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

BackgroundThe Cavalieri estimator is used for volume measurement of brain and brain regions. Derived from this estimator is the Area Fraction Fractionator (AFF), used for efficient area and number estimations of small 2D elements, such as axons in cross-sectioned nerves. However, to our knowledge, the AFF has not been combined with serial sectioning analysis to measure the volume of small-size nervous structures. New methodUsing the nigrostriatal dopaminergic system as an illustrative case, we describe a protocol based on Cavalieri's principle and AFF to estimate the volume of its somatic, nuclear, dendritic, axonal and axon terminal cellular compartments in the adult mouse. The protocol consists of (1) systematic random sampling of sites within and across sections in regions of interest (substantia nigra, the nigrostriatal tract, caudate-putamen), (2) confocal image acquisition of sites, (3) marking of cellular domains using Cavalieri's 2D point-counting grids, and 4) determination of compartments’ total volume using the estimated area of each compartment, and between-sections distance. ResultsThe volume of the nigrostriatal system is ~0.38 mm3, with ~5% corresponding to perikarya and cell nuclei, ~10% to neuropil/dendrites, and ~85% to axons and varicosities. Comparison with existing methodsIn contrast to other methods to measure volume of discrete objects, such as the optical nucleator or 3D reconstructions, it stands out for its versatility and ease of use. ConclusionsThe use of a simple quantitative, unbiased approach to assess the global state of a system may allow quantification of compartment-specific changes that may accompany neurodegenerative processes.