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.

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Figures

Figure 2.

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.

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).

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References

    1. 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
    1. Barton RA, Harvey PH (2000) Mosaic evolution of brain structure in mammals. Nature 405: 1055-1058. - PubMed
    1. 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.
    1. Clark DA, Mitra PP, Wang SS (2001) Scalable architecture in mammalian brains. Nature 411: 189-193. - PubMed
    1. de Winter W, Oxnard CE (2000) Evolutionary radiations and convergences in the structural organization of mammalian brains. Nature 409: 710-714. - PubMed

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