The Rapid Adjustment Of Ciliary Neurotrophic Factor On Glutamate Ionotropic Receptors In Hippocampal Neurons Caused By Intracellular Calcium Concentration Increases

AbstractThe Rapid Effect of CNTF on the Increase of [Ca2+] in Primary Cultured Hippocampus Induced by Ionic-Receptor of Glutamate 98Grade of pathophysiology, Qingdao Medical CollegePostgraduate: Wu YangyangTutor: Prof. Cui RuiyaoCiliary Neurotrophic Factor(CNTF) is a new neurotrophic factor purified from ciliary nerve node of chicken embryo. It shares the same family with IL-6,IL-11,ect. CNTF has many biological activities such as facilitating pre-neuron growing, differentiation and mature, stimulating neurons survival, promoting neurons rehabilitation, preventing neurons degeneration, protecting glia and preventing neurons from injury of stress. The research in mechanism of biological activities of CNTF has been on gene's way. CNTF links to the α subunit of CNTF receptor, then links to gp130 and leukemia inhibitory factorβ, which induces tyrosine phosphorylation of the jak-tyk family and phosphorylation of two transcription factors. Then STAT3, STAT89/88 is activated, which go into nucleus and combine to DNA sequence of STAT91 to control transcription and synthesis of protein. It has been reported that CNTF can regulate the transmitter releasing at pre-and post-synapse of nerve terminals, which occurred in very short time. Recently research has shown that CNTF can inhibit the increase of [Ca2+]i in primary cultured hippocampus in very short time. It is impossible that the rapid action of CNTF occurs through gene's way. Perhaps there is another way through which biological activities of CNTF can happen immediately.Primary cultured hippocampus was involved in our research. We used the image analyzing system of Ca2+ according to the theory of double-wavelength fluorescence imaging described by Grynkiewicz et al as our main method. Under different condition we determined the change of the concentration of [Ca2+]i and analysis the mechanism of the activities of CNTF.The main results are as follow:1. Primary cultured hippocampus cell2. Two antagonists of ionic-receptor of Glu acted on hippocampus neuron and resulted in the change of [Ca2+]i 2.1 From 0~115μM AMPA induced increase of [Ca2+]i of hippocampus neurons. The increase of [Ca2+]i was dose-correspondence of AMPA. The suitable concentration of AMPA is 2.3μM, which leaded to the Ratio of 0.8±0.14.2.2 From 0~1000μM NMDA induced increase of [Ca2+]i of hippocampus neurons. The increase of [Ca2+]i was dose-correspondence of NMDA. The suitable concentration of NMDA is 2.3μM, which leaded to the Ratio of 1.27±0.26.3. CNTF acting on resting hippocampus neuron alone couldn't cause the change of [Ca2+]i.4. Incubated with CNTF(1μg/ml；10μg/ml；50μg/ml) for 5min, NMDA(100μM), AMPA(2.3μM) caused the increase of [Ca2+]i hippocampus neuron4.1 Incubated with CNTF (1μg/ml；10μg/ml；50μg/ml) for 5min, the increase of [Ca2+]i caused by AMPA (2.3μM) was not different from the control.4.2 Incubated with CNTF (1μg/ml；10μg/ml；50μg/ml) for 5min, the increase of [Ca2+]i caused by NMDA (100μM) was lower than the control.5. Incubated with CNTF (10μg/ml) for 3 min, the increase of [Ca2+]i caused by NMDA (100μM) was also lower than the control.6. Incubated with PTX (100ng/ml) for 24 hours, the inhibition of CNTF disappeared.It can be concluded that CNTF can inhibit the increase of [Ca2+]i of primary cultured hippocampus neuron caused by glutamate in a very shout time (<5min), in which the NMDA receptor is involved. AMPA receptor is not effective in the rapid action of CNTF. The rapid action of CNTF is related to the linkage to G protein. This rapid action happens ahead of the gene signal transmit way, and regulates the gene's way.