A study for morphology and the reactivity of rats hippocampal slice in vitro culture

Objective:To explore the method of culturing rat hippocampal slice in vitro and to observe the changes of neurons' morphology and reactivity in cultured slice.Methods:Organotypic hippocampal slices were prepared by taking brains from 6-9 day old wistar rats.After the hippocampi was isolated and chopped into 400μm thickness,slices were transferred to Millipore inserts and placed in six-well trays in CO_2 incubators at 37℃.Then the cultured hippocampal slices were observed for the changes of morphology by macroscopic observation and using inverted phase contrast microscope;Expression of Fos protein in cultured hippocampal slice was detected by immunocytochemistry for observation of the internal response of hippocampal neurons to the damage;The electrophysiological activity of hippocampal neurons were detected by using patch clamp technique. Results:(1)The number of neurons in cultured hippocampal slices was increased,and the thickness of cultured hippocampal slices was decreased markedly along with the culturing period in vitro.After being cultured for 4 weeks,the thickness of hippocampal slices was decreased to about 150μm.(2)With the treatment of pilocarpine to induce seizure-like activity,the number of neurons which expressing Fos protein was increased in CA1 area of the cultured hippocampal slice.(3)With the application of pathch clamp technique,the changes of the ion currents of pyramidal neurons in CA1 area were recorded by the whole-cell recording after culturing for 1 week,2 weeks,3 weeks and 4 weeks respectively.Conclusion:Cultured hippocampal slice in vitro could retain satisfactory liveness and function for at least 4 weeks.