Cortical Cell and Neuron Density Estimates in One Chimpanzee Hemisphere

Cortical cell and neuron density estimates in one chimpanzee hemisphere

Christine E. Collinsa,b, Emily C. Turnera, Eva Kille Sawyerc, Jamie L. Reeda, Nicole A. Younga,d, David K. Flahertye, and Jon H. Kaasa,1

aDepartment of Psychology, Vanderbilt University, Nashville, TN 37240; bEli Lilly and Company, Indianapolis, IN 46285; cNeuroscience Graduate Program, Vanderbilt University, Nashville, TN 37240; dDepartment of Neuroscience, Neuroscience Research Institute, The Ohio State University College of Medicine, Columbus, OH 43210; and eVanderbilt University Medical Center, Flow Cytometry Shared Resource, Nashville, TN 37232

Contributed by Jon H. Kaas, December 8, 2015 (sent for review November 10, 2015; reviewed by Patrick R. Hof and Chet C. Sherwood) The density of cells and neurons in the neocortex of many mammals considerably lower in primary motor cortex (M1), with more pro- varies across cortical areas and regions. This variability is, perhaps, nounced differences appearing in larger-brained primates. As most pronounced in primates. Nonuniformity in the composition of there is a relationship between neuron density in cortex and cortex suggests regions of the cortex have different specializations. average neuron size (1, 11), and larger neurons have larger Specifically, regions with densely packed neurons contain smaller dendritic arbors with more synaptic contacts (12), larger neurons neurons that are activated by relatively few inputs, thereby pre- are more suitable for integrative functions, whereas smaller neu- serving information, whereas regions that are less densely packed rons preserve information and more faithfully reflect the sources have larger neurons that have more integrative functions. Here we of their activation (13–15). present the numbers of cells and neurons for 742 discrete locations Here, we extend our observations on areal neuron densities in across the neocortex in a chimpanzee. Using isotropic fractionation strepsirrhine galagos and anthropoid monkeys to the full neo- and flow fractionation methods for cell and neuron counts, we cortical sheet of a chimpanzee (Pan troglodytes). We provide the estimate that neocortex of one hemisphere contains 9.5 billion cells 2 most detailed assessment of cortical neuron densities across the and 3.7 billion neurons. Primary visual cortex occupies 35 cm of cortical sheet for any species, having dissected the chimpanzee surface, 10% of the total, and contains 737 million densely packed cortex into 742 tissue pieces, providing roughly six times more neurons, 20% of the total neurons contained within the hemisphere. detail than has ever been reported for the cortex of any primate Other areas of high neuron packing include secondary visual areas, species (1) and nearly 100 times more evaluations than a typical somatosensory cortex, and prefrontal granular cortex. Areas of low quantitative study (10). This comprehensive approach allows us levels of neuron packing density include motor and premotor cortex. These values reflect those obtained from more limited samples of to demonstrate significant regional differences in neuron densities cortex in humans and other primates. that may be small enough to be lost in the noise of more limited studies. Although this has not yet been attempted for a human primate neocortex | neuron density | visual cortex | isotropic fractionator | brain, direct comparisons will be possible in future studies. flow fractionator Results The total surface area of the cerebral cortex of the right hemi- he present study is part of our ongoing effort to determine sphere of this chimpanzee was 341 cm2, although that could be a how human brains are both similar and different from the T slight underestimate resulting from a loss of some neocortex brains of our closest living primate relatives (e.g., refs. 1 and 2). along the outer margins of the cortical sheet. The neocortex Present-day chimpanzees and bonobos diverged from the line of contained an estimated 9.51 billion cells (count includes all cell hominins that led to modern humans some 6 million years ago types identified by DAPI; e.g., glial, neuronal, etc.), of which (3). The modern chimpanzee brain is similar in size to our ear- about 3.71 billion, or 39%, were neurons (only cells that stain liest hominin ancestors (4). In contrast, the brains of later positive for neuronal nuclear antigen). The cell and neuron hominins rapidly increased in size about 1.5 million years ago, resulting in modern humans having brains about three times the size of early hominins and present-day chimpanzees. Similarly, Significance the neocortical sheet of one cerebral hemisphere is almost three times larger in humans (average, ∼975 cm2 in humans; e.g., refs. Chimpanzees are our closest relatives, and understanding the 5 and 6) than in chimpanzees (∼341 cm2). This increase also organization of their brains can help us understand our own appears to be reflected in an increase in the number of areas that evolution. Here we present a detailed examination of cell and are architectonically and physiologically modified for different neuron densities across the chimpanzee cortex. We found simi- specialized functions. Such specializations are reflected in a wide larities to other mammals, including primary sensory areas with range of neuron types and laminar and sublaminar patterns of high neuron densities and a trend of decreasing neuron densities regional and areal organization in larger primate brains, espe- along the posterior to anterior axis of the cortex. However, we cially those of humans (7). It would be surprising if average also found a prefrontal region with anomalously high neuron neuron size and neuron packing densities did not vary across density that disrupts the trend of decreased neuron densities in areas, especially in large-brained primates. However, the claim frontal brain regions. The data reported here allow valuable has been made that neuron densities are uniform across cortical comparisons among the brains of our close relative and those of areas and mammalian species, except for a twofold increase in humans and other primates. primary visual cortex (V1) in some primates (8, 9). This con- Author contributions: C.E.C. and N.A.Y. designed research; C.E.C., E.C.T., E.K.S., N.A.Y., clusion is contrary to the results of several previous studies (e.g., and D.K.F. performed research; E.C.T., E.K.S., and J.L.R. analyzed data; and C.E.C., N.A.Y., ref. 10), especially those of our recent reports on neuron den- and J.H.K. wrote the paper. sities across the cortical sheet in several primate species (1, 2). Reviewers: P.R.H., Mount Sinai School of Medicine; and C.C.S., The George The primates examined to date (baboons, macaques, and gala- Washington University. gos) had much higher neuron densities in V1. Neuron densities The authors declare no conflict of interest. were also higher than average in other sensory areas, and 1To whom correspondence should be addressed. Email: [email protected].

