may contribute to variation in regional volume, such as proliferation, death, and number and packing density of neurons we estimated volumes bilaterally in an expanded set of bxd recombinant inbred strains taken from the mouse brain library. we generated matched microarray data for the cerebral cortex in the same large panel of strains and in parental neonates to efficiently nominate and evaluate candidate genes. using these databases in combination with other bioinformatic tools we have identified positional candidates on these qtl intervals. the size of the cerebral cortex varies widely within human populations, and a large portion of this variance is modulated by genetic factors. volume of the neocortex varies widely, and is a heritable trait.
 the size of the cerebral cortex varies widely within human populations, and a large portion of this variance is modulated by genetic factors. the discovery and characterization of these genes and their variants can contribute to an understanding of individual differences in brain development, behavior, and disease susceptibility. here we use unbiased stereological techniques to map quantitative trait loci that modulate the volume of neocortex.  we estimated volumes bilaterally in an expanded set of bxd recombinant inbred strains taken from the mouse brain library. we generated matched microarray data for the cerebral cortex in the same large panel of strains and in parental neonates to efficiently nominate and evaluate candidate genes. volume of the neocortex varies widely, and is a heritable trait. genome-wide mapping of this trait revealed two qtls – one on chromosome at ± mb and another at chr. we generated both neonatal and adult neocortical gene expression databases using microarray technology. using these databases in combination with other bioinformatic tools we have identified positional candidates on these qtl intervals.  this study is the first to use the expanded set of bxd strains to map neocortical volume, and we found that normal variation of this trait is, at least in part, genetically modulated. these results provide a baseline from which to assess the genetic contribution to regional variation in neocortical volume, as well as other neuroanatomic phenotypes that may contribute to variation in regional volume, such as proliferation, death, and number and packing density of neurons 