Quantitative determination of neuronal size and density using flow cytometry

Abstract

Background: Recent anthropomorphic disturbances are occurring at an increasing rate leading to organisms facing a variety of challenges. This change is testing the information processing capacity (IPC) of all animals. Brain function is widely accepted to be influenced by a variety of factors, including relative size, number of neurons and neuronal densities. Therefore, in order to understand what drives an animals IPC, a methodological approach to analyze these factors must be established.

New method: Here we created a protocol that allowed for high-throughput, non-biased quantification of neuronal density and size across six regions of the brain. We used the Isotropic Fractionator method in combination with flow cytometry to identify neuronal and non-neuronal cells in the brains of adult rats.

Comparison with existing methods: The results obtained were comparable to those identified using stereological counting methods.

Results: By employing this new method, the number of nuclei in a specific brain region can be compared between replicate animals within an experiment. By calibrating the forward scatter channel of the flow cytometer with size standard beads, neuronal and non-neuronal nuclear sizes can be estimated simultaneously with nuclei enumeration. These techniques for nuclear counting and size estimation are technically and biologically reproducible.

Conclusion: Use of flow cytometry provides a methodological approach that allows for consistency in research, so that information on brain morphology, and subsequent function, will become comparable across taxa.

Keywords: Flow cytometry; High-throughput; Isotropic fractionator; Neuron glia ratios; Neuronal density; Neuronal size.