Maternal immune activation model
All animal experiments were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All protocols were approved by the Institutional Animal Care and Use Committee at Kumamoto University, and every effort was made to minimize the number of animals used and their suffering.
Pregnant C57BL/6N mice were purchased from Charles River Laboratories Japan, Inc. at E8 or E9. The animals were kept in a temperature- and humidity-controlled, specific pathogen-free environment with a 12 h light/dark cycle (lights on at 7:00) at the Center for Animal Resources and Development of Kumamoto University. Food and water were provided ad libitum. SFB infection was prevented by 0.5 mg/ml vancomycin (FUJIFILM Wako Pure Chemical Corporation) in drinking water throughout the experiment, starting from the arrival of the animals. The fecal pellets of the dams were collected at the time of arrival and weaning to confirm the absence of SFB throughout pregnancy and lactation. All experimental pregnant dams were tested for the absence of SFB. Each pregnant dam received a single intraperitoneal injection of poly(I:C) (20 mg/kg, Sigma Aldrich) at E9, E10, E11, E12, E13, E14, E15, or E16. The control animals received PBS injections. The dams were kept undisturbed until giving birth. The litters were kept with the mother and then group-housed with the sex-matched litters after weaning at 3 weeks of age. For the histological experiment, the mouse brains were collected at P0 or P1. The brains were isolated immediately after the decapitation of the pups without anesthesia.
PCR Analysis Of The Fecal DNA
Bacterial genomic DNA was isolated from the fecal pellets of mice using a QIAamp Fast DNA stool kit (QIAGEN). The PCR analysis was conducted using the following primers, forward: 5′-TGTGGGTTGTGAATAACAAT-3′ (779F), reverse: 5′-GCGGGCTTCCCTCATTACAAGG-3′ (1380R) targeting the SFB 16S rRNA gene, and forward: 5′-CGGCAACGAGCGCAACCC-3′ (Bac1114F), reverse: 5′-CCATTGTAGCACGTGTGTAGCC-3′ (Bac1275R) targeting the total bacterial 16S rRNA gene37. After running 40 cycles of PCR with 40 ng of input DNA using Ex Taq enzyme (Takara), the PCR products were analyzed either by agarose electrophoresis or by a microchip electrophoresis system (MCE-202 MultiNA, Shimadzu). The absence of SFB was confirmed by the absence of an amplicon of 200 bp by SFB primers. The amplicon of 145 bp by primers for total commensal bacteria served as an experimental control for each sample.
Immunohistochemistry
Brains were removed at P0 or P1 and fixed with 4% paraformaldehyde phosphate buffer solution (FUJIFILM Wako Pure Chemical Corporation) overnight at 4°C. The brains were sectioned at 30 µm thickness with a cryostat and stored in a section storage solution (PC101, FD NeuroTechnologies, Inc.) until the date of staining. The free-floating sections were washed with PBS and sequentially incubated with a permeabilizing solution containing 0.2% Triton X-100 in PBS for 30 min at room temperature, blocking solution containing 0.2% Tween-20 and 5% goat serum in PBS for 1 h at room temperature, and primary antibodies overnight at 4℃. After overnight incubation with primary antibodies (1:2,000; rabbit anti-Tbr1 antibodies (ab31940, Abcam), 1:50; mouse anti-Satb2 antibodies (ab51502, Abcam)), sections were rinsed with PBS and incubated for 1 h in secondary antibodies (1:2,000; goat anti-mouse IgG1 Alexa Fluor 568 (A-21124, Invitrogen), 1:2,000; donkey anti-rabbit IgG Alexa Fluor 647 (ab150075, Abcam)). The primary and secondary antibodies were diluted with a solution containing 0.05% Tween-20 and 3% goat serum in PBS. The sections were rinsed with PBS and mounted on glass slides. Images of stained sections were acquired using a confocal microscope (FV3000, Olympus) with a 10× objective lens. Sixteen coronal sections every 180 µm, which at least covered an anterior slice without corpus callosum and a posterior slice with hippocampal formation through the cortex, were stained in each mouse. The broken slices during the staining process were removed from the analysis, and the slice numbers analyzed are shown in Supplementary Table S1. The cortical area was manually analyzed for cortical malformations reported previously, which included protrusion, intrusion, and ectopic expression of cortical markers5.
Prepulse Inhibition Test
The startle response and prepulse inhibition test system (O'HARA & CO., LTD.) was used. The PPI test was conducted when offspring were 9–11 weeks of age. Mice were brought to the test room 1 h before the start of the test for habituation. Mice were placed in the test chambers with white noise of 65 dB for 5 min, and then the test was started. The test was composed of 3 blocks with 94 trials of pulse presentation in total. The intervals between trials were randomized between 10 and 20 seconds. The first block was composed of 3 trials of 115 dB pulses for acclimatization. The second block was the PPI block. In the PPI block, trials were a prepulse sound (0, 70, 74, 78, 82, and 86 dB)-pulse (115 dB) paired stimulus or a no prepulse-no pulse pair. Each stimulus was presented 10 times in a randomized order, resulting in 70 trials in total. The delay between prepulse and pulse was 100 ms. In the third block, pulse sounds (0, 70, 80, 90, 100, 110, 120) were presented 3 times for each, making up 21 trials. Percentage PPI was calculated from the second block as {[(response amplitude of trial without prepulse) – (response amplitude of trial with prepulse)]/(response amplitude of trial without prepulse)} X 100. The third block was used to assess the startle response abilities of the animals.
Social Interaction Test
A social interaction test was conducted when offspring were 11–16 weeks of age following the PPI test. Mice were brought to the test room 1 h before the start of the test for habituation. The test was conducted using the three-chambered social interaction test system (O'Hara & CO., LTD.) at 10 lux inside the chamber. In this system, the field, which is 61 cm wide and 40 cm deep, is divided into left, central, and right equal-sized chambers by 2 partitions. Each partition has an opening (5 cm wide and 3 cm high) at the bottom center so that mice can freely move between different chambers. The mouse position was automatically monitored by the software. First, each mouse was acclimatized to the chambers just before the test by placing it into the central, left, and right chambers in this order with partition openings closed. Then tests were started. Tests were composed of 3 sessions called the empty session, the stranger session, and the familiar-stranger session. In the empty session, locomotor activity was evaluated by placing the mice in the chamber without stimulant mice for 10 min. Next, in the stranger session, an interaction cage with a novel mouse was placed at the upper left corner of the left chamber, and social interaction was recorded for 10 min. Last, another interaction cage was placed at the upper right corner of the right chamber for another 10 min of recording in the familiar-stranger session. The interaction cages were quarter-circular with a diameter of 10 cm. The inner and outer lines placed 3.7 cm and 5 cm from the cage, respectively, served as the border of the social interaction zone. Crossing the inner line toward the interaction cage indicated the beginning of the interaction, and crossing the outer line outward indicated the end of the interaction. The interaction ratio in the familiar-stranger session was calculated as {(interaction time with the stranger)/[(interaction time with the stranger) + (interaction time with the familiar)]} X 100. The mice with less than 50 s of social interaction in the stranger session were excluded from the analysis of the interaction ratio because those mice are not considered suitable to assess social novelty preference.
Statistical analysis
Statistical analyses were carried out using python programs. All results are presented as the mean ± standard error of the mean (SEM). Two-way analysis of variance (ANOVA) was used to assess the PPI and startle response differences between groups. Unpaired two-tailed Student’s t test was used for statistical comparisons between groups to assess differences in the 3-chamber social interaction test. In all cases, P < 0.05 was considered significant.