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
|School||Huazhong University of Science and Technology|
|Keywords||Primary cultured Purkinje cells Suckling mice Whole-cell patch clamp Single channel recording method Common Focus Determination of intracellular calcium concentration Mice Cerebellar cortex Microdialysis technique Glutamate Aspartate Glycine Mass Spectrometry|
The first part of the NPC neonatal rat Purkinje cells in primary culture and cell Ca -2 the / sup current and Ca 2 sup> dynamics characteristics Objective To establish a simple and effective NPC neonatal rat Purkinje cells in primary culture methods, Preliminary studies of the cell electrophysiological properties and cultured cells marked fluorescent dye Fluo-3/AM for measured under confocal microscopy-intracellular Ca 2 sup> concentration, relatively NPCs-/ - suckling mice and wild type neonatal rat cultured cells the difference between the observed indicators. Method blunt dissection Health 24h neonatal rat cerebellum using low concentrations of trypsin plus mechanical blowing play single cell suspension then DMEM/F12 containing 10% fetal bovine serum culture 24 hours after the replacement of 1% N 2 sup> 1% T3 1% glutamine the DMEM/F12 maintain basic fluorescence intensity value in the determination of the Purkinje cell culture immunofluorescence staining ,5-7d ,3-5d confocal microscope after the change of the fluorescence intensity value and adding a the KCL solution of (60umol / l) and NBQX (AMPA receptor blockers), the use of the corresponding software processing derived-intracellular Ca 2 sup> concentration measuring results, 7 - 9d single-channel method of whole cell patch clamp recording calcium current. Results of the culture typical neuronal morphology, cytology identified positive rate (gt; 70%), and record to the calcium channel current and NPCs-/ - neonatal rat cultured cells Ca 2 sup> high concentrations in the wild-type suckling mice. Conclusions drawn easily, simple operation, good growth state in cultured Purkinje cells, high activity, can be used electrophysiological and the of Ca 2 sup> dynamics of. Focus; intracellular Ca 2 sup> concentration determination of the second part of the mass spectrometry determination of five-week-old the NPC mouse cerebellar cortex of three kinds of amino acid content of the purpose of the determination of the five-week-old the NPC mouse cerebellar cortex amino acid content comparing NPC- / - mice and wild-type mice brain amino acid content differences. Collected using microdialysis technique amino acids in the mouse cerebellar cortex extracellular fluid mass spectrometry determination of dialysate glutamate (Glu), aspartic acid (Asp) and glycine (Gly) content. The results of mass spectrometry can be fast and accurate determination of the content of amino acids in the cerebellar cortex, NPC-/ - mice and wild-type mouse cerebellum three kinds of amino acid content was no significant difference. Conclusion The five-week-old NPC-/ - compared with wild-type mice, three kinds of amino acids in no significant change of amino acid neurotransmitters may not be involved in the pathological changes of this period NPC-/ - mice.