The Influence To Rat Ethology And AMPA Receptors Expression In Central Nervous System By Chronic Immobilization Stress And The Regulative Effect Of Xiao Yao San

Stress is a series of neuroendocrine responses which is marked with sympathetic nerves excitement and increase of pituitary -adrenal gland cortex secretion when organisms is expose to stressor stimulation, it's very important for individual survival. Mammals respond to appropriate stress with characteristic changes in their behaviour as well as in autonomic and neuroendocrine parameters aimed at reinstating their disturbed homeostasis. Among such so-called coping strategies, alterations of the hypothalamic–pituitary–adrenal (HPA) axis play a crucial role. However, excessive, especially chronic stress, causes physical, psychological disorders in vary degrees, and even causes body diseases, can be regarded as a pathological process resulting from the reaction of the body to external forces and abnormal conditions that tend to disturb the organism's homeostasis. Since multiple systems relate to chronic stress, and different stressor, different individuals and different circumstances might lead to different results, it's so difficult for prevention and treatment to chronic stress diseases, whereas Chinese medicine, which with a concept of whole adjustment, treatment based on syndrome differentiation and individualized diagnosis, has shown greater superiority. The present project was supported by National Natural Science Foundation of China (NO.30672578), and we focus on the experimental study on the central neural system mechanisms of chronic stress response and the role of regulative function of Xiao yao san in chronic stress response.Experimental studyMost excitatory signalling in mammalian brain occurs at synapses that use glutamate as a neurotransmitter. Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS), and is the agonist of two distinct categories of glutamate receptors, metabotropic glutamate receptors and ionotropic glutamate receptors. Ionotropic glutamate receptors have been divided in three classes, according to their pharmacological, molecular, and electrophysiological properties: alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainite (KA), and N-methyl-D-aspartate (NMDA) receptors. The AMPA receptors are the principal molecular units for fast excitatory synaptic transmission in the central nervous system. Glutamate binding to postsynaptic AMPA-type glutamate receptors induces depolarizations that cause neuronal firing. This AMPA receptor-mediated neurotransmission contributes to the brain's moment-to-moment activities, including cognitive responses and motor actions. Furthermore, changes in AMPA receptor function at synapses contribute to the plasticity that underlies learning and memory. GRIP binding to the unphosphorylated PDZ motif is proposed to anchor GluR2-containing receptors at synapses, The last four amino acids of stargazin bind to the PDZ domains of a number of synaptic scaffolding proteins, this PDZ interaction mediates synaptic targeting of surface receptors, phosphorylation of the PDZ-binding motif of stargazin is a regulator of synaptic levels of AMPA receptors and synaptic strength. C-fos is a member of immediate early genes (IEG), which belong to a class of genes that be regarded as tracing marker of neurocyte function.Therefore we chose GluR1, GluR2, two subunit of AMPA receptor, two proteins interacting with AMPA receptor subunits, GRIP1 ((glutamate receptor-interacting protein 1) and Stargazin (voltage-dependent calciumchannel gamma-2 subunit,Cacng2) and c-fos as the research targets, chronic restraint as chronic stress model, using ELISA, immunohistochemistry and RT-PCR methods, to observe Xiaoyaosan's influence on rats'behaviors, stress related hormones as well as AMPA receptors and its'interacting proteins mRNA expression, and to investigate the central nervous mechanisms and the role of regulative function of Xiao yao san to chronic stress responses and chronic stress impairments. The results were as the following.Behavioral changes: After 21 days immobilization stress, the rats displayed unhealthy changes, Xiaoyaosan could improve the unhealthy statement. The body weight increase of the rats in model group decreased significantly. At the first day of open-field test, the central grille time, total cross number of the rats in model group increased significantly, at the 21st day, the central grille time increased significantly, but the total cross number, stand number and modify number decreased significantly. At the 14th day of elevated plus-maze test, the open arm entry (OE) and OE%, open arm time (OT) and OT%, open and close arm entry and head dipping of the rats in model group decreased significantly. At the 21st day in Y-maze test, the error number, total reaction time of the rats in model group decreased, with significant increased correct reaction rate. Xiao yao san showed significant improvement to body weight, open-field test and elevated plus-maze test, whereas without significant effect to Y-maze test.ELISA results: In immobilization stress 21 days rats, the concentration of serum CORT increased, with statistical significance, Xiao yao san could regulate it towards normal level. Immunohistochemistry findings: In immobilization stress 21 days rats, the number of c-fos positive cells increased in CA1, CA3 region of hippocampus and basolateral amygdale (BLA); the number of GluR1 positive cells and immunoreaction products increased in CA1 and DG region of hippocampus; the number of GluR2-positive cells decreased in CA3 and DG region of hippocampus, so did the immunoreaction products in CA1, CA3 and DG region of hippocampus. In BLA, neither GluR1 nor GluR2 expression changes with statistical significance. Xiao yao san could decrease the number of c-fos positive cells in hippocampus and BLA as well as GluR1 positive cells and immunoreaction products in hippocampus, and could increase the number of GluR2 positive cells and immunoreaction products in hippocampus.RT-PCR results: In immobilization stress 21 days rats, both of the mRNA expression of GRIP1 and Stargazin decreased in CA1 region of hippocampus, whereas without statistical significant changes in CA3, DG region of hippocampus and BLA. Xiao yao san could increase the mRNA expression of GRIP1 and Stargazin.Results presented above suggest: (1) Under the same chronic stress, different behavioral tests may induce different results; (2) In the state of chronic immobilization stress, the expressional tendency of AMPA receptors subunits GluR1 and GluR2 was different in hippocampus; (3) Hippocampus and amygdala played different role in chronic restraint stress response; (4) The most sensitive target region of the chronic immobilization stress works on synaptic plastisity might be CA1 region of hippocampus; (5) The regulative effect of Xiao yao san shows regional-selective, time-dependent and double-way (up and down) regulative characterization.