2.4 Autoradiography

Another set of the same brains was used to visualize oxytocin receptor (OTR) density in the hippocampus (figure 2). We evaluated OTR because receptor density is relatively stable in adults (Ophir et al., 2013) and because receptors are often regarded as the targets of selection (Ketterson and Nolan Jr, 1999). To visualize OTR, we labeled brain tissue with ¹²⁵I labeled ornithine vasotocin analog ([¹²⁵I]-OVTA); NEX 254, PerkinElmer; Waltham, MA) using our established protocol for autoradiography (Ophir et al., 2009). The radiolabeled slides were exposed to Biomax MS film (GE Healthcare, Buckinghamshire, UK) for 72 hours along side ¹²⁵I microscale standards (American Radiolabeled Chemicals, St. Louis, MO).

Films were developed and digitized using a Microtek Scanner (Microtek, Santa Fe Springs, CA) and then scored using ImageJ. Localization of OTR expression in the hippocampus was identified using the rat brain atlas (Paxinos et al., 2009) and cresyl violet stained sections as guides. We measured optical density for the hippocampus for each bilateral section. We also measured nonspecific binding on each section by measuring the background levels of cortex (bilaterally) in areas that do not express OTR. OTR density was estimated by converting the optical density of exposed film into disintegrations per minute (dpm) in tissue equivalence (TE) estimated from 1mg in rat brain (dpm/mg TE). The optical density for each section was then averaged and adjusted to represent specific binding by subtracting nonspecific binding from total binding.