Neurites were traced using NeuronJ following the developers' instructions. After the neurites are traced, they were labeled as primary (emanating directly from the soma), secondary (branching from a primary) or tertiary (branching from a secondary). The neurites could be labeled as axon or dendrite; however, assignment of the axon/dendrite labels requires the use of fluorescent markers that are specific to axonal and dendritic regions within the cell. Since many users are tracing neurons that are either labeled with an antibody to neuronal specific Tubulin Beta3 (which labels all neurites) or expressing a fluorescent protein such as EGFP, it is not possible to accurately assign axon and dendrite labels based on staining. Therefore, our program use the primary/secondary/tertiary labels to perform calculations.
Once labels were assigned, the neurite tracings appeared color coded by type (Fig. 2A-E). Neurites were then assigned to clusters, with each cluster comprised of a primary neurite and all its associated branches. After tracing was completed and clusters had been assigned, a text file containing neurite length measurement data was generated for each neuron traced and a snapshot of the tracings overlaid on the neuron was saved as a TIFF file (e.g. Fig. 1A-E and 2A-D). For the analysis Table 2, 124-145 neurons from each treatment group were traced. Because some of these were stage 2 neurons, the analysis of stage 3 neurons (Fig. 2) was performed on 95-112 neurons.