740–745 | PNAS | January 19, 2016 | vol. 113 | no. 3 www.pnas.org/cgi/doi/10.1073/pnas.1524208113 Downloaded by guest on September 30, 2021 counts for individual pieces of tissue varied considerably (Fig. 1). chimpanzees is somewhat uncertain, the location shown in Fig. 1 Some of this variation may be random, possibly reflecting biases conforms to expectations. This estimate of V2 comprises 28.69 cm2 based on tissue distortions during flattening or other processing of cortical surface, or 8% of the total neocortical surface. The factors. Nevertheless, there were large, statistically significant total cells in V2 were just more than 788 million, of which 396 regional and areal differences that were reflected by the com- million, or 50%, were neurons. The average cell densities in V2 parisons of mean counts (see Fig. 2 A and B for statistics). were 27 million per square centimeter of surface (Fig. 2B), or 115 million cells per gram of tissue. The average neuron density Visual Areas of Cortex. The estimated surface area of V1 from the in V2 was 14 million neurons per square centimeter of cortical right hemisphere of the present chimpanzee was 35.04 cm2, surface (Fig. 2A), or 59 million neurons per gram of cortical about 10% of the total neocortical surface. This estimate was tissue, a drop of 7 million neurons per square centimeter or 30 based on the piece of cortex that was separated along the margin million neurons per gram from V1, but still higher than other of the lunate fissure, which marks the rostral border of V1 (16, cortical regions. Neuron densities in V2 were 1.2, 2.1, and 2.2 17). We compensated for a slight error in that cut by adding a times greater than those in somatosensory, motor, and premotor narrow margin of tissue from the larger main piece, as shown in cortices, respectively. red in Fig. 1 B and C. The estimated volume of V1 (5.30 cm3)is Cortex just rostral to V2 is expected to contain a number of close to previous estimates of 5.52 cm3 (18) and 4.64 cm3 (19), as visual areas that have been identified in macaque monkeys and well as those from the other hemisphere of our chimpanzee other primates, including visual areas V3 and V4 and dorso- based on measures from serial brain sections (see ref. 20; 4.8 cm3). medial visual area (21, 22). These and other caudal visual areas Cell and neuron packing densities were greatest in V1. At only would all have higher than average cell, and especially neuron, 10% of the neocortical surface, V1 contained just more than 1.13 packing densities, although not as high as V1 and V2. These data billion cells, of which 737 million, or 65%, were neurons. Cell support the conclusion that retinotopically organized visual areas densities in V1 averaged about 32 million cells per square cen- in chimpanzees have high neuron packing densities. timeter of surface (see Fig. 2B, which shows estimated means) or 138 million cells per gram of tissue. The average neuron density Somatosensory Areas. Somatosensory areas had higher than av- was 21 million neurons per square centimeter of cortical surface erage neuron packing densities relative to nonprimary sensory (Fig. 2A), or 89 million neurons per gram of cortical tissue. Cell areas. Anterior parietal cortex of chimpanzees and other an- and neuron densities varied across the 77 pieces of V1, but most thropoid primates includes areas 3a, 3b, 1, and 2 (23), with area had neuron densities near or higher than 20 million per square 3b being the primary tactile area. The region designated as so- centimeter. The packing densities of neurons in V1 were 1.2, 2.1, matosensory cortex in Fig. 1 likely contains most of areas 3b, 1, 3.3, and 3.5 times greater than neuron densities in secondary and 2. As we separated rostral from caudal cortex along the visual cortex (V2) and somatosensory, motor, and premotor depth of the central sulcus, our “somatosensory cortex” likely cortices, respectively. Total cell densities were less variable includes the caudal half of area 3a, with the rostral half included acrosscortex,butwerestill1.3,1.5,1.3,and1.4times in motor cortex. The most lateral part of somatosensory cortex greater than in V2, somatosensory, motor, and premotor areas, may have been excluded from our designated block, which was respectively. 16.6 cm2 of surface, or 4.86% of neocortex. Cell densities in the The higher cell and neuron packing densities distinguished V1 somatosensory block averaged 22 million cells per square cen- from all other areas of cortex. Although the full extent of V2 in timeter of surface (Fig. 2B), or 91 million cells per gram of tissue.

