Animals

Six- to 16-week-old male C57BL/6J mice of similar body weights were used for all experiments. The animals were housed and maintained in a controlled environment at 22–24 °C and 55% humidity with 12-h light/dark cycles and fed regular rodent chow and tap water ad libitum. Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (#SNU-220701-1-2).

Stereotaxic viral delivery

Nine- to 10-week-old wild-type male mice that showed stable hierarchies in their cages were used for viral delivery. The animals were anesthetized with isoflurane and secured in a stereotaxic frame (Stoelting Co., Wood Dale, IL, USA). Holes the size of the injection needle were drilled into the skull, and bilateral injection with 0.7 μl (titer 1013 GC/ml) of adeno-associated virus, serotype DJ (AAVDJ) and 5 (AAV5), were administered on each side. The injection syringe (Hamilton Company, Reno, NV, USA) delivered the AAVDJ and AAV5 at a constant volume of 0.1 μl/min using a syringe pump (Stoelting Co.). The needle was left in place for 3 min after each injection to minimize the upward flow of the viral solution after raising the needle. The injection coordinates of the prelimbic (PL) cortex of the medial prefrontal cortex (mPFC) were AP: +2.43, ML: ±0.20, DV: -1.80 from bregma. AAVDJ-GFAP-mCherry was purchased from the Korea Institute of Science and Technology (Seoul, Korea), and pXac2.1-gfaABC1D-hM3Dq-mCherry plasmid was purchased from Addgene (#92284; Watertown, MA, USA) and packaged in AAV5 (AAV5-gfaABC1D-hM3Dq-mCherry) by UPenn Vector Core (Philadelphia, PA, USA). Animals injected with virus were used for behavioral assessment 3–4 weeks post-injection. Animals injected with AAVDJ or AAV5 but showing no detectable viral expression in the target region were excluded from analyses.

Chronic restraint stress

Mice were individually placed into 50-ml polypropylene conical tubes with a nose-hole for ventilation (3 h/day) for 21 consecutive days. After restraint, the mice were returned to their home cages. One day after the last restraint session, the mice were weighed and used for further experiments. The mice that underwent chronic restraint stress were designated RS mice, and the unstressed control mice were designated control mice (CTL mice).

Behavioral studies

On all behavioral testing days, animals were moved to the test room and left to habituate there for at least 1 h. The light conditions in the test room were maintained at the same intensity (100 lx) as the animal rooms under daylight conditions.

Tube test

The tube test was conducted as previously described. We used a transparent Plexiglas tube 30 cm long with a 3-cm inner diameter. Six-week-old male mice were housed in groups of 4 for at least 2 weeks before the tube test. Before the main test, each mouse was trained to go through the tube for 10 trials over two consecutive days. On the test day, pairs of mice were released into the opposite ends and met in the middle of the tube. The tube test was performed for 2 min. If no winner or loser was decided within 2 min, the test was repeated. Between trials, the tube was cleaned with 70% ethanol. Within each cage, the four mice were randomly assigned such that each mouse would meet every other mouse in the group only once, resulting in six matches per cage. All 6 pairs of mice were tested daily in a round robin design, and the ranks were determined by the total number of wins. Only cage mates who maintained stable ranks for 3 or 4 days were used for further experiments. The behaviors were videotaped, and both pushing and resistance behaviors were counted. The test mouse was matched 4 times against mice from the other group. The winning rate of each test mouse was calculated by counting its total number of wins.

Tube test with chemogenetic mPFC astrocyte stimulation

We injected AAV5-gfaABC1D-hM3Dq-mCherry. Clozapine N-oxide (CNO) (4936; Tocris, Minneapolis, MN, USA) was dissolved and diluted in normal saline solution with 0.5% DMSO (6 mg/kg). After 3 weeks of virus injection, we performed 2 days of tube training and basal tube test for 3 consecutive days without CNO injection. The following day, CNO was intraperitoneally injected into test mice. Then, we performed the tube test 2, 6, and 24 h after injection.

Open field test (OFT)

The apparatus consisted of a brightly illuminated 40 cm x 40 cm square arena surrounded by a wall 40 cm high. Mice were individually placed in the center of the arena, and their locomotion activity was monitored by an automatic system for 10 min. Total distance and time spent in the center zone per minute were analyzed by an automated video tracking system (SMART; Panlab SL, Barcelona, Spain). The activity chamber was cleaned with 70% ethanol after each use to eliminate any olfactory cues from the previously tested mouse.

