Comparison of semi-automatic and automatic data acquisition methods for studying three-dimensional distributions of large neuronal populations and axonal plexuses

Neuroanatomy is in need of high throughput methods for reliably recording the distribution of tissue elements across large brain regions. We compared two methods for recording the spatial distribution of identified neuronal elements such as tracer labelled cell bodies or axonal plexuses. The methods compared were computerized image-combining microscopy (semi-automatic method), which is a user controlled method providing feedback during digitization, and digital camera technology with image analysis software (automatic method). Both methods were applied to biotinylated dextran amine labelled axonal plexuses and FluoroRuby labelled neuronal cell bodies, in the pontine nuclei of the rat. Coordinates were assigned to the labelled elements using both methods. The ensuing distribution patterns were compared, section by section, and in three-dimensional reconstruction. The experienced investigator, using the semi-automatic method, could detect individual axons, fragments of axons, weakly labelled elements, and overlapping cell bodies, better than the automatic system. Nevertheless, both methods detected the overall distribution of the labelled axons and cells investigated. Automatic methods provide opportunities for efficient large-scale data acquisition of labelled neuronal elements.