C NEUROSCIENCE B V1 V2 D Somatosensory P Motor Premotor Frontal

2 2 Million cells/cm Million neurons/cm < 18 25 - 27 34 - 36 < 6 10 - 12 16 - 18 19 - 21 28 - 30 37 - 39 6 - 8 12 - 14 18 - 20 22 -14 31 - 33 > 40 8 -10 14 - 16 > 20

Fig. 1. (A) Lateral view of intact, adult female chimpanzee brain. (B) Complete dissection map, illustrating the 742 pieces of tissue dissected from the chimpanzee cortex. Total cell density in millions of cells per square centimeter of cortical surface is illustrated on the cortex flat map, with the darkest shading indicated areas of high cell density and lighter shading indicating low cell density. The total cell density includes all types of cells in cortex; that is, neurons, glial cells, and epithelial cells. Areal boundaries are estimated for V1, V2, somatosensory (areas 3b, 3a, 1, and 2), motor (areas 4 and 6), and premotor cortex. (C) Complete dissection map illustrating the 742 pieces of tissue dissected from the chimpanzee cortex. Total neuron density in millions of cells per square centimeter of cortical surface is illustrated on the cortex flat map, with the darkest shading indicating areas of high neuron density and lighter shading indicating low neuron density.

Collins et al. PNAS | January 19, 2016 | vol. 113 | no. 3 | 741 Downloaded by guest on September 30, 2021 A Motor and Premotor Areas. Our block of tissue pieces designated as motor in Fig. 1 likely contains most of M1, including the 2 cortex of the rostral bank of the central sulcus and cortex of the **** * caudal half of the precentral gyrus (17, 23–25). The more rostral * *** block of pieces likely includes dorsal and ventral divisions of 2 * * ** premotor cortex. Our motor cortex block consisted of 24.97 cm *** * of cortical surface with 625 million cells, of which 163 million, or 27%, were neurons. Cell packing densities averaged 25 million *** * cells per square centimeter of cortical surface (Fig. 2B), or 91

face area (millions/ cm ) area face ** * r million cells per gram of tissue. Neuron packing densities aver-

y su aged 7 million neurons per square centimeter of surface (Fig. 2A), or 24 million neurons per gram. Nearly all of the individual

ons b –

r pieces of M1 had low neuron densities, in the 6 7 million range. Thus, M1 was characterized by low neuron packing densities and moderate cell packing densities. Our premotor block included 24.46 cm2 of cortical surface, with 636 million cells and 172 million neurons, for a composition of 26% neurons. Cell densities in premotor cortex averaged 25 million per square centimeter of cortical surface (Fig. 2B), or 88

