| With the rapid changing of society and the increasing pressure of competition all around, negative stress; especially negative psychological stress has been a crucial factor threatening people's health. Negative psychological stress beyond one can bear is always the source of many diseases, particularly mental disorders, i.e. depression. Thus, it is extremely important for prevention and cure of stress-related mental disorders to investigate the central mechanisms of negative psychological stress and the biological effect of anti-stress medicine. However, it has not fully understood for the central circuits involved in psychological stress and the mechanisms of the anti-stress medicine, which is one of the most important issues in neuroscience and psychiatric medicine to be solved.Extracellular signal-regulated kinase1/2 (ERK1/2), is one of the most important members of mitogen-activated protein kinase (MAPK) family; and its signal transduction pathways is critical to the regulation of growth, degeneration, differentiation and survival of neurons. Recent study reported that the signal conduction molecule, ERK1/2, plays an important role in stress-induced activity of neurons in brain; and phosphorylated-ERK1/2 (p-ERK1/2), its active form, has been used as a new marker for the activity of neurons in functional morphology study. And cAMP response element binding protein (CREB), a neucleoprotein belonging to structure-relevant transcription factor CREB family, can regulate the transcription of genes that contain cAMP response element (CRE). As a key element in many intracellular signal pathways, this nuclear factor has extensive biological function including regulation of learning and memory. CREB is a potential transcription activator, and its expression and/or changes of activity may affect the regulation of many downstream target genes, i.e. brain-derived neurotrophic factor (BDNF); thereby affects the total curative effect of anti-mood disorders medicine.In present study, we firstly established a chronic restraint stress model in the rat. By using open-field test, Morris water maze, Western blot and immunohistochemistry, we then investigated the changes of exploring behavior, space memory ability and the level of p-ERK1/2 and phosphorylated CREB (p-CREB) in the brain of rats underwent chronic restraint stress. And at the same time we investigated the effect of pretreatment with a selective serotonin reuptake inhibitor (SSRIs), citalopram, on the above changes of rats. The results might provide clues for clarifying the central mechanisms of negative stress, and offer a new way to prevent and cure stress-related mental diseases in clinic. Objective1. Effect of administration and pretreatment with SSRIs on the exploring behavior and space memory of rats subjected to chronic restraint stress.2. Changes of level of p-ERK1/2 and total ERK1/2 in prefrontal cortex (PFC) of rats underwent chronic restraint stress.3. Effect of pretreatment with SSRIs on the level of p-ERK1/2 and p-CREB in stress related brain areas of rats underwent chronic restraint stress.Methods1. Effect of administration and pretreatment with SSRIs on the exploring behavior and space memory of rats underwent chronic restraint stressMale Sprague-Dawley (SD) rats were used and divided randomly into 4 groups: normal control group (NC, N=6), restraint stress group (RS, N=6), citalopram only group (OC, N=6) and citalopram prophylaxis group (CP, N=6). Daily intraperitoneal injection (i.p.) of 1 ml normal saline was done in RS group for 7 consecutive days, 10 mg/ (kg?d) citalopram, in OC and CP groups. And then the animals in RS and CP groups were subjected a restraint. The procedure as follows: to keep the rat in tailor-made container to confine its activity from 9:00 to 15:00 everyday for 21 d. During the stress time, the rats were not allowed to eat and drink. And i.p. normal saline to RS group and citalopram to CP group everyday before stress, at the same time i.p. citalopram to OC group in the same dose as above. The animals in NC and OC groups were not subjected to restraint but also subjected to food and water deprivation. All rats received open-field test before the end of the whole managements. And all rats were trained in Morris water maze for space memory at the last week of the experiment and were formally tested for space memory before the end of the whole managements.2. Changes of level of p-ERK1/2 and total ERK1/2 in PFC of rats underwent chronic restraint stressMale Sprague-Dawley (SD) rats