Ultrashort Feedback Control Of Corticotropin-releasing Hormone Secretion During Traumatic Stress In Rat's Hypothalamus

The main characteristics of stress response are a series of physiological and psychological changes from activation of hypothalamus-pituitary-adrenal (HPA) axis. It is very complicated to control the activation of HPA axis in central nervous system. Synthesis and release of corticotropin-releasing hormone (CRH) from hypothalamus plays a key role in the regulation of the HPA axis. CRH controls the stimulating level of HPA axis. The existence of ultrashort feedback control of CRH secretion during stress was first suggested by Ono et al in 1985. CRH-containing axon may contact with CRH-producing neurons in synapse formation. CRH type 1 receptor (CRH1R) is expressed in hypothalamic neurons. It is suggested from morphology that there is a possibility of feedback regulation of CRH receptors in CRH-producing neurons.In this study the model of traumatic stress in rats was established, the changes of CRH, CRH1R and c-fos distribution and expression were observed and determined with immunocytochemistry, western blotting , RT-PCR in vivo; Hypothalamic neurons from fatal rats were and irritated with CRH. Using Fura-2/AM ratio fluorescence imaging analysis was applied to detect intracelluar Ca2+, immunocytochemistry and western blotting the distribution and content of PKA, CREB, CRH in virto.The main results and conclusions are as follows:1. The model of impact injury of right lateral superior chest of rats with unilateral femur fracture was established. The model has theadvantages of simple manning, controllable physical parameters, accuracy and repeatability of impacting site and constant severity of wound.2. Hypothalamic neurons in vitro was well and stables cultured. The neurons grew most fast during days 1-3, and reached confluent monolayer and grew up synapse during days 7-10.3. CRH caused an increased intracellular concentration of Ca2+ with marked difference between every expermental group and control group. The L-type Ca2+ channel inhibitor nifedipine or the PKA inhibitor H89 can reduce the CRH-induced increase in cytosolic Ca +, indicating that L-type Ca2+ channel and PKA signal pathway may play an impoatant role in CRH-induced [Ca2+]i.4. Normally PKA is mainly distributed in the cytoplasm of neuron. After stimulation of CRH, however, it appeared also around the nucleal membrane, and even in the nucleus. The concentration of PKA increased, indicating that binding of CRH to its cognate receptors was associated with the activation of PKA signal pathway.5. CRH may cause the increase of phosphorylated CREB in the neuron of hypothalamus, while CP-154526, H98, Nif can significantly inhibit the production of phosphorylated CREB. Combining with CRH CP-154526, H89 and Nif may cause the increase of activated PKA production and [Ca2+]i in the neuron of hypothalamus, indicating both of these two signal transduction pathways may increase the content of phosphorylated CREB , thus regulate transcription activation of the neuron in hypothalamus.6. CRH may cause macked increase of CRH content in cultured hypothalamic neuron, while CP-154526, H89 and Nif may greatly inhibit the increase of CRH content, indicating PKA signal pathway and Ca2+ pathway play an important role in combination of CRH with CRH1R and stimulating hypothalamic neuron to cause its self gene expression.7. Traumatic stress induced an increased intracellular concentration of CRH and CRH1R in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of rat hypothalamus, while CP-154526, H89 or nifedipine significantly inhibit the expression of CRH and CRH1R.