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.