Equal Numbers of Neuronal and Nonneuronal Cells Make the Human Brain an Isometrically Scaled-Up Primate Brain FREDERICO A.C. AZEVEDO,1 LUDMILA R.B. CARVALHO,1 LEA T. GRINBERG,2,3 JOSE �� MARCELO FARFEL,2 RENATA E.L. FERRETTI,2 RENATA E.P. LEITE,2 WILSON JACOB FILHO,2 ROBERTO LENT,1 AND SUZANA HERCULANO-HOUZEL1* 1Instituto de Cie ��ncias Biome ��dicas, Universidade Federal do Rio de Janeiro, Cidade Universita ��ria, Ilha do Funda ��o 21941-590 Rio de Janeiro, Brazil 2Grupo de Estudos em Envelhecimento Cerebral da Faculdade de Medicina da Universidade de Sa ��o Paulo, Sa ��o Paulo, Brazil 3Instituto Israelita de Ensino e Pesquisa Albert Einstein ABSTRACT The human brain is often considered to be the most cogni- tively capable among mammalian brains and to be much larger than expected for a mammal of our body size. Al- though the number of neurons is generally assumed to be a determinant of computational power, and despite the wide- spread quotes that the human brain contains 100 billion neurons and ten times more glial cells, the absolute number of neurons and glial cells in the human brain remains un- known. Here we determine these numbers by using the isotropic fractionator and compare them with the expected values for a human-sized primate. We find that the adult male human brain contains on average 86.1 8.1 billion NeuN-positive cells (���neurons���) and 84.6 9.8 billion NeuN- negative (���nonneuronal���) cells. With only 19% of all neurons located in the cerebral cortex, greater cortical size (repre- senting 82% of total brain mass) in humans compared with other primates does not reflect an increased relative num- ber of cortical neurons. The ratios between glial cells and neurons in the human brain structures are similar to those found in other primates, and their numbers of cells match those expected for a primate of human proportions. These findings challenge the common view that humans stand out from other primates in their brain composition and indicate that, with regard to numbers of neuronal and nonneuronal cells, the human brain is an isometrically scaled-up primate brain. J. Comp. Neurol. 513:532���541, 2009. �� 2009 Wiley-Liss, Inc. Indexing terms: human brain size neuron numbers glia/neuron ratio evolution comparative neuroanatomy It is repeatedly stated in the literature and in neuroscience textbooks that the human species is an unusually enceph- alized primate species, whose brain, five to seven times larger than expected for a mammal of its body size (Jerison, 1973 Marino, 1998), contains 100 billion neurons and about ten times more glial cells (Kandel et al., 2000 Ullian et al., 2001 Doetsch, 2003 Nishiyama et al., 2005 Noctor et al., 2007). The supposedly unusual scaling of the human brain, however, derives from comparisons across orders (Jerison, 1973) and, even when restricted to primates, regards only the brain���body relationship (Marino, 1998) rather than addressing how its cellular composition compares with that expected from other primates. Moreover, to our knowledge, the widespread numbers on the cellular composition of the human brain have never been supported by experimental studies. The high anisotropy and large size of the human brain hinder stereological determina- tion of cell numbers and their distribution in the brain as a whole. Estimates of the cellular composition of the human brain are available only for some structures, such as the cerebral cortex (von Economo and Koskinas, 1925 Shariff, 1953 Pakkenberg, 1966 Pakkenberg and Gundersen, 1997 Pelvig et al., 2008), cerebellum (Lange, 1975 Andersen et al., 1992), and some subcortical nuclei (Pakkenberg and Gun- dersen, 1988). Such studies have estimated the number of Grant sponsor: FAPERJ (to S.H.-H., R.L.) Grant sponsor: CNPq (to S.H.-H., R.L.) Grant sponsor: Pronex (to R.L.) Grant sponsor: CAPES (to R.E.P.L.) Grant sponsor: IIEP-Albert Einstein (to L.T.G.) Grant sponsor: Alexander von Humboldt Foundation (to L.T.G.). The last two authors contributed equally to this work. *Correspondence to: Suzana Herculano-Houzel, Instituto de Ciencias�� Biomedicas, �� Universidade Federal do Rio de Janeiro, Av. Brigadeiro Trompowski s/n, Ilha do Fundao �� 21941-590 Rio de Janeiro-RJ, Brasil. E-mail: suzanahh@gmail.com Received 6 June 2008 Revised 15 September 2008 Accepted 16 De- cember 2008 DOI 10.1002/cne.21974 Published online in Wiley InterScience (www.interscience.wiley.com). The Journal of Comparative Neurology 513:532���541 (2009) �� 2009 Wiley-Liss, Inc.

