A report on digitised neuronal tracing method to study neurons in their entirety

Conventional camera lucida (CL) aided neuronal tracing technique for studying neural plasticity is a demanding procedure. Stereo Investigator-Neurolucida enabled neuronal tracing system is not accessible to all researchers. This necessitates alternate simple and less challenging digitised neuronal tracing methods. This report describes a novel digitised neuronal tracing method using widefield microscopy, and its effectiveness is compared with the traditional camera lucida aided neuronal tracing method. Golgi-Cox stained hippocampal cornu ammonis area-3 (CA3) pyramidal neuron photomicrographs were serially captured at a depth of every 2µm in the z-axis by a wide field microscope from the point of appearance to the disappearance. These images were stacked along the axis perpendicular to the image plane to reconstruct the neuron in its entirety, digitally traced and dendritic quantification was performed using open source software. The same neurons were manually traced using camera lucida, and Sholl analysis was done manually to quantify the dendritic arborisation pattern. The dendritic quantification data were not significantly different in both methods. Hence, the technology-enabled, less demanding, and equally accurate neuronal tracing can be adopted instead of manual tracing and analysis of neurons. • A simple digitised neuronal tracing method is described. • It is fast, rigorous, and comparable to traditional tracing techniques. • Helps the researcher to repeatedly probe data to reduce errors.