were used and divided randomly into 2 groups: normal control group (NC, N=3) and restraint stress group (RS, N=3). The method is as same as above. All rats were immediately killed at the end of the experiment. Investigate the changes of level of p-ERK1/2 and total ERK1/2 in PFC of rats underwent chronic restraint stress.3. Effect of pretreatment with SSRIs on the level of p-ERK1/2 and p-CREB in stress related brain areas of rats underwent chronic restraint stressMale SD rats were used and divided randomly into 3 groups: normal control group (NC, N=6), restraint stress group (RS, N=6), and citalopram prophylaxis group (CP, N=6). The method is the same to 1. All rats were killed at the end of the study. Investigate the changes of level of p-ERK1/2 and total ERK1/2 in stress related brain areas of rats underwent chronic restraint stress.Results1. Compared with NC group, the rats in RS group had a lower horizontal activity in open-field test, and had more central time. The difference had statistical significance (P<0.05); but there were no significant changes in OC group. Compared with RS group, the rats in CP group had a higher horizontal activity in open-field test, and had less central time, the difference had statistical significance (P<0.05).Compared with NC group, the rats in RS group had less target quadrant time and stage cross time in Morris water maze, the difference had statistical significance (P<0.05); but there were no significant changes in OC group. Compared with RS group, the rats in CP group had more target quadrant time and stage cross time in Morris water maze, the difference had statistical significance (P<0.05).2. The protein level of p-ERK1/2 in RS group was lower than that in NC group, the difference had statistical significance (P<0.05), but the protein level of total ERK1/2 had no significant changes in the two groups.3. The distribution of p-ERK1/2 positive neurons was extensive in brain of NC group, and we take our focus on some stress-related regions. There were many p-ERK1/2 positive neurons in PFC, medial amygdaloid nucleus (MeA), posteromedial cortical amygdaloid nucleus (PMCo), lateral septal nucleus, ventral part (LSV) and bed nucleus of the stria terminalis (BNST) in NC group. There were less p-ERK1/2 positive neurons in the above regions in RS group, and drum dyeing of neurons was thinner, dendrites were dyed less, the difference had statistical significance (P<0.05). Compared with RS group, the number of p-ERK1/2 positive neurons was much more in CP group in the above regions, and drum dyeing of neurons was thicker, dendrites were dyed much more, the difference had statistical significance (P<0.05).The changing pattern of p-CREB positive neurons in NC group was similar to p-ERK1/2. There were many p-CREB positive neurons in PFC at the superficial layer of the cortex. There also were considerable p-CREB positive neurons in dentate gyrus (DG), CeA, MeA and PMCo. There were less p-CREB positive neurons in the above regions in RS group, and the drum dyeing was thinner, the difference had statistical significance (P<0.05). Compared with RS group, there were more p-CREB positive neurons in the above regions in CP group, the difference had statistical significance (P<0.05).Conclusion1. The explore behavior and space memory of rats subjected to chronic restraint stress were greatly damaged. Pretreatment with citalopram can significantly relieve the harmful effect of stress on the behavioral performance of rats, and play a role in anti stress. However, citalopram itself does not affect the performance of rat in open-field test and Morris water maze.2. The level of p-ERK1/2 in PFC of rats underwent chronic restraint stress was much lower, but the level of total ERK1/2 had no significant change, indicating that the above regions might be associated with negative stress-related central mechanisms.3. The levels of p-ERK1/2 and p-CREB were changed in PFC, CeA, MeA, PMCo, DG, LSV and BNST of rats underwent chronic restraint stress. This result demonstrate the above regions may be relevant to stress-related central regulation, and ERK1/2 and CREB signal transduction pathways may participate in this regulation process. The level of p-ERK1/2 and p-CREB can be upgraded by pretreatment with citalopram to rats, what suggest citalopram may produce anti-stress effects through ERK1/2 and CREB signal conduction pathways. The results may be helpful for the selection of anti-stress medicine in clinic. |
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