A total of 254 sympathetic axons that satisfied the histological criteria for morphometry were located in the initial screening for staining quality and specimen integrity. Of that inventory, 160 axons were randomly selected for digitization, and individual sympathetic terminal arbors were then subsequently traced, digitally reconstructed, and evaluated quantitatively using a Neurolucida (MicrobrightField Inc., Williston, VT, USA; RRID:nig-0000-10294) workstation employing a Zeiss Axio Imager Z2 microscope (Carl Zeiss Microimaging, Gottingen, Germany) equipped with DIC optics and both a 40× dry and a 63× water immersion long working distance objective.
For each of the digitized sympathetic arbors, the following standardized morphometric measures captured with the Neurolucida software were analyzed for all neurites: total arbor length, parent axon length (using the Strahler Analysis algorithm), varicose neurite length, total number of terminal branches, highest branch order, and two-dimensional terminal field size (using the Convex Hull algorithm [cf. Powley et al., 2012 (link), 2013a (link), 2013b (link), 2014 (link)]).
For each of the digitized sympathetic arbors, the following standardized morphometric measures captured with the Neurolucida software were analyzed for all neurites: total arbor length, parent axon length (using the Strahler Analysis algorithm), varicose neurite length, total number of terminal branches, highest branch order, and two-dimensional terminal field size (using the Convex Hull algorithm [cf. Powley et al., 2012 (link), 2013a (link), 2013b (link), 2014 (link)]).
