| ObjectivePost-traumatic stress disorder (PTSD), an anxiety disorder, develops after a life-threatening, traumatic experience, and is characterized by symptoms that often endure for years including intrusive memories (flashbacks), avoidance of stimuli associated with the trauma, numbing of general responsiveness, and increased arousal. Currently, PTSD with the characteristics of high incidence, long course, difficult to be cured and seriously impact on people's physical and mental health. So they have attracted a lot of attention. The exact mechanism of PTSD is still elusive. Negative feedback of the enhanced hypothalamic-pituitary-adrenal (HPA) axis is a putative neuroendocrinological hallmark of PTSD.As a kind of neurotransmitter, serotonin (5-hydroxytryptamine,5-HT) plays an important role in controlling the complex neuronal communication, such as negative feedback of the HPA axis via the serotonergic receptor. It was proposed that serotonergic type 1A (5-HT 1 A) receptor has an important effect on mood and anxiety modulation, but keeps the role of serotonin receptor on PTSD contradicative. It is well established that extracellular signal-regulated kinase 1 and 2 (ERK1/2) play an important role in many cell types, including neurons.Single-prolonged stress (SPS) was shown to induce enhanced inhibition of the hypothalamic-pituitary-adrenal (HPA) axis. And SPS paradigms have been extensively applied in the investigation of PTSD. In this study, we built animal PTSD model using SPS-method, and observed the behavioral changes and determined the hydrocortisone level in rats blood serum. To investigate the effect of 5-HT1A receptor on GR and CRF, we exposed rats to SPS with or without prior treatment with the receptor antagonist and observed the GR level in the hippocampus and CRF level in the hypothalamus. Also, we determined the changes of phosphorylated ERK1/2 (pERKl/2) and c-fos levels in the mPFC of SPS rats, and assessed the effect of PD98059, an inhibitor of the ERK pathway.Methods1, Behavioral changes of PTSD ratsWistar rats were divided randomly into control group and model group. The model of PTSD was prepared by SPS. The behavioral changes of rats were observed with the open field test, elevated plus-maze, and Morris water maze. Hydrocortisone level in rat blood serum was determined with chemiluminescence. And the freezing behavior was measured too.2, Relationship between the activity of 5-HT1A receptor and GR in hippocampus and CRF in hypothalamus in PTSD rats.Rats were randomly divived into normal group, sham group (vehicle administration), SPS group (SPS+vehicle administration), and blockade group (SPS+ WAY100635 administration). GR levels in the hippocampus and CRF levels in the hypothalamus by immunohistochemistry, Western blotting and RT-PCR3, Change of ERK in mPFC of PTSD ratsRats were randomly divided into three groups:control group, SPS group, and PD98059 group. The SPS group rats and PD98059 group rats were treated with SPS procedure, and respectively injected solvent or PD98059 into the bilateral mPFC 30 min before exposure to SPS. Brains were removed for immunohistochemical staining and Western blotting of pERKl/2. And RT-PCR was employed to detect the c-fos mRNA. Results1, Behavioral changes of PTSD rats(1) Results of OF testThe result showed that detention time in central grille and erect quantity of model rats were less than that of control rats (P<0.05). There were no significantly difference in other index.(2) Results of EMP testPercentage of the number of entry into open arms and percentage of time spent were significantly decreased in SPS rats compare to control rats (P＜0.01).(3) Results of MWM testCompared to control group, a striking increase of the latency was observed in SPS rats (P<0.01).(4) Results of freezing behavior The model rats showed freezing behavior when they were faced contextual fear condition again. However, the control rats showed free movement when faced the fear condition again. The percentage of freezing of control rats was less than model rats (P＜0.01).(5) Hydrocortisone level in blood serumHydrocortisone level in model rats blood serum was less than that of control rats (P＜0.01).2, Relationship between the activity of 5-HT1A receptor and GR in hippocampus and CRF in hypothalamus in PTSD rats.(1) Results of immunohistochemical analysis of GR and CRFThere was no statistically significant difference between normal and sham groups. Compared to the normal and sham groups, expression of GR and CRF increased in SPS group (P＜0.01), which were partially blocked by WAY100635 (P<0.05).(2) GR expression detected by Western blotting Similar to the immunohistological staining, there was GR expression in the hippocampus area when rats were under normal conditions. SPS significantly increased the GR expression (P<0.01) which is partially decreased by WAY100635 (P＜0.01).(3) GR and CRF mRNA expression detected by RT-PCRThere was both GR and CRF expression in the normal condition. Compared with the normal group, sham had no effect on the GR and CRF mRNA expression, however, SPS significantly increased their expression (P<0.01) which were partially decreased by WAY100635 (P<0.05).3, Change of ERK in mPFC of PTSD rats(1) Result of pERK1/2 immunohistochemistry stainingThe pERKl/2 protein was located in cytoplasm, and positive cells were brown. In control group, the OD of pERKl/2 positive cells was little, and that of SPS rats were significantly increased (P<0.01), and after the rats were bilaterally infused the PD98059, the pERKl/2 positive cells in the mPFC was significantly abolished (P< 0.01).(2) Result of Western blotting analysis pERKl/2The density of pERKl/2 bands had a significant increase in SPS rats (P<0.01). After the rats were bilaterally infused the PD98059, the SPS-induced ERK1/2 hyperphosphorylation in the mPFC was significantly abolished (P<0.01).(3) Result of RT-PCRVery low level of c-fos mRNA were detected of control rats. However, the expression of c-fos mRNA had a significant increase in SPS rats (P＜0.01). After the rats were bilaterally infused the PD98059, the SPS-induced c-fos mRNA increasing in the mPFC was significantly abolished (P＜0.01).Conclusions1, SPS rats exhibit behavioral abnormalities that mimic the symptoms of PTSD.2, Activity of 5-HT1A receptor is involved in the alteration of GR and CRF in SPS rats.3, SPS promoted the phosphorylated ERK in mPFC and ERK may be related to signal transduction pathway in single-prolonged stress.
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