PMID: 35063768 DOI: 10.1016/j.bbrc.2022.01.022
2. Methods
2.1. Animals
This study was approved by the Institutional Animal Care and Use Committee at Soochow University (Suzhou, Jiangsu, China). Twenty-four female and six male C57BL/6 mice in breeding age were purchased from Zhaoyan Laboratory (Taicang, Jiangsu, China) for producing next generation of mice. On postnatal day (PND) 21, the offspring mice were separated from dams and housed 4–5 per cage by gender. Four male and four female mice were specifically used as the stranger mice, which were trained to stay calmly in the enclosure before social interaction test. All the mice were raised with free access to food and water in a controlled environment (room temperature 21–22 °C, 12/12 h light/dark cycle, and light on at 7 a.m.).

2.2. Anesthesia
A retired anesthesia machine (Datex-Ohmeda, Inc.) was employed to supply a consistent concentration of anesthetic gas. A sealed plastic box (20 L × 20 W × 6 H cm) was used as the anesthetizing chamber, which was drilled with three holes for gas inflow, gas outflow and gas monitoring. An electric heater was placed underneath the anesthetizing chamber to keep neonatal mice warm during anesthesia. A gas analyzer (Datex-Ohmeda, Inc.) was applied to adjust gas concentrations. On postnatal day 6, the neonatal mice were randomly assigned into two groups. Twenty-eight mice (17 males and 11 female) received 3.0% sevoflurane in 60% oxygen for 2 h (Sevo), and thirty-one mice (11 males and 20 female) inhaled merely 60% oxygen for 2 h (Oxyg). Sex of each mouse and amount of each group were not identified until weaning on PND 21. These subject mice were tested for social interaction behavior at one- and two-month-old.

Another battery of neonatal mice were treated with the air condition (control) or 60% oxygen (oxyg) or 3.0% sevoflurane in 60% oxygen (sevo) for 2 h on PND 6, and then killed for harvesting brain tissues 24 h after treatment. Sevoflurane anesthesia was strictly performed by the protocols of previous studies [11,12], in which all neonatal mice could spontaneously breath during general anesthesia, and their arterial blood pressure and blood gas analysis showed within normal limits. The vapor for releasing sevoflurane was turned off at the end of anesthesia, and the residual anesthetic was washed out with 60% oxygen for 15 min. Finally, these pups were smeared with own bedding and sent back to their dams.

2.3. Social interaction paradigm
Social interaction test is performed with the three-chambered social box, with three chambers (40 L × 20 W × 22 H cm) and two enclosures (7 ID × 15 H cm). The floor is painted grey to provide a high contrast with the testing mice. Grid bars of the enclosure allow direct contacting between the subject and stranger mice. A novel video-tracking system was developed by hanging two video-cameras right above two enclosures. Thereby, two video-images were integrated into one with the montage effect in ANY-maze program (Stoelting Co., USA). The subject mouse initiates social interaction with the stranger mouse by nose-to-nose or nose-to-tail sniffing, thus the animal's head is tracked by the ANY-maze program.

2.4. Social interaction test
First of all, the stranger mice were transferred into behavioral room and hidden 2 m away from social apparatus. Each subject mouse was taken into behavioral room about 45 min before social interaction test. In the first session (Habituation, 10-min), the subject mouse was gently placed into the middle chamber, and allowed to freely explore in three chambers. In the second session (Sociability, 10-min), the subject mouse was guided into the middle chamber and transiently confined there. An unfamiliar conspecific (Stranger 1) was introduced into one enclosure, the subject mouse was allowed to explore in three chambers and sniff at two enclosures containing Stranger 1 or not. In the third session (Preference for social novelty 10-min), the subject mouse was again confined into the middle chamber. Another unfamiliar conspecific (Stranger 2) was introduced into the other enclosure, and the subject mouse was allowed to explore in three chambers and sniff at two enclosures containing Stranger 2 or Stranger 1. Placement of Stranger 1 on left and right side were balanced between trials, and two stranger mice were the same gender as the subject mice.

Sociability is characteristic of the mouse taking more time sniffing its conspecific mouse compared with an inanimate object. Preference for social novelty is characteristic of the mouse taking more time sniffing an unfamiliar mouse compared with a familiar one. Four parameters were measured for judging social choice, including 1) time sniffing at the enclosure, 2) number of sniffs, 3) time exploring in the chamber, and 4) number of entries. Sniffing time at the enclosure was primary outcome, number of sniffs at the enclosure and time exploring in the chamber were secondary outcomes. In social interaction test, “at the enclosure” is defined as the head of mouse entering an area about 3 cm around the enclosure, as described in similar social study [13]. And “in the chamber” is defined as the head of mouse entering into the chamber.

2.5. Immunoblotting analysis
The brain tissues of neonatal mice were harvested on dry ice at 24 h after treatment. Next, the cortex and hippocampus were homogenized on ice using the immunoprecipitation buffer plus protease inhibitor. And then, the lysates were centrifuged at 15,000 rpm for 30 min at 4 °C. After that, the lysates were quantified for total protein by the bicinchoninic acid (BCA) protein assay kit (MultiSciences Biotech Co., Ltd. Cat: PQ0012, Lot: A91041). Finally, western blot was performed by the protocols to analyze protein levels in cortex and hippocampus. Neuroligin-1 antibody (1:1000; Santa Cruz Biotechnology, Inc.) was used to detect neuroligin-1 (101 kDa). PSD-95 antibody (1:1000; Cell Signaling Technology, Inc.) was used to detect PSD-95 (95 kDa). Anti–β-actin (1:5000; Sigma) was used to detect β-actin (42 kDa).

2.6. Statistical analysis
Data were expressed as Mean ± SD. Statistical analyses were performed by using GraphPad Prism 5.0 (San Diego, USA). Data representing social behavior of testing mice were normally distributed by Kolmogorov-Smirnov test. Data of each mouse from the left or right side were mutually exclusive, and two-tailed paired t-test was used to determine side preference, which was supported by other social studies [14,15]. Student's t-test was used to assess differences in the levels of Neuroligin-1 and PSD95 expression in cortex and hippocampus of mice. P values less than 0.05 (∗), 0.01 (∗∗) and 0.001 (∗∗∗) were considered statistically significant.