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.