Isotropic fractionator: a simple, rapid method for the quantification of total cell and neuron numbers in the brain - PubMed (original) (raw)
Isotropic fractionator: a simple, rapid method for the quantification of total cell and neuron numbers in the brain
Suzana Herculano-Houzel et al. J Neurosci. 2005.
Abstract
Stereological techniques that estimate cell numbers must be restricted to well defined structures of isotropic architecture and therefore do not apply to the whole brain or to large neural regions. We developed a novel, fast, and inexpensive method to quantify total numbers of neuronal and non-neuronal cells in the brain or any dissectable regions thereof. It consists of transforming highly anisotropic brain structures into homogeneous, isotropic suspensions of cell nuclei, which can be counted and identified immunocytochemically as neuronal or non-neuronal. Estimates of total cell, neuronal, and non-neuronal numbers can be obtained in 24 h and vary by <10% among animals. Because the estimates obtained are independent of brain volume, they can be used in comparative studies of brain-volume variation among species and in studies of phylogenesis, development, adult neurogenesis, and pathology. Applying this method to the adult rat brain, we show, for example, that it contains approximately 330 million cells, of which 200 million are neurons, and almost 70% of these are located in the cerebellum alone. Moreover, contrary to what is commonly assumed in the literature, we show that glial cells are not the majority in the rat brain.
Figures
Figure 2.
The total number of neurons in dissected brain regions (top; cortex, cerebellum, olfactory bulb, and remaining areas) is estimated by determining the proportion of DAPI-stained nuclei in isotropic suspensions (left) that are also NeuN positive (center; right, merged images). All of the photomicrographs are shown at the same magnification. Scale bars, 50 μm. Notice that the proportion of NeuN-positive nuclei is distinct among the four regions of interest. “Cortex” refers to gray and white matter of neocortex, hippocampus, and adjacent paleocortices lateral to the olfactory tract; “Cerebellum” refers to cerebellar cortex, deep nuclei, and cerebellar white matter, including the cerebellar peduncles. “Remaining areas” include accessory olfactory nuclei, basal ganglia, diencephalon and optic chiasm, and brainstem. Smaller, precise regions can also be quantified using the isotropic fractionator, provided constant criteria are used for dissection (e.g., specific cortical areas and gyri, subcortical nuclei, basal ganglia, diencephalon, midbrain, pons, and medulla). In addition, dissections can also be performed from vibratome sections.
Figure 1.
A, The isotropic fractionator is a 10-step method that requires little material and allows rapid determination of the total number of cells (2 h from steps 2-8) and total numbers of neuronal and non-neuronal cells (24 h from steps 2-10) in aldehyde-fixed brain tissue. Fr, Fraction. B, C, Appearance of isotropic suspensions of DAPI-stained nuclei prepared from unfixed (B) or paraformaldehyde-fixed (C) adult rat cortex, shown at the same magnification. Scale bars, 50 μm. Although complete dissociation of unfixed tissue leads to nuclear destruction (B), fixed, dissociated nuclei are intact and have preserved morphology (C).
Similar articles
- How to count cells: the advantages and disadvantages of the isotropic fractionator compared with stereology.
Herculano-Houzel S, von Bartheld CS, Miller DJ, Kaas JH. Herculano-Houzel S, et al. Cell Tissue Res. 2015 Apr;360(1):29-42. doi: 10.1007/s00441-015-2127-6. Epub 2015 Mar 5. Cell Tissue Res. 2015. PMID: 25740200 Free PMC article. - Validation of the isotropic fractionator: comparison with unbiased stereology and DNA extraction for quantification of glial cells.
Bahney J, von Bartheld CS. Bahney J, et al. J Neurosci Methods. 2014 Jan 30;222:165-74. doi: 10.1016/j.jneumeth.2013.11.002. Epub 2013 Nov 12. J Neurosci Methods. 2014. PMID: 24239779 Free PMC article. - Automatic isotropic fractionation for large-scale quantitative cell analysis of nervous tissue.
