After the end of CPA testing, all mice were perfused with 0.9% saline, followed by ice-cold solution of 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) (pH 7.4). The brains were removed and fixed in 4% PFA overnight. The brains were cut in 50-μm coronal sections using a vibratome (Leica, USA) and collected in PBS. To examine the injection site and the expression of each tracer or virus, brain slices were rinsed in PBS for three times (5 min for each wash) and incubated with 4′,6-diamidino-2-phenylindole (C1002, Beyotime Biotechnology, USA) diluted into 0.5 μg/ml for 10 min at room temperature. To do immunohistochemistry experiments, brain slices were rinsed in PBS for three times (5 min for each wash) and incubated with blocking solution containing 10% normal goat serum and 0.3% Triton X-100 in PBS for 2 hours at 4°C. For the analysis of c-Fos expression or Arc expression in FG-labeling BLA neurons, brain slices were incubated with rabbit anti–c-Fos antibody (#5348, Cell Signaling Technology, USA) diluted 1:500 or rabbit anti-Arc/Arg 3.1 antibody (#I56003, Synaptic Systems, Germany) diluted 1:1000 overnight at 4°C. Subsequently, they were rinsed in PBS for three times (5 min for each wash), followed by application of Alexa Fluor 594–conjugated goat anti-rabbit antibody (Jackson ImmunoResearch Laboratory, USA) diluted 1:200 for 2 hours at 37°C. For the analysis of c-Fos and Arc expression in FG-labeling BLA neurons, brain slices were incubated with guinea pig anti–c-Fos antibody (#226004, Synaptic Systems, Germany) diluted 1:500 overnight at 4°C. Then, they were rinsed in PBS for three times (5 min for each wash) and incubated with goat anti-guinea pig immunoglobulin G (IgG) antibody (Vector, US) diluted 1:200 for 1 hour at 37°C, followed by Alexa Fluor 488–conjugated streptavidin (Jackson ImmunoResearch Laboratory, USA) diluted 1:1000 for 1 hour at 37°C. Subsequently, they were rinsed in PBS for three times (5 min for each wash) and incubated with anti-Arc/Arg 3.1 antibody (#I56003, Synaptic Systems, Germany) diluted 1:1000 overnight at 4°C. Then, they were rinsed in PBS for three times (5 min for each wash) and incubated with goat anti-rabbit IgG antibody (Vector, USA) diluted 1:200 for 1 hour at 37°C, followed by cyanine 3–conjugated streptavidin (Jackson ImmunoResearch Laboratory, USA) diluted 1:1000 for 1 hour at 37°C. Last, immunolabeled sections were rinsed in PBS for three times (5 min for each wash), mounted on glass slides, and imaged by confocal microscopy (Nikon AIR MP, Japan). All c-Fos and Arc antibodies were dissolved into 10% normal goat serum and 0.3% Triton X-100 in PBS, and other antibodies were dissolved into 10% normal goat serum in PBS.

A series of slices containing the BLA were imaged by confocal microscopy with a 20× immersion lens and collected at a resolution of 1024 × 1024 pixels. The same laser and scanning settings were used for all confocal images within an experiment to allow comparison across groups. In general, coronal sections from five to eight mice were used for quantitative analysis. Five to eight images of each mouse and one image of each slice were averaged to determine a value for the slice. Series of images were captured from the confocal microscope and converted to 8-bit gray scale images, and then the area and mean gray values of white color clusters were measured using the Image-Pro Plus 6.0 software. Quantification of c-Fos, FG, and c-Fos + FG labeling neurons was estimated in the form of optical density with the same threshold. The positive neurons were defined with large nuclei stained diffusely and staining above basal background. Quantitative analysis of Arc labeling neurons and quantitative analysis of c-Fos + Arc + FG labeling neurons were performed as described above.

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