Applying the area fraction fractionator (AFF) probe for total volume estimations of somatic, dendritic and axonal domains of the nigrostriatal dopaminergic system in a murine model

Abstract

Using the nigrostriatal dopaminergic system as a case example, we describe a protocol based on Cavalieri's principle to estimate the total volume of its somatic, nuclear, dendritic and axonal domains in the adult mouse. This protocol requires serial section analysis, a confocal laser scanning microscope (CLSM), and a stereological software combining the serial section manager (SSM) tool and the area fraction fractionator (AFF) probe. The protocol consists of the following steps: 1) systematic random sampling (SRS) of sites across sections for relevant regions of interest (ROI), which in this study included the substantia nigra pars compacta (SNc) and pars reticulata (SNr), the nigrostriatal tract, and the caudate-putamen complex (CP), 2) high resolution image acquisition of all SRS sites within and across sections, 3) loading of images on the previously defined SRS sites, 4) marking of all cellular structures of interest (cSOI) in the images, including perikarya and cell nuclei, dendrites, axonal segments and varicosities, using Cavalieri's point-counting grids, and 5) estimation of cSOI total volume using the estimated area of each cSOI in every brain section, and the distance between sections. The added volume for all cSOI comprising the nigrostriatal dopaminergic system, in this murine model is around 0.37 mm3, with approximately 5% corresponding to perikarya and cell nuclei, 10% to neuropil/dendrites in substantia nigra, and 85% to axonal segments and varicosities within the CP. The use of a simple quantitative approach to assess the global state of a system may allow quantification of compartment-specific changes that may accompany neurodegenerative processes.