酶处理急性分离技术用于研究果蝇成虫中枢神经细胞钾电流

Enzymatic dissociation technique for studying potassium currents in acutely isolated maturing drosophila central neurons

目的 建立急性分离果蝇成虫中枢神经细胞技术，并记录分析其钾电流。方法 取S、R或H品系果蝇成虫3—5只，分离出果蝇脑组织。胶原酶消化处理后，加入10％的胎牛血清果蝇培养基终止酶反应。巴氏吸管吹打，细胞悬液静置1h后用于电生理实验。全细胞电压钳记录并分析其外向钾电流。结果 酶消化法成功急性分离出果蝇成虫中枢神经细胞。85％以上为Ⅱ型神经细胞，少部分为Ⅰ型和Ⅲ型神经细胞。记录到快失活与持续复合型、慢失活型、快失活和持续型四种全细胞钾电流。结论 本技术可成功急性分离三品系果蝇成虫中枢神经细胞，并记录到四种钾电流。

Objective To develop a method for studying acutely isolated maturing Drosophils central neurons（MDCNs）and to assess their petossium eurrents. Methods Three to five adult Drosophila from S, H or R strain were dissected to expese the brains. Then the brains were incubation with collagenase for some time. The tissue was then suspended in culture medium to end the enzymatic trentments.Then the tissue was gently passed to dissociate the cells.The resulting suspension was plated for lh prior to electrophysiological examination. Whole-cell recording were made to assess the potassium currents in the central neurons.Results The enzymatic method was succeed to isolate acutely MDCNs. Type Ⅱ ceils comprised beyond 85% of the total populatien,other ceils were type Ⅰ or type Ⅱ ceils. Four phenotypes potassium currents were found. They were not only the inactivating currents but also a sustained component, a slow inactivation time course, a fast inactivating potassium currents and the sustained component. Conclusion The enzymatic dissociation procedure was succeeded to isolate MDCNs acutely and record the four phenotypes potassium currents.