Mean number of neu million per gram of tissue. The average neuron packing density was 6 million neurons per square centimeter of cortical surface (Fig. 2A), or 23 million neurons per gram of cortical tissue. Thus, cortex in the premotor block had low levels of neuron packing, with little variability across tissue pieces, and closely matched the low neuron packing densities across motor areas. B ** ** * Prefrontal Cortex. Prefrontal cortex generally consists of the cortex * * rostral to premotor cortex, and it has several subdivisions including

) mc /snoillim( aera e aera /snoillim( mc ) 2 * * a large dorsolateral region of granular frontal cortex and adjoining * * regions of orbital frontal and medial frontal cortex (26–28). Areal * ** * variations in neuron densities across prefrontal cortex have previously been shown in macaques (29). Overall, the prefrontal block of tissue denoted in Fig. 1 B and C has pieces of tissue with higher cell packing densities and neuron packing densities than those in

c either motor or premotor cortex. Most of this cortex with higher

f rus y rus cellandneuronpackingdensities would be considered granular frontal cortex. Pieces of cortex with lower values were located along

b s the margins of frontal cortex, including medial frontal and orbital

c frontal regions. However, we distinguished a dorsomedial block of

cortex within the presumptive region of granular frontal cortex (Fig. rebmun naeM rebmun 1C) as having higher neuron densities than other prefrontal regions. Pieces of cortex in this block had average neuron packing densities of 11 million per square centimeter. Totalcelldensities averaged 30 million per square centimeter, or 106 million per gram, with 36% neurons. This high-density block of tissue covered 13.11 cm2 with 515 cells and 181 million neurons.

The Anterior to Posterior Pattern. To examine whether the chimpanzee brain showed uniform cell and neuron packing densities Fig. 2. Pairwise comparison results of estimated marginal means based on across cortex (or a linear decrease in density beyond V1 from the original scale of the dependent variable, either neuron (A)orcell(B) posterior to anterior), we examined densities in pieces of cortex density in square centimeter, with mean differences significant at P < 0.05 across the anterior-to-posterior (A-P) dimension, using linear and robust SE bars shown. All significant pairwise comparisons are high- regression and curve estimation, testing multiple model func- lighted by the significance bars. (A) V1 estimated marginal means of neuron tions. Recently, there have been a number of descriptions of density are significantly higher than all other cortical regions, whereas V2 is neuron packing densities across the cortical sheet in monkeys significantly higher than motor, premotor, and somatosensory blocks. So- and other mammals that revealed a trend from low packing matosensory predicted values are also significantly higher than motor and densities to high neuron packing densities from anterior to poste- premotor blocks. Motor and premotor cortices do not significantly differ – from one another, but are both significantly lower than all other areas of rior cortex (11, 30 33). When we assessed how cell and neuron cortex. (B) V1 and the frontal region estimates show no significant differ- densities varied in the selected cortical regions across the A-P di- ences in cell density, but each region independently contains higher means mension, using generalized linear modeling with robust estimators, than every other area. Somatosensory, premotor, and motor cortex do not most cortical areas showed significant differences in estimated significantly differ from one another. means of neuron and cell density (Fig. 2 A and B). In addition, the locations of samples within cortical regions were better predictors than A-P coordinates alone of cell density (cortical area, P = 6.4 × − Neuron numbers averaged 10 million per square centimeter 10 10;A-P,P = 0.818) and of neuron density (cortical area, P = − (Fig. 2A), or 41 million neurons per gram of tissue. The so- 1.0 × 10 4;A-P,P = 0.835). matosensory section of cortex contained 362 million cells, of When the density values for individual pieces were plotted by A-P which 164 million, or 45%, were neurons. location and color-coded for tissue block of origin, the average curve

742 | www.pnas.org/cgi/doi/10.1073/pnas.1524208113 Collins et al. Downloaded by guest on September 30, 2021 for neuron density had higher values in posterior cortex and a downward slope to lower values in anterior cortex (Fig. 3A). Al- A though an A-P gradient is apparent in the array of values for individual pieces, it clearly does not correspond to a simple linear A-P

gradient. This type of plotting allows V2 values to mix with V1 values 2 to contribute to a steep rise in the posterior slope that obscures the clear difference between V1 and V2. In addition, the prefrontal granular region contains high neuron packing values with a fringe of lower values. However, motor and premotor regions contain low values, the somatosensory region has moderate values, and much of temporal and posterior parietal regions have moderate values. A similarpattern,withlessvariation,isapparentforallcells(Fig.3B). For the densities of cells and neurons, we found that cubic models provided the best correlations (Fig. 3 A and B), all with P values <0.0001. These results, including increased densities in frontal regions of cortex, support deviations from linear or uniform patterns across the A-P dimension. Discussion In the present study, we flattened the neocortex of one cerebral hemisphere of a chimpanzee into a sheet, divided the sheet into

