Experimental design. Male rats in the stress group began twice-daily inescapable electric shock in an enclosed box on 3 consecutive days. After 1 week of stress, the body weight changes, dexamethasone suppression and behavior in contextual fear conditioning, open field, elevated plus maze, hot plate, and Morris water maze were tested. Meanwhile, tissues from the hippocampus and prefrontal cortex were collected immediately for detection of apoptosis rate, concentration of Glu and GABA and morphological evaluation. HPA: hypothalamic-pituitary-adrenal; Glu: glutamate; GABA: gamma-aminobutyric acid.

Effects of stress exposure on changes of body weight, dexamethasone suppression test, freezing behavior, and latency to flinch or raise hind paws in hot-plate test. Rats were randomly assigned to 2 groups and subjected to an enclosed box with or without electric foot shock stress a total of 6 times during 3 days. (A) Rats were weighed before and after the stress. The body weight changes were calculated as percentages of change over the body weight before stress (stress group: n=8, sham group: n=10). (B) Enhancement of HPA axis inhibition after stress. The dexamethasone suppression test indicated significantly enhanced inhibition of the plasma corticosterone increase in the stressed rats (n=8 for each group). Vehicle-sham, no stress exposure with vehicle (saline containing 5% ethanol) injection. (C) After 1 week of stress, rats of all groups were re-exposed to the stress context for 3 min without stimulus presentation (stress group: n=8, sham group: n=10). (D) Comparison of latency to flinch or raise hind paws in hot-plate test (stress group: n=8, sham group: n=10). Data were normalized to respective observation periods. The results are expressed as mean ±SEM. * p<0.05, ** p<0.01 compared with the sham group.

The anxiety-like behavior and locomotor activity after exposure to electric foot shock stress in enclosed box in rats. (A) The path track of sham group in the open field. (B) The path track of stressed group in the open field. (C–F) Measured 1 week after the exposure to stress, rats in the stressed group showed a decrease in locomotor activity and an increase in anxiety index. Data were normalized to the respective observation periods. * p<0.05 compared with the sham group (stress group: n=8, sham group: n=10). The results are expressed as mean ±SEM. * p<0.05, ** p<0.01 compared with the sham group.

Anxiety-like effects of stress on the behavior in EPM test. (A): The path track of the sham group in the EPM test. (B): The path track of the stressed group in the EPM test. (C): Anxiety-like effects of stress on the percentage of entries into open arms (% number entry into open entries). (D): Anxiety-like effects of stress on the percentage of time spent (% open arm time). (E) Anxiety-like effects of stress on the number of rearings. (F) Anxiety-like effects of stress on the number of head dips. Data were collected from 2 groups (stress group: n=8, sham group: n=10). The results are expressed as mean ±SEM. * p<0.05 compared with the sham group.

Comparison of escape latency and spatial probe test between animals in stress and sham groups during Morris water maze test. Rats were tested for 5 consecutive days. (A) The path track of escape latency to the platform for the rats of sham and stressed groups during 5 days. (B) Comparison of the escape latency to the platform in 60 s for the animals in stress and sham groups for 5 days for escape test. (C) The path track of target crossing for the rats of sham and stress groups during 5 days after removing the platform. (D) Comparison of the times of crossing platform in 120 s after removing the target in stress and sham groups for 5 days in spatial probe test. The results are expressed as mean ±SEM. * p<0.05, ** p<0.01 compared with the sham group (stress group: n=8, sham group: n=10).

Morphological evaluation of HE staining, Nissl staining, and silver impregnation in CA1, CA3, and DG subfields of hippocampus and prefrontal cortex after stress. In HE staining of hippocampus, normal histology and morphology (A–D) of hippocampus were seen in the sham group (magnification: 100× and 400×, respectively). No injured neurons were observed. In the stress group (E–H), injured neurons in CA1, CA3, and DG subfields in the hippocampus were noted after stress. In hippocampal morphological evaluation of Nissl staining in CA1, CA3, and DG subfields, normal histology and morphology (A–D) of the hippocampus in the sham group (magnification 100× and 400×, respectively). No injured neurons were observed. In the stress group (E–H), damaged neurons in CA1, CA3, and DG subfields of the hippocampus were noted after stress. In the silver-impregnated sections of the hippocampus, healthy cells of sham rats (A–D) are stained as deep brown, and the nerves are in a line (B, D). The stressed rats show dark argyrophilia of pyramidal cells, mainly in the CA3 subfield (G). In addition, some degenerating cells were seen in the DG and CA1 subregions (F, H). The array of fiber is tangled (E, H). While in morphological evaluation of the HE staining, Nissl staining, and silver impregnation in prefrontal cortex, normal histology and morphology (A, B, E, F, I, J) of neurons were seen in the sham group, with no injured neurons (magnification 100× and 400 ×, respectively). In the stress group (C–D, G–H, K–L), damaged neurons are seen spreading after stress. Scale bars =200 μm. The neuronal density in 1-mm sections of hippocampal CA1, CA3, and DG subfields and prefrontal cortex were also calculated after stress. A: CA1, B: CA3, C: DG, D: prefrontal cortex. (n=6 per group, * p<0.05 compared with the sham group). The results are expressed as mean ± SEM. * p<0.05, ** p<0.01 compared with the sham group.

Schematic view of the role of physiological, pathological, and neurochemical changes in abnormal behaviors after severe stress in the present study. The severe stress induced hippocampal hyperactivation and prefrontal cortex hypoactivation, which resulted in the abnormal behaviors in rats. The possible mechanisms mainly refer to neuronal injury and apoptosis caused by the imbalance of glutamate and GABA.

Analysis of the apoptotic rate in prefrontal cortex and hippocampus by flow cytometry and TUNEL after stress. Flow cytometry pictographs: viable cell population (Annexin V⁻/PI⁻) is in region 3; early apoptotic cell population (Annexin V⁺/PI⁻) is in region 4; late apoptotic and necrotic cell populations (Annexin V⁺/PI⁺) are in region 2; and necrotic cell population (Annexin V⁻/PI⁺) is in region 1. (A) Cell population in the prefrontal cortex of sham group. (B) Cell population in the prefrontal cortex of stress group. (C) Cell population in the hippocampus of sham group. (D) Cell population in the hippocampus of stress group. (E) Comparison of the apoptotic rate in prefrontal cortex between stress and sham groups (* p<0.05 compared with the sham group). (F) Comparison of the apoptotic rate in the hippocampus between stress and sham groups (n=7 per group, * p<0.05). (G) Photomicrograph of neuronal apoptotic cells (TUNEL-positive cells) in the hippocampus (CA1, CA3, DG) and prefrontal cortex between stress and sham groups (see triangles) (n=6 per group, * p<0.05, ** p<0.01). The results are expressed as mean ±SEM. (Bar size 200 μm, magnification ×400).

The changes in concentration of Glu and GABA and ratio of GABA/Glu in hippocampus and prefrontal cortex after the stress. (A) Glu analysis. (B) GABA analysis (C) Ratio of GABA/Glu (n=6 per group, * p<0.05). The results are expressed as mean ±SEM. * p<0.05, ** p<0.01 compared with the sham group.