Brain-Wide Mapping of Axonal Connections: Workflow for Automated Detection and Spatial Analysis of Labeling in...

Axonal tracing techniques are powerful tools for exploring the structural organization of neuronal connections. Tracers such as biotinylated dextran amine (BDA) and Phaseolus vulgaris leucoagglutinin (Pha-L) allow brain-wide mapping of connections through analysis of large series of histological section images. We present a workflow for efficient collection and analysis of tract-tracing datasets with a focus on newly developed modules for image processing and assignment of anatomical location to tracing data. New functionality includes automatic detection of neuronal labeling in large image series, alignment of images to a volumetric brain atlas, and analytical tools for measuring the position and extent of labeling. To evaluate the workflow, we used high-resolution microscopic images from axonal tracing experiments in which different parts of the rat primary somatosensory cortex had been injected with BDA or Pha-L. Parameters from a set of representative images were used to automate detection of labeling in image series covering the entire brain, resulting in binary maps of the distribution of labeling. For high to medium labeling densities, automatic detection was found to provide reliable results when compared to manual analysis, whereas weak labeling required manual curation for optimal detection. To identify brain regions corresponding to labeled areas, section images were aligned to the Waxholm Space (WHS) atlas of the Sprague Dawley rat brain (v2) by custom-angle slicing of the MRI template to match individual sections. Based on the alignment, WHS coordinates were obtained for labeled elements and transformed to stereotaxic coordinates. The new workflow modules increase the efficiency and reliability of labeling detection in large series of images from histological sections, and enable anchoring to anatomical atlases for further spatial analysis and comparison with other data.