2.4. Data and statistical analyses
All data are expressed as mean ± standard error of the mean (SEM). All
statistical analyses were performed using JMP software (version 16.2.0,
SAS Institute, Cary, NC, USA). The significance criterion for all
statistical tests was set at P < 0.05. A researcher
blinded to the experimental conditions recorded behavioral responses
using a Microsoft Excel–based Visual Basic program that recorded the
duration and number of key presses (Kiyokawa et al. , 2015;
Kiyokawa et al. , 2016). In Experiments 1 and 3, the duration of
freezing (an immobile posture with cessation of skeletal and vibrissae
movements, except for those related to respiration) and investigation
(sniffing towards the other enclosure at a nose-wire mesh distance of
less than 1 cm, including poking of the snout into the wire mesh and
climbing up the wire mesh), and frequency of walking (number of steps
taken with the hind paws) of the subjects were recorded during the
acclimation and experimental period. The data were analyzed using
multiple analysis of variance (MANOVA) followed by Dunnett’s post hoc
test. In Experiment 2, the frequency of walking in each 5-min bin was
recorded. After Mauchly’s test of sphericity, the data were analyzed by
two-way repeated analysis of variance (ANOVA).
For immunohistochemical analyses, six sections of each region were
captured at 4× (the dmBNST, MePD, plCoA, and pmCoA) or 10× (the other 12
brain regions) magnification using a microscope equipped with a digital
camera (DP30BW, Olympus, Tokyo, Japan). A researcher blinded to the
experimental conditions randomly selected three sections of the left
hemisphere and three sections of the right hemisphere for each nucleus.
The number of Fos-ir cells was determined within a 0.5 × 0.5 mm square
of each nucleus using ImageJ software (version 1.53t). When the
designated area was smaller than the boundaries of a 0.5-mm square, only
the cells within the nucleus were counted. Although some sections were
lost due to technical issues, more that 5 subjects in every group were
analyzed. In all regions, the density (Fos-ir cells/0.25
mm2) was analyzed using the non-directional Student’st-test.