Histology

Within 24–48 h of behavioral testing, rats were sacrificed by rapid decapitation. Brains were removed and frozen in −45°C isopentane and then stored at −80°C until being sectioned on a microtome. Coronal sections of ∼40 µm were taken throughout the entirety of the cannula tracts visible in the brain tissue. The 40 µm coronal slices were mounted on charged microscope slides and stained with Neutral Red (1%). Slides were photocaptured and analyzed to confirm the placement of the cannula injector tip in the mPFC. Out of 223 surgeries (see Fig. 1A for placements), there were a total of 10 misplaced cannula with the distribution as follows: Experiment 1 (n = 4), Experiment 2 (n = 2), Experiment 3 (n = 1), and Experiment 4 (n = 3).

A subset of animals underwent the same surgical and cannula clearing procedures mentioned previously (see Stereotaxic surgery) and were used to visualize the spread of muscimol in the mPFC. Rats were infused with fluorophore-conjugated muscimol (Muscimol BODIPY-TMR-X, Fisher Scientific) at the same volume, dose, and infusion rate mentioned previously (see Drug Infusion) (Allen et al. 2008). The fluorescent muscimol was diluted to a concentration of 2 µg/µL into a solution made of half PBS and half DMSO to ensure dissolution (Hallock et al. 2013). The animals were sacrificed by rapid decapitation 15 min after infusion of the fluorescent muscimol in order to match the spread to what the experimental animals received immediately prior to behavioral testing. The brains were removed and flash frozen in −45°C isopentane, stored at −80°C, and then coronal slices were sectioned at 60 µm. The slices were mounted on charged microscope slides and counterstained with ProLong Diamond with DAPI (Life Technologies). The stained slices incubated in a cool, dark room at room temperature for 3 d before being visualized with a confocal microscope (ZEISS LSM 880) in the Bioimaging Center at the Delaware Biotechnology Institute. A digital plate from the Paxinos and Watson (2007) rat brain atlas was overlaid on the image to visualize the spread (see Fig. 1B).