Mapping Melanocortin Receptor Neurons

Subjects were injected with a lethal dose of sodium pentobarbital (Beuthanasia-D, 0.25 ml i.p., Schering, Union, NJ, United States) and sacrificed by rapid decapitation. Brains were extracted, frozen in optimum cutting temperature compound (VWR Scientific Products) and stored at −80°C. Brains were later sliced coronally on a cryostat at 14 μm thickness and sections were mounted on Colorfrost Plus slides (Fisher Scientific, Waltham, MA, United States). Some studies in rats and mice have provided an extensive analysis of range of the distribution of melanocortin receptor expressing neurons. Here, we limited our focus to the forebrain and midbrain with tissue collected from slices containing the prefrontal cortex through to slices containing the dorsal raphe nucleus. Slides were stored at −80°C until RNAscope procedure treatment.