cells in the human cerebral cortex as 3, 7, 14, 19���23, or 21���26 billion neurons and, very recently, 28���39 billion glial cells (Pelvig et al., 2008), and the number of cells in the human cerebellum has been estimated as 70 or 101 billion neurons (Lange, 1975 Andersen et al., 1992) and fewer than 4 billion glial cells (Andersen et al., 1992). From such studies, the total number of neurons in the human brain might be inferred to fall anywhere between about 75 and 125 billion plus an undeter- mined number of neurons in the brainstem, diencephalon, and basal ganglia that may or may not be comparatively small. Additionally, no evidence is found to support the common quote of ten times more glial cells than neurons in the human brain. The glia:neuron ratio in subcortical nuclei can be as high as 17:1 in the thalamus (Pakkenberg and Gundersen, 1988), but, given the relatively small combined number of glial cells reported for the cerebral (Pelvig et al., 2008) and cerebellar (Andersen et al., 1992) cortices, the only possible explanation for the quote of ten times more glial than neuronal cells in the entire human brain would be the presence of nearly one trillion glial cells in the remaining structures. We have recently determined the cellular scaling rules that apply to the brain of a number of rodent and primate species and found that, whereas the rodent brain increases in mass faster than it gains neurons (defined as NeuN-positive cells) across species, suggesting that the average neuronal cell size increases in larger rodent brains (Herculano-Houzel et al., 2006), the primate brain increases in mass linearly with in- creases in its number of neurons across species, suggesting that the average neuronal cell size does not increase signifi- cantly with brain size (Herculano-Houzel et al., 2007). The power laws relating body mass, brain mass, and number of neurons for rodent and primate species allowed us to predict that, if built according to the cellular scaling rules that apply to rodents, a brain of 100 billion neurons should weigh over 45 kg and belong to a body of 109 tons. In contrast, if built accord- ing to the scaling rules that apply to primates, this brain of 100 billion neurons should weigh 1.45 kg and belong to a body of 73 kg, values that approach those observed in humans, sug- gesting that the human brain is indeed constructed according to the same rules that apply to other primates. We thus set out to determine the total cellular composition of the human brain with the aid of the same method (the isotropic fractionator Herculano-Houzel and Lent, 2005) and relying on the same criterion of NeuN labeling to identify ���neurons��� and ���nonneuronal cells��� in order to evaluate how its composition compares with the expected composition of a primate brain of its size. While supporting several indepen- dent stereological estimates, our results challenge the values so often cited in the literature and suggest that, with regard to brain cellular composition, humans are just scaled-up, large primates. MATERIALS AND METHODS Human material All brains were obtained from the Brain Bank of the Brazilian Aging Brain Study Group (Grinberg et al., 2007), located at the University of Sao �� Paulo Medical School (FMUSP). The project was approved by the Ethics Committee for Research Projects Analysis (CAPPesq) of FMUSP, Research Protocol number 285/04. Informed consent for removal of the brains was pro- vided by next of kin, who also responded to the Clinical Dementia Rating Scale (CDR) semistructured interview and to the Informant Questionnaire on Cognitive Decline in the Elderly���Retrospective Version (IQCODE Jorm and Jacomb, 1989 Morris, 1993). Four brains from 50-, 51-, 54-, and 71- year-old males, deceased from nonneurological causes and without cognitive impairment (CDR 0, IQCODE 3.0), were analyzed. The brain of the 71-year-old male was included in the analysis because it contained a similar number of cells and an even slightly higher number of neurons than the other brains. The corpses remained at 4��C until the brains were removed from the cranium less than 24 hours after death and fixed immediately. Fixation and dissection Brains were fixed by perfusion with 4 liters of 2% phosphate-buffered paraformaldehyde through the basilar ar- tery and the internal carotids, followed by immersion for 36 hours in the same fixative. Fixation for less than 48 hours was critical to allow for antibody recognition of NeuN, while still being enough to guarantee that the nuclei remained intact throughout the homogenization procedure. The meninges and major blood vessels were removed, and the brains were split sagitally into two hemispheres. After dissecting each cerebel- lar hemisphere by cutting the cerebellar peduncles at the surface of the brainstem, each hemisphere was cut into 1-cm- thick coronal sections, and the cerebral cortex was separated from the remaining regions (basal ganglia, diencephalon, mesencephalon, and pons, named collectively ���rest of brain,��� or RoB) by cutting through the white matter along the surface of the striatum in each section. In three of the four brains analyzed, one of the hemispheres of the cerebral cortex had the gray matter dissected away from the underlying white matter by careful shaving of the gray matter around the gyri with a scalpel until the white matter was exposed. The me- dulla was excluded because of inconsistency in the inferior section level among cases during the autopsy procedure. After fixation, the three main regions of interest (cerebellum, cerebral cortex, and RoB) were stored in phosphate-buffered saline (PBS pH 7.4) at 4��C and subjected individually to the isotropic fractionator method (Herculano-Houzel and Lent, 2005). Each structure was cut into smaller pieces that could be homogenized in a tissue grinder and counted in 1 day, and partial results were added together. Determining the total number of cells in each brain typically required about 4���6 weeks. Isotropic fractionator The isotropic fractionator method has been described else- where (Herculano-Houzel and Lent, 2005). Briefly, it consists of a chemomechanical dissociation of fixed biological tissue in a saline detergent solution (1% Triton X-100, 40 mM sodium citrate) using 40���200-ml glass tissue grinders, followed by intense agitation of the suspension containing all nuclei in the original structure, in order to achieve isotropy. After adding the fluorescent DNA marker 4 -6-diamino-2-phenylindole di- hydrochloride (DAPI) to the suspension, the density of nuclei is quantified by use of a hemocytometer under a fluorescence microscope (Fig. 1A,C). The total number of nuclei is calcu- lated by multiplying the density of nuclei by the total suspen- sion volume and heretofore is referred to as ���total number of cells��� in each structure. The Journal of Comparative Neurology 533 THE HUMAN BRAIN AS A SCALED-UP PRIMATE BRAIN