Novel applications of common stereology software to represent the complete distribution, density and spatial organization of anterogradely labelled fibers in neuroanatomical tract-tracing studies

In a recent study we have adapted for the first time available software used in spatial neuronal organization studies to represent the complete fiber distribution of a brain projection [Leite-Almeida H, Valle-Fernandes A, Almeida A. Brain projections from the medullary dorsal reticular nucleus: an anterograde and retrograde tracing study in the rat. Neuroscience 2006;140:577–95]. Here we describe the technique in detail using the injection of biotinylated-dextran amine (BDA) in the rat cuneate nucleus and its projection through the pyramidal decussation as an example.Camera-lucida-like computer drawings were produced with StereoInvestigator software. Using high magnification lens, rat brain sections were screened and lines superimposed with virtually all fibers and terminal boutons present in each brain section. Additionally, in each screening field of the section, all focal planes were inspected and lines depicted only on focused fibers and/or fiber segments. Later, NeuroExplorer software was used to interpret fiber depth information and obtain the three-dimensional pathway of each fiber bundle along the rostrocaudal extension of each section.This computer methodology presents several advantages over classical camera-lucida hand-made drawings: (i) the software is prepared to work interchangeably in different magnifications without misplacements, which allows the representation of virtually all labelled fibers and, therefore, results in a much more accurate fiber location in each brain section; (ii) three-dimensional information of all individual fibers and fiber bundles is recorded; (iii) the method is more flexible allowing, for example, to colour differently labelled fibers according to their profiles (e.g. terminal or ramified fiber arborizations and terminal boutons).