three main parts, and then further divided the large pieces into 742 surface by Mean (millions/cm ) number of neurons area small pieces of tissue. Tissue blocks were individually processed for estimates of total neuron numbers using the rapid and accurate flow fractionation method (34, 35), and estimates of total cell number were obtained using the isotropic fractionation method (36). The results are shown in cell or neuron number per square centimeter of cortical surface because the number of neurons in the vertically defined cortical columns that extend across the depth of the cortex has been considered basic to cortical function (37). The results indicate that areas of neocortex in chimpanzees differ greatly in B packing density, such that visual areas V1 and V2 have the highest neuron density, and the motor and premotor areas are among those that have the lowest densities. Perhaps surprisingly, a region of

granular prefrontal cortex had higher neuron densities than sur- 2 rounding cortical regions. These clear differences in cell and neuron densities, when considered together with the rapidly accumulating evidence from other primates, and even nonprimate species (38, 39), should dispel any notion that the neocortex is uniform in this respect. The present results have functional implications for the neocortex in chimpanzees and invite comparisons with the neocortex of other primates, especially with humans, as the closest biological extant relative of chimpanzees and bonobos. NEUROSCIENCE

Comparisons with Other Primates. Our results allow detailed comparisons with similar maps of flattened neocortex in macaques and baboons (1, 2, 40). In these primates, V1 had the highest neuron densities, as much as three to six times that of most cortical regions. This is unsurprising, as larger numbers of neurons per cortical col- umn have been previously reported in V1 of monkeys (8), and more

recently reproduced (9). However, those authors argued for the surface by Mean number of cells (millions/cm ) area “basic uniformity” in neuron numbers across the depth of cortex for other areas of cortex, and across mammalian species. Here, we show that neuron numbers vary across the chimpanzee cortical sheet, with high values also in V2, somatosensory cortex, and part of frontal granular cortex, and low values in motor and premotor cortex. Most notably, neocortex in macaques and baboons also reflects this general pattern. In a more limited study of flattened cortex of a galago, V1 also had much higher neuron densities than other areas (1), and M1 of owl monkeys, squirrel monkeys, and galagos has been shown Fig. 3. The cell and neuron densities of all 742 pieces are plotted based on their to have low neuron densities in comparison with other cortical re- anterior–posterior position in the flattened cortex, and designated to a cortical gions when studied with our current methods (2). Such a detailed area(showninsmallmap).Neuron(A)andcell(B) packing densities follow the comparison with neocortex of humans is not yet possible, but same pattern, in which densities are highest in the most posterior positions and lower in more anterior positions, with the exception of a block of frontal cortex meaningful comparisons of neuron densities using another approach that contains higher densities. For both cell and neurons, although the correla- indicate impressive similarities with our present results. By using tion is low, cubic models provide a better correlation (neurons: R2 = 0.424; cells: thick frontal sections of a human brain and comparing neuronal R2 = 0.066) compared with linear (neurons: R2 = 0.303; cells: R2 = 0.011) or densities across anterior to posterior slices of cortex, Ribeiro et al. quadratic (neurons: R2 = 0.414; cells: R2 = 0.035) models. Most outliers are cell- or (30) reported very high neuron densities for posterior slices including neuron-dense pieces in the posterior part of the brain.