Azevedo FA, Andrade-Moraes CH, Curado MR, Oliveira-Pinto AV, Guimarães DM, Szczupak D, Gomes BV, Alho AT, Polichiso L, Tampellini E, Lima L, de Lima DO, da Silva HA, Lent R. Azevedo FA, et al. J Neurosci Methods. 2013 Jan 15;212(1):72-8. doi: 10.1016/j.jneumeth.2012.09.015. Epub 2012 Sep 24. J Neurosci Methods. 2013. PMID: 23017980 - The search for true numbers of neurons and glial cells in the human brain: A review of 150 years of cell counting.
von Bartheld CS, Bahney J, Herculano-Houzel S. von Bartheld CS, et al. J Comp Neurol. 2016 Dec 15;524(18):3865-3895. doi: 10.1002/cne.24040. Epub 2016 Jun 16. J Comp Neurol. 2016. PMID: 27187682 Free PMC article. Review. - Variability in neuron densities across the cortical sheet in primates.
Collins CE. Collins CE. Brain Behav Evol. 2011;78(1):37-50. doi: 10.1159/000327319. Epub 2011 Jun 17. Brain Behav Evol. 2011. PMID: 21691046 Review.
Cited by
- Saxitoxin potentiates human neuronal cell death induced by Zika virus while sparing neural progenitors and astrocytes.
Souza LRQ, Pedrosa CGDS, Puig-Pijuan T, da Silva Dos Santos C, Vitória G, Delou JMA, Setti-Perdigão P, Higa LM, Tanuri A, Rehen SK, Guimarães MZP. Souza LRQ, et al. Sci Rep. 2024 Oct 1;14(1):22809. doi: 10.1038/s41598-024-73873-z. Sci Rep. 2024. PMID: 39354036 Free PMC article. - In vivo neuropil density from anatomical MRI and machine learning.
Akif A, Staib L, Herman P, Rothman DL, Yu Y, Hyder F. Akif A, et al. Cereb Cortex. 2024 May 2;34(5):bhae200. doi: 10.1093/cercor/bhae200. Cereb Cortex. 2024. PMID: 38771239 - SOX2 and SOX9 Expression in Developing Postnatal Opossum (Monodelphis domestica) Cortex.
Baričević Z, Pongrac M, Ivaničić M, Hreščak H, Tomljanović I, Petrović A, Cojoc D, Mladinic M, Ban J. Baričević Z, et al. Biomolecules. 2024 Jan 5;14(1):70. doi: 10.3390/biom14010070. Biomolecules. 2024. PMID: 38254670 Free PMC article. - Different ways of evolving tool-using brains in teleosts and amniotes.
Estienne P, Simion M, Hagio H, Yamamoto N, Jenett A, Yamamoto K. Estienne P, et al. Commun Biol. 2024 Jan 12;7(1):88. doi: 10.1038/s42003-023-05663-8. Commun Biol. 2024. PMID: 38216631 Free PMC article. - Accurate classification of major brain cell types using in vivo imaging and neural network processing.
Das Gupta A, Asan L, John J, Beretta C, Kuner T, Knabbe J. Das Gupta A, et al. PLoS Biol. 2023 Nov 9;21(11):e3002357. doi: 10.1371/journal.pbio.3002357. eCollection 2023 Nov. PLoS Biol. 2023. PMID: 37943858 Free PMC article.
References
- Andersen BB, Korbo L, Pakkenberg B (1992) A quantitative study of the human cerebellum with unbiased stereological techniques. J Comp Neurol 326: 549-560. - PubMed
- Barton RA, Harvey PH (2000) Mosaic evolution of brain structure in mammals. Nature 405: 1055-1058. - PubMed
- Brizzee KR, Vogt J, Kharetchko X (1964) Postnatal changes in glia/neuron index with a comparison of methods of cell enumeration in the white rat. Prog Brain Res 4: 136-149.
- Clark DA, Mitra PP, Wang SS (2001) Scalable architecture in mammalian brains. Nature 411: 189-193. - PubMed
- de Winter W, Oxnard CE (2000) Evolutionary radiations and convergences in the structural organization of mammalian brains. Nature 409: 710-714. - PubMed
Publication types
MeSH terms
LinkOut - more resources
Full Text Sources
Other Literature Sources