1.Regulation Of NMDA Receptor-mediated Current By Corticotropin-Releasing Hormone In Cultured Hippocampal Neurons 2.Regulation Of Neurite Development And SGK Expression By Corticotropin-Releasing Hormone In Cultured Hippocampal Neurons

The hippocampus is one of the basal structures of the limbic system. It plays a fundamental role in learning and memory process and in negative feedback regulation of the HPA axis during stress. Dysfunction of the hippocampus could result in some of the vegetative and endocrine abnormalities, as well as cognitive and memory deficits. The hippocampus has been found that numerous neuronal cell bodies and projections that contain CRH are distributed in hippocampus. Corticotropin-releasing hormone (CRH) is the primary factor driving stress-induced adrenocorticotropin secretion from the anterior pituitary and capable of integrating the neuroendocrine, behavioural, autonomic and immune response to stress. At present, the study on the regulation of hippocampus functions by CRH remains elusive.In the first part of present study, we examined the direct modulation of CRH on NMDA-mediated current in cultured rat hippocampal neurons by using the whole-cellpatch-clamp technique. We also conducted experiments to investigate whether the effectsof CRH could have occurred via CRH receptors and through an AC/PKA- orPLC/PKC-dependent pathway.In the second part, we observed the effects of CRH on neurite development incultured hippocampal neurons. We also studied on the regulation of mitotic kinesin likeprotein 1 (MKLP1) by CRH.In the third part, we studied on the regulation of Serum- and glucocorticoid-regulatedprotein kinase (SGK) expression by CRH in cultured hippocampal neurons. And themechanism of this effect was further explored.Main results:1. Regulation of NMDA-mediated current by CRH in cultured hippocampal neurons.1) Both CRHR1 and CRHR2 expressed in cultured rat hippocampal neurons showed by immunofluorescence analysis.2) Whole-cell patch-clamp technique showed that CRH can reduce NMDA currents in hippocampal neurons. This effects of CRH was in a dose-dependent manner. CRHR1 but not -R2 mediated the inhibitory effect of CRH on NMDA currents.3) CRH was found to induce cAMP production by RIA and increase phosphorated PLC-βexpression by Western blotting analysis. 4) NMDA currents were still reduced by co-application of PKA inhibitor H89 or AC inhibitor SQ 22536 and CRH. PLC inhibitor U73122, PKC nonselective inhibitor chelerythrine and IP3R antagonist 2APB substantially blocked the CRH-induced decrease of NMDA currents, respectively. Application of the PKCα/βinhibitor Go6976 partly blocked the CRH-induced decrease of NMDA currents. Dialysis with the Ca²⁺ chelator BAPTA also mostly prevented CRH-induced depression of NMDA currents.2. Regulation of neurite development by CRH in cultured hippocampal neurons.1) CRH was found to increase the total dendritic branch length (TDBL) in cultured hippocampal neurons by transfection with enhanced green fluorescent protein (EGFP). CRH was also found to induce microtubule-associated protein (MAP2) expression by immunofluorescence analysis. And CRHR1 but not -R2 mediated the effects of CRH.2) Real-time PCR showed that CRH up-regulated MKLP1 mRNA expression dose-dependently, which can be blocked by nonselective CRH receptor antagonist astressin or specific CRHR1 antagonist antalarmin, not by specific CRHR2 antagonist astressin 2B. Urocortinâ…¡, the specific CRHR2 agonist had no effect on MKLP1 expression. The results demonstrated that CRH-R1 but not -R2 mediated the regulatory effect of CRH on MKLP1 mRNA expression.3. Regulation of SGK expression by CRH in cultured hippocampal neurons.1) Immunofluorescence analysis showed CRH can significantly increase the frequency of SGK-positive cells. Western blot analysis also showed that CRH enhanced SGK protein expression in a dose-dependent manner in the period of 24 h treatment.2) Immunofluorescence analysis and Western blot analysis showed that the effects of CRH were reversed by antalarmin but not by astressin 2B. The results showed that CRHR1 but not -R2 mediated the regulated effect of CRH on SGK protein.3) Treatment of cells with H89 exhibited a similar effect as SQ22536, completely blocking CRH action on SGK expression. And forskolin could significantly increase SGK protein expression. Treatment of cells with U73122 or G(o|¨)6976 did not block CRH-induced SGK expression at protein level.Conclusions:In conclusion, these results suggest that in hippocampal neurons, CRH acts on CRH-R1 to suppress NMDA receptor-mediate currents, which is dependent on PLC/IP3R/Ca²⁺ and PLC/PKC signaling pathways, and to stimulate SGK protein expression via a mechanism that is involved in AC/PKA signaling pathways. In addition, CRH also can regulate neurite development and MKLP1 mRNA expression.