Elevated plus maze (EPM)

The behavioral apparatus consisted of two open arms (width 5 cm x length 30 cm) and two closed arms (width 5 cm x length 30 cm) elevated 60 cm above the floor and dimly illuminated. Mice were placed individually in the center of the maze facing an open arm and allowed to freely explore for 5 min. The time spent in each arm was analyzed using a video tracking system. The maze was cleaned with 70% ethanol after each test to prevent olfactory influence from the previously tested mouse.

Forced swim test (FST) and tail suspension test (TST)

We conducted the FST by placing each animal individually in a transparent cylinder filled with water (23–25 °C; depth 15 cm) for 5 min. Immobility in the FST was defined as a state in which the mouse made only the movements necessary to keep its head above the surface. The TST was performed by hanging each animal from the top of a square box for 5 min. Both trials were videotaped and immobility time was analyzed using a video tracking system.

The experimental protocols for the OFT, EPM, FST, and TST were designed to take into account how potential stress from a previous test could affect mouse behavior in other tests. The behavioral assessments were performed in the following sequence to minimize such stress effects: OFT, EPM, FST, and TST.

Immunohistochemistry

Whole brains were saline-perfused, fixed in 4% paraformaldehyde in 0.1 M PBS overnight at 4 °C, and dehydrated with 30% sucrose for 3 days. Coronal sections with a thickness of 50 μm were incubated in cryopreservation solution at -20 °C until immunohistochemical staining was performed. The sections were blocked in a blocking solution containing 5% normal donkey serum (Jackson Immunoresearch, Bar Harbor, ME, USA), 2% BSA (A7638, Sigma-Aldrich, St. Louis, MO, USA), and 0.1% Triton X-100 (0694, VWR Life Science, Solon, OH, USA) for 1 h at room temperature. The sections were then incubated with rabbit S100β (ab52642, 1:500; Abcam) antibodies overnight at 4 °C in blocking solution. After being washed with 0.1 M PBS containing 0.1% Triton X-100, the sections were incubated for 2 h with FITC-conjugated secondary antibodies (711-095-152, 1:200; Jackson Immunoresearch) in blocking solution at room temperature, washed 3 times, and then mounted on gelatin-coated slide glass using Vectashield (Vector Laboratories, Inc., Burlingame, CA, USA). Fluorescent images of the mounted sections were obtained with a confocal microscope (LSM800; Carl Zeiss, Jena, Germany).

Pearson’s correlation analysis and K-means clustering

K-means clustering analysis is one of the simplest and most popular unsupervised machine learning algorithms. It aims to separate scattered data into k clusters in which each data point belongs to the cluster with the nearest mean (centroid). This method has been successfully employed in various biological fields to identify and classify complex biological features [40,41,42,43].

We plotted individual mouse immobility values into a 2-dimensional space by defining Mn (tFST, tTST), in which M means mouse and n indicates the mouse number. We first performed a Pearson’s correlation analysis to verify whether the immobility values in the FST and TST correlated with each other. The FST immobility values of 72 data, including all CTL (n = 36) and RS (n = 36), showed a significant positive correlation with the TST immobility values. We performed a K-means classification analysis to divide the immobility values into two groups (k = 2). Classification was started from two random points for every cluster, and we performed 1000 iterative calculations to optimize the positions of the centroids in each cluster. After 1000 iterations, the centroids of each classified cluster were determined, and we compared the centroids with the values measured from experiments. Centroids of each classified group were similar to experimental values, and we then verified classification accuracy. The data were classified into ‘Normal’ and ‘Depressed’ groups with 93.1% classification accuracy. Among the 36 CTL data, 33 were correctly classified as ‘Normal’ and 3 were incorrectly classified as ‘Depressed’ (91.7% classification accuracy). Among the 36 RS data, 34 were correctly classified as ‘Depressed’ and 2 mice were incorrectly classified as ‘Normal’ (94.4% classification accuracy). Next, we performed K-means classification using the FST and TST immobility values obtained from the non-RS and RS mice after tube testing. The k value was again 2, and the classification iteration was 1000. Both Pearson’s correlation and K-means classification analyses were performed in MATLAB (R2019a).

Statistics

Statistical significance was determined using the two-tailed Student’s t-test and paired t-test for comparisons between two groups. For multiple comparisons, one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison tests was used. All data are presented as mean ± s.e.m., and differences were considered statistically significant when the p-value was less than 0.01.