Pregnant Sprague-Dawley rats purchased from Samtako Bio Korea (Osan, Korea) were housed at a temperature of 23℃ under a controlled 12:12 light: dark cycle with ad libitum food and water. All experiments were carried out with the approval of the Animal Care and Use Committee at the Chungnam National University (CNU-00789) and were consistent with the ethical guidelines of the National Institutes of Health.

The protocol for AIS used was adapted from previous publications.^8,9,10) Pregnant rats were subjected to AIS on gestational day 15 (G15) by taping four limbs to metal mounts attached to a board for 45 minutes at 09:00 and 18:00 hour under bright light (Fig. 1A).

In the case of CIS, the following protocol was established. Pregnant rats were exposed to CIS, once each day for 2 hours, from day 15–19 of gestation.¹¹⁾ To prevent the habituation of the animals to the daily exposure to these procedures, restraint time was randomly shifted to different times (09:00 to 18:00 hour) of the day. For immobilization stress, the head fix restrainer (#JD-R-14 Φ 60×180mm, Jeung Do Bio & Plant CO., LTD, Seoul, Korea) was used (Fig. 1B).

Fifteen postnatal day 15 (P15) SD rats were divided into three groups: (1) control (n=5), (2) AIS offspring (n=5), and (3) CIS offspring group (n=5) received 15-mg/kg NMDA administration on P15. After NMDA administrations, the offspring in each group were observed for 75 minutes. The onset of a spasm was defined by high degree of flexion (i.e., hand and trunk flexion, forelimb, hind limb, and hip flexion). We observed the latency from NMDA administration to the onset of the spasm, and the total number of spasms during observational period.

Five animals per group were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally) and perfused transcardially with heparinized phosphate-buffered saline (PBS) followed by perfusion with 4% paraformaldehyde in PBS. Each brains were removed, immersed in the same fixative overnight, and then cryoprotected in 10%, 20%, and 30% sucrose serially. The brains were then embedded in Frozen Section Compound (#3801480, Leica Microsystems, Nussloch, Germany) and frozen in 2-methyl butane rapidly, which had been precooled to its freezing point with liquid nitrogen. Frozen coronal sections (35 µm thick) were obtained using a Leica cryostat (Leica Microsystems, Nussloch, Germany). Parallel free-floating sections were subjected to endogenous peroxidase blocking with 1% H₂O₂ in PBS, followed by treatment with a blocking buffer (1% fetal bovine serum in PBS and 0.3% Triton X-100) for 30 minutes and incubation with glutamic acid decarboxylase isoform 67 (GAD67, 1:400; #MAB5406, Millipore, Billerica, MA, USA) and K+/Cl− cotransporter (KCC2, 1:200; #07-432, Millipore, Billerica, MA, USA) primary antibodies. Immunohistochemical staining of the tissue sections for KCC2 and GAD67 were performed using the avidin-biotin peroxidase complex method, which has been described previously.¹⁰⁾ After washing with PBS, the tissues were exposed to biotinylated anti-rabbit/mouse IgG and streptavidin peroxidase complex (Vector Laboratories, Burlingame, CA, USA). Immunostaining was visualized with diaminobenzidine (DAB) and mounted using Polymount (Polysciences, Inc., Warrington, PA, USA).

The NIH image program (ImageJ, National Institute Health, Bethesda, MD, USA) was used to assess the immunohistochemical signals quantitatively with densitometric measurements. The quantitative data were then analyzed using nonparametric 1-way analysis of variance. The Newman-Keuls method was used for post hoc analysis. A P value less than 0.05 was considered statistically significant. All statistical analyses were conducted using the Prism 5 program (GraphPad, San Diego, CA, USA).

In this study, we used a rat model of prenatal exposure to AIS and CIS as well as postnatal NMDA-triggered spasms. After NMDA administration, the phenotype of the spasms that were observed in a P15 rat was characterized by a continuously twisting tail and a high degree of flexion. We observed that the NMDA-triggered spasms occurred earlier, more frequently, and more intensely in infant rats (P15) that were exposed to prenatal stress (Fig. 2). The frequency of spasms (mean±standard deviation [SD], times/hr) was significantly increased in infant rats exposed to either prenatal AIS (119.0±15.0) or CIS (202.3±88.6) compared to rats in the control (46.0±3.6) group (Fig. 2A). In addition, the latency to the onset of spasms (mean±SD, seconds) was significantly decreased in the prenatally AIS (826.7±67.3) and CIS (817.0±97.5) exposed rats compared with controls (1,205.3±170.4) (Fig. 2B).

We examined the expression of KCC2 following prenatal exposure to AIS or CIS (Fig. 3). In the control group, KCC2 expression increased as expected as a function of age. In both prenatal-stressed groups, KCC2 was expressed at significantly lower levels than the control group in cortical layers II and III on P15. The density of KCC2 of prenatally stressed groups was also decreased in immunohistochemical staining analysis (Fig. 3M).

We checked the expression of GAD67 following prenatal exposure to AIS and CIS (Fig. 4). In the control group, GAD67 expression developed as expected as a function of age. In both prenatal-stressed groups, GAD67 was expressed at significantly lower levels than the control group in cortical layers II and III in P15 rats. It demonstrated that the density of GAD67 was decreased in the prenatally stressed groups (Fig. 4M).