2.3.3. Experiment 3
Fear conditioning and the fear expression tests were performed as described above, with the exception that the subjects were intraperitoneally administered either naloxone (Nal group: n = 9) or saline (Sal group: n = 10).
After the fear-expression test, each subject was returned to its home cage and kept in the colony room. At 50 min after the fear-expression test (i.e., 60 min after the first tone delivery), each subject was deeply anesthetized using sodium pentobarbital and perfused intracardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer. The brain was dissected, immersed overnight in the same fixative, and then placed in 30% sucrose/phosphate buffer for cryoprotection. The avidin-biotin-peroxidase method was used to identify immunoreactive (ir) cells, as described previously (Kobayashi et al. , 2013; Kobayashi et al. , 2015). Briefly, six serial 30-μm sections containing the prelimbic cortex (PL), infralimbic cortex (IL), and posterior complex of the anterior olfactory nucleus (AOP; Bregma + 3.24 mm), anterior cingulate cortex (ACC), nucleus accumbens (NAc) shell, and NAc core (Bregma + 2.28 mm), dorsomedial bed nucleus of the stria terminalis (dmBNST) and ventral bed nucleus of the stria terminalis (vBNST; Bregma - 0.12 mm), insular cortex (IC), paraventricular nucleus of the hypothalamus (PVN), and anterior cortical amygdala (aCoA; Bregma - 1.80 mm), anterior subdivision of the medial amygdala (MeA; Bregma - 2.04 mm), posterodorsal subdivision of the medial amygdala (MePD), and posteroventral subdivision of the medial amygdala (MePV; Bregma -3.12 mm), or posterolateral cortical amygdala (plCoA) and posteromedial cortical amygdala (pmCoA; Bregma -4.44 mm) were collected (Fig. 1). After incubation in 0.3% H2O2, the free-floating sections were incubated in citrate buffer (B442, LSI Medience Corporation, Tokyo, Japan) at 60°C for 2 h. The sections were then incubated with anti-c-Fos antibody (1:7500, RRID: AB_2247211, Cat# 2250, Cell Signaling Technology, Danvers, MA, USA) for 65 h at 4°C, followed by incubation with VECTASTAIN Elite ABC reagent (PK-6100, Vector Laboratories). Finally, the sections were developed using diaminobenzidine solution with nickel intensification.