果蝇晚三龄幼虫脑神经元的快速分离与鉴定

目的探索果蝇晚三龄幼虫脑神经元急性分离的新方法，并对分离制备的脑细胞进行形态学观察和电生理学记录。方法解剖果蝇晚三龄幼虫脑组织，用胶原酶化学消化并辅以机械微振荡法分离制备单个细胞，用果蝇细胞培养液适当孵育。在倒置相差显微镜下对其进行形态学观察和分类，结合全细胞膜片钳技术对其电生理学特征进行初步研究。结果成功制备出三类大小和形态各异、活性较好的果蝇脑神经元．未见神经胶质细胞；以三型细胞为电生理学研究对象，记录到五种类型的全细胞外向钾电流。结论新建立的果蝇晚三龄幼虫脑细胞急性分离方法简便易行，稳定可靠。

Acute Isolation and Identification of Cerebral Neurons from the Late Third Instar Larvae of Drosophila Melanogaster

OBJECTIVES: To develop a new method for acute isolation of the cerebral neurons from late third instar larvae of Drosophila melanogaster. METHODS: The dissociated cells were characterized by morphological observation and whole-cell voltage-clamp recording. The brains were dissected from late third instar larvae, torn into small fragments, and then were digested in the calcium-magnesium-free PBS solution endowed with collagenase for 45-60 minutes. Single cell was obtained by micro-shaking the digested fragments for 5 to 10 s until the clumps of the tissue were not invisible. Preparation of dispersed cells was incubated in the culture media of Drosophila for thirty minutes at room temperature (20 +/- 1) degrees C. RESULTS: All neurons studied were categorized into three types according to morphological observation: large (> 8 microm) round type I neuroblast-like cells (7%), small (2-5 microm) type II cells (77%). and intermediate-sized type II cells (16%). Neurogliocytes were not found. The electrophysiological properties of three types of neurons were investigated by whole-cell voltage-clamp recording technique. Five types of outward potassium currents were detected readily. CONCLUSION: Morphological and electrophysiological investigation showed that the method for acute isolation of Drosophila neurons is simple, available and stable.