Collins et al. PNAS | January 19, 2016 | vol. 113 | no. 3 | 743 Downloaded by guest on September 30, 2021 V1, and very low values for anterior slices in frontal and prefrontal order sensory areas have larger neurons with larger arbors. The cortex. It is not clear yet from these results whether V1 and V2 neuron density values in cortex illuminate this hypothesis in detail differ, whether M1 and premotor cortex are specifically low in by indicating average levels of information preservation and in- neuron number, or whether a granular region of prefrontal cortex tegration for areas across the cortical sheet for chimpanzee. with higher neuron densities exists. It is also uncertain whether pri- However, neurons of quite different sizes may occur in the same mary somatosensory cortex is higher in primates in general, as area and play different functional roles. For example, V1 contains expected from its well-developed layer of granular cells, or whether both small granular cells in layer 4 and large Meynert cells in granular primary auditory cortex values are higher in any primate. layers 5 and 6. However, the average neuron size should suggest a As for the nonprimates that have been studied, areal differences in dominant role for a region or area. mean densities may be less pronounced, but they vary considerably One of the important findings of the present study was that a across cortical areas with high densities in V1 and primary so- dorsal part of granular prefrontal cortexinthechimpanzeehada matosensory cortex (S1) in mice (39) and rats (41). Others have region of higher neuron density. As the term “granular frontal cortex” reported higher neuron densities in S1 than M1 and V2 in rats (42), implies, this cortex contains small neurons in layer 4. However, large and higher values in visual areas and S1 than M1 in cats (10). pyramidal neurons have also been described in prefrontal cortex, suggesting such neurons receive and sum many inputs (12). Although Does Neuron Packing in Chimpanzee Cortex Reflect a Developmental all parts of granular prefrontal cortex would seem suitable for pre- Pattern of Cortical Neurogenesis? In a series of publications, Finlay serving information, the dorsomedial part seems more specialized for and colleagues have presented evidence that an anterior to posterior this function. Notably, granular frontal cortex is thought to be a pattern of cortical development that is seen in primates and other specialization of the primate brain (26) that appears to be important mammals results in a matching gradient of neuron densities from low – in working memory (50). Our present results are consistent with the to high across the cortex (e.g., refs. 11 and 31 33). Our chimpanzee conclusion that granular frontal cortex is not uniform in function (28), results roughly reflect such a pattern, with at least four exceptions. and the region of particularly dense neuron packing in frontal cortex These exceptions include the sharp increase in neuron densities in V1 – may be a specialization of primates that occurs to a lesser extent in from V2 at the V1 V2 border, the very low densities in motor and Old World macaques (1) and baboons (40), is especially marked in premotor cortex, the increased neuron densities in anterior so- chimpanzees, and is likely in humans. matosensory cortex, and the higher than expected neuron densities in dorsofrontal cortex of “granular” frontal cortex. These exceptions do Materials and Methods not argue against the developmental gradient having an important Experimental procedures were all approved by the Vanderbilt Institutional role in creating neuron density differences across the cortex, but the Animal Care and Use Committee. One adult female chimpanzee brain was exceptions do indicate that other factors are also involved. obtained for this study from the Texas Biomedical Research Institute. The age Such additional factors may include areal differences in neuron of the chimpanzee was estimated to be 53 y. The animal was humanely death during development (31). Direct evidence for this possibility killed because of myocarditis (heart failure). The neocortex is expected to comes from V1 of macaques after loss of visual inputs to the brain have few age-related changes in a chimpanzee of this age (51, 52). Shortly during fetal development. In such monkeys, parts of V1 fail to de- before death, her body weight was 34.8 kg. The brain was flushed with 0.9% velop normally histologically (43, 44), and these abnormal parts had PBS, removed from the skull, and shipped overnight in the same solution. On cell densities reduced by about 25% (43). In view of the de- arrival, the brain weight was 344 g. The brain was bisected into right and left velopmental gradient theory of regional differences in neuron den- hemispheres. The right neocortex was flattened and used in this report, and the sities, it may just be a fortunate circumstance for primates that V1 left hemisphere was sectioned and histologically processed for other studies. evolved in caudal neocortex. Higher neuron densities in V1 function To determine neuron and cell number per unit of cortical surface area across all parts of the cortical surface, we first manually flattened the neocortex into a to precisely preserve visual information, whereas motor and pre- sheet. The unfixed cortex of the right hemisphere was separated from the un- motor cortex, as well as parts of prefrontal cortex, evolved within derlying structures and most of the white matter. A cut was made in the depth of more anterior cortex to contain extremely low neuron densities as a the central sulcus to separate the most anterior region, and another cut was made result of larger neurons. This makes neurons in these anterior re- to separate primary visual cortex from the rest of the caudal cortex, creating three gionsbettersuitedtointegrateinformation from many sources of separate cortical pieces for flattening. This was done to preserve the integrity of activation. Alternatively, more modular features of cortical devel- the tissue during flattening. Sulci were carefully opened, and the cortical sheet opment could have played a prominent role in the evolution of such was then unfolded under gentle pressure (Fig. 1A). Excess white matter was striking differences in neuron densities across cortex. removed. After postfixing in 4% (wt/vol) paraformaldehyde for 2 wk, each sheet was cut into small pieces, ∼5mm2 in surface area, resulting in 742 pieces that Functional Implications. Differences across cortical areas in neu- were photographed, weighed, numbered, and assigned to a cortical area when ron packing densities imply there is an inverse relationship with possible (Fig. 1 B and C). Pieces were assigned to cortical areas based on their average neuron size (45). Larger neurons take up more space expected relation to sulcal landmarks and their myelin content, as primary sensory areas appear dark relative to the surrounding cortex when viewed on a and require more glial and other support cells that vary in size. light box because of their myelin-dense composition. The surface area of each Smaller neurons have smaller dendritic arbors and are connected piece of cortex was measured from the photograph, using ImageJ (NIH). Each by fewer inputs (46). Overall, small cortical neurons are better piece of cortex was individually disassociated to a solution, where cell mem- designed for preserving information from a small number of branes were ruptured but cell nuclei remained intact, as previously described activating inputs, whereas large neurons are better suited for (34, 36). We determined the number of cells in each tissue piece, using the iso- integrating information from a larger number of activating inputs tropic fractionator method (36). The flow fractionator method was used to esti- (47, 48). Thus, V1 has densely packed small neurons (granular mate of the total number of neuron nuclei labeled with the anti-NeuN antibody cells) in layer 4 that are activated by just a few neurons in the andDAPIineachtissuepiece,aspreviouslydescribedindetail(1,2,34,35,40). dorsal lateral geniculate nucleus, and they activate other neurons IBM-SPSS software (version 22) was used to test for deviations from the in V1 that are, with few exceptions, small pyramidal neurons with normal distribution (Kolmogorov-Smirnov test), to test for nonlinearities small apical arbors contacted by relatively few inputs (46, 48). (linear regression), and to compare cell and neuron densities between se- The high neuron densities for V1 of primates have been postu- lected cortical areas (generalized linear modeling). Significance was considered for P < 0.05. Statistical analysis included using Huber-White robust lated as a mechanism for preserving the high visual acuity of SEs through the generalized linear modeling procedures specifying “robust” primates (49). Motor cortex is known for its large pyramidal cells covariance matrix estimators (also called Huber-White or sandwich estima- and lack of small layer 4 granular cells, which promotes integration tors) in SPSS software. Robust SEs are corrections to help account for viola- from more sources of information. Primary sensory areas generally tions of the assumption of independence between cell counts on tissue have smaller neurons with smaller dendritic arbors, whereas higher- pieces from a single chimpanzee brain. The resulting P values can be used to

