Major neurological and psychiatric diseases have been reported to be associated with significantly changed glia densities, but considerable confusion surrounds the question of the true numbers and ratios of neurons and glial cells in the brains of humans and animals. Traditionally and per """"""""textbook knowledge,"""""""" it was thought that glial cells outnumber neurons by a ratio of between 10:1 and 50:1. Recently introduced new counting techniques have challenged this belief and postulate a ratio of approximately 1:1, reducing the number of glial cells in the human brain from previous estimates by as much as 4.9 trillion (from 5 trillion to a mere 100 billion). The new, much lower estimates of glial cells are primarily based on a novel methodological approach, the isotropic fractionator (IF). However, this technique has not been calibrated or validated. It is therefore uncertain whether it produces true numbers or may possibly be biased towards larger particles (neuronal nuclei) and may underestimate smaller ones (glial nuclei). The fractionator technique homogenizes whole brains (or dissected parts thereof), then takes samples of the cell nuclei in solution, and uses antibody labeling to distinguish neuronal nuclei from other nuclei such as glia in sample solutions. While conceptually elegant, it remains to be calibrated against known numbers and ratios of neurons and glial cells. Therefore, we propose a series of calibrations that will unambiguously determine the presence or absence of any biases in the isotropic fractionator (IF) technique. We will probe a variety of human and non-human primate brain regions (primarily white matter tracts) to determine whether the fractionator accurately estimates numbers of glial cells. The same brain regions will be examined by measuring DNA content (which can directly reflect cell numbers), and by histology, using systematic random sampling to independently estimate glial numbers and ratios. Preliminary data indicate that the histological approach is most suitable to provide a gold standard for comparison. Modern stereological sampling techniques will identify the ratios of cell types at the ultrastructural level. This work will clarify true neuron-glia ratios in the bain and other parts of the CNS. Validation of the new fractionator method will be crucial for understanding neurological and psychiatric diseases with reported imbalances of glia cell densities, such as schizophrenia, autism, bipolar disease, and depression.

Public Health Relevance

Interaction of glia and neurons is crucial for brain function, as well as brain health and disease. Altered glia densities have been reported in schizophrenia, autism, bipolar disease, and depression. It is necessary to employ reliable and validated tools to quantify numbers of glial cells and neurons to understand how imbalances in numbers and ratios of neurons and glial cells contribute to these diseases.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Small Research Grants (R03)
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Cellular and Molecular Biology of Glia Study Section (CMBG)
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Morris, Jill A
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University of Nevada Reno
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von Bartheld, Christopher S; Bahney, Jami; Herculano-Houzel, Suzana (2016) The search for true numbers of neurons and glial cells in the human brain: A review of 150 years of cell counting. J Comp Neurol 524:3865-3895
Herculano-Houzel, Suzana; von Bartheld, Christopher S; Miller, Daniel J et al. (2015) How to count cells: the advantages and disadvantages of the isotropic fractionator compared with stereology. Cell Tissue Res 360:29-42
von Bartheld, Christopher S; Wouters, Fred S (2015) Quantitative techniques for imaging cells and tissues. Cell Tissue Res 360:1-4
Bahney, Jami; von Bartheld, Christopher S (2014) Validation of the isotropic fractionator: comparison with unbiased stereology and DNA extraction for quantification of glial cells. J Neurosci Methods 222:165-74