Primary Culture For The Purkinje Cell Of Npc Neonatal Mouse And The Cultured Cell For Ca²⁺ Dynamics Characteristic And Determining The Amino Acid In Mouse Cerebellum

Part OnePrimary culture for the cerebellum Purkinje cell of NPC neonatal mouse and the cultured cell for the characteristics of Ca²⁺ current and intracellular Ca²⁺ dynamicsObjective To establish an easier and better primary cell culture method suitable for Purkinje cell of NPC neonatal mouse, and research the cytoelectrophysiological characteristics of the cultured cells preliminarily. The cultured cells also used to determine the intracellular Ca²⁺ concentration under Confocal microscope by labeling with immunofluorescence dye of Fluo-3/AM.Methods Cerebellum of neonatal mouse born in 24 hours is dissected bluntly to obtain suspended solution of single cells by low concentration of trypsin digestion and mechanical dissociation,then cells are cultured in DMEM/F12 media containing 10% fetal bovine serum. The media are changed into DMEM/F12 with 1% N²⁺1%T3+1%glutamine after24 hours for maintaining culture. Cytoimmunofluorescent dyeing is carried out after 3-5 days, while the intracellular fundamental Ca²⁺ fluorescence intensity and the change of fluorescence intensity after adding potassium chloride(60umol/l) and NBQX(AMPA receptor blockage) are determining under Confocal microscope after 5-7days,also Ca²⁺ current be recorded by single channel method with whole cell patch clamp after 7-9 days.Results the Purkinje cells cultured in this method showed typical neurons characteristic, cytological evaluation illustrated high positive rate(>70%)and Ca²⁺ current can be recorded. The intracellular Ca²⁺ concentration of cultured cells of NPC-/- mouse is higher than the wild type neonatal mouse.Conclusions Materials are easily obtained for this culture with convenient operation , good growth status of cultured primary purkinje cells and high activity are suitable for electrophysiological and Ca²⁺dynamics research.Part TwoDetermining 3 amino acid neurotransmitters in cerebellum cortex of NPC mouse of five weeks by mass spectroscopyObjective To determine the contents of amino acid in cerebellum cortex of Balb/C mouse of five weeks and compare the discrepancy difference between NPC-/- mouse and wild type mouse.Methods The sample was collected by microdialysis. Three amino acid neurotransmitters: Asp,Glu and Gly in the microdialysate were determined by mass spectroscopy. Results The mass spectroscopy method is quick and accurate to measure the contents of amino acid in the cerebellum cortex of the mouse , but the contents of three amino acid in NPC-/- mouse and wild type mouse without significant difference.Conclusion Compare with the wild type mouse, the contents of three amino acid in NPC-1mouse alter little, the amino acid neurotransmitters might not participant in the pathological change of NPC-/- mouse during this period.