744 | www.pnas.org/cgi/doi/10.1073/pnas.1524208113 Collins et al. Downloaded by guest on September 30, 2021 help describe differences within the one brain, but they do not directly allow fixed factor (see Fig. 1C for regional bin map), and A-P coordinates (x-value inferences about the population of chimpanzees. from the centroid calculation) were binned for use as the components of the Cell and neuron numbers per square centimeter of cortical surface were second fixed factor. Model main effects were tested and evaluated for the obtained for each piece of cortex and color coded for cells (Fig. 1B) or neurons relative contribution of each factor to the variance in mean density. Multiple (Fig. 1C). In addition, each dissected piece of tissue was assigned coordinates comparisons were adjusted by the Bonferroni method, and robust estimators, along the A-P axis by generating centroid measures for each piece, using NIH as addressed earlier, were used in this analysis to account for violations of ImageJ software. Cell and neuron numbers per square centimeter of surface independence between the samples from a single chimpanzee. area versus the A-P locations were plotted and used in linear regression curve-fitting estimations to assess whether cell and neuron densities were uniform or increased or decreased in the A-P dimension with linear, ACKNOWLEDGMENTS. We thank Laura Trice, Kallie Yeoman, and Feyi Aworunse for laboratory assistance. Flow cytometry experiments were quadratic, and cubic regression models. performed in the Vanderbilt University Medical Center (VUMC) Flow Cytometry The results were tested for deviations from the normal distribution Shared Resource. The VUMC Flow Cytometry Shared Resource is supported (Kolmogorov-Smirnov test), and generalized linear regression modeling by the Vanderbilt Ingram Cancer Center (P30 CA68485) and the Vanderbilt was then used to compare cell and neuron densities between selected cortical Digestive Disease Research Center (DK058404). This work was supported areas. Selected cortical areas defined by experts were the components of one by a grant from the G. Harold and Leila Y. Mathers Foundation (to J.H.K.).

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