Mapping c-Fos Expression in Noradrenergic Regions

Sections were analyzed under bright-field through a slide scanner (FSX-100 Inverted Microscope, Olympus). To refine our scope, sections were first non-quantitatively analyzed to identify regions with notable changes in c-Fos expression. Noradrenergic regions and areas with unambiguous c-Fos expression were then manually counted with observers blinded to the treatment. A cell was considered c-Fos+ if a cell expressed a black nucleus and excluded cells that expressed nuclei that were light/medium brown (which may or may not be c-Fos+). This level of stringency ensured that we only identified c-Fos+ cells and thereby excluded the possibility of identifying false positive cells. Regions of interest were identified using the rat brain atlas¹² and counted using ImageJ. Three representative sections were taken across the rostral/caudal axis for each region per animal, and this sampling strategy is based on a recent study that showed that the distribution of LC projections had no specific organization across anterior/posterior or medial/lateral axes^{13 (link)}.