从晚期果蝇蛹的大脑原发性神经文化

Summary

这个视频演示从后期果蝇蛹大脑中的主要神经文化的准备。生活文化的视图显示突起生长的钙水平使用FURA – 2和成像。

Abstract

在这段视频中，我们展示了从后期果蝇蛹大脑中的主要神经文化的准备。程序开始在70-78小时puparium形成后，从动物的大脑去除。木瓜蛋白酶，在无血清生长介质中的几个洗短暂的潜伏期后孤立的大脑。在盖玻片上一个媒体下降5 UL每个大脑的机械分离的过程说明。在解离附近的SOMA的有丝分裂后神经元的轴突和树突sheered但神经细胞开始再生电镀的几个小时内进程。图像显示，在2天的现场文化。神经元继续阐述在第一周在文化进程。使用GAL4线驱动组织特异性表达GFP或RFP，如荧光标记，可以识别特定的神经元的人口文化。全细胞记录已经证明，培养的神经元形成胆碱能和GABA能突触的功能，自发主动。一个简短的视频段说明使用FURA – 2作为钙指示剂监测自发性钙瞬变和一碟培养的神经元的尼古丁诱发的钙反应的培养神经元的钙动态。这些蛹脑文化是一个有用的模型系统，在其中的遗传和药理工具可以被用来确定中央突触的形成和功能的内在和外在因素影响。

Protocol

培养前一天的准备工作： 设为无菌解剖解决方案。 使无菌DMEM培养液，并保持在4 ° 10毫升分装在2个星期。 在1个月的50或100μL分装无菌DDM2补充和冻结。 使错那县/层粘连蛋白。 大衣盖玻片。 可选：请无菌中国建材和存储长达4个月的冻结。 在培养一天一，准备在层流罩酶解决方案（ES） 把解剖解决方案（DS…

Discussion

收获胚胎/产后的啮齿类动物的大脑的神经细胞可以生长在原代细胞培养，他们延长突起，形成功能性突触连接。这些文化的编制方法是建立在啮齿类动物的神经元文化的研究起到了关键作用，在确定基因和环境因素参与调节突触的形成和功能的银行家和Goslin，（1991）。虽然从各种不同的物种昆虫的神经元也可以培养，从果蝇（Rohrbough等，2003）所示，形成文化功能的突触连接的唯一的昆虫神经元。神经母细胞收?…

Materials

Material Name	Tipo	Company	Catalogue Number	Comment
Concanavalin A		Sigma	C-2010	To 2.5 ml DS, add 25 mg Concanavalin A bottle. This is 10 mg/ml concentration. Make aliquots of 90 ul and store at -20 C, no longer than 3 months.
Laminin		Sigma	L-2020	Add 1ml DS to 1mg Laminin bottle. This is 0.5 mg/ml concentration. Make aliquots of 10 ul and store at -20 C, no longer than 3 months.
Coverslips		Bellco Biological Glassware	1943-00012	12 mm glass coverslips
ConA + Laminin solution				Stock solution, from Con A stock and laminin stock, for coverslip coating: Add in 5 ml DS, 83.5 ul of ConA (167 ug/ml) and 8.35 ul of Laminin (0.835 ug/ml). Mix. Make aliquots of 100 ul and store at -20 C, not longer than a month.
Dissecting Solution	Buffer			For 500 ml: 400 ml Ultra filtered water + 25 ml Stock Solution A + 14 ml Stock Solution B + 3.0 g (33.3 mM) D (+)-Glucose (Sigma G-8270) + 7.5 g (43.8 mM) Sucrose (Sigma S-0389). Adjust pH to 7.4 with 1N NaOH (around 2 ml). Bring final volume to 500 ml with ultra filtered water. Decant into a clean glass bottle and autoclave. Label “Dissecting Solution” and store at 4?C.
Dissecting Solution	Buffer			For 500 ml: 400 ml Ultra filtered water + 25 ml Stock Solution A + 14 ml Stock Solution B + 3.0 g (33.3 mM) D (+)-Glucose (Sigma G-8270) + 7.5 g (43.8 mM) Sucrose (Sigma S-0389). Adjust pH to 7.4 with 1N NaOH (around 2 ml). Bring final volume to 500 ml with ultra filtered water. Decant into a clean glass bottle and autoclave. Label “Dissecting Solution” and store at 4?C.
Solution B: HEPES	Buffer	Sigma	H-3375	20.97g (9.9mM). Add ultra filtered water up to 200 ml. Mix until dissolve and bring final volume to 250 ml. Place in a clean bottle and autoclave. Label “Solution B” and store at 4 C.
Solution A	Buffer			For 500 ml: 80.0g (137 mM) NaCl (Sigma S-9625) + 4.0g (5.4mM) KCl (Sigma P-4504) + 0.24g (0.17mM) Na2HPO4 (Sigma S-0876) + 0.3g (0.22 mM) KH2PO4 (Sigma P-5379). Weigh out all ingredients and mix until dissolved with 400 ml ultra filtered water. Bring final volume to 500 ml. Place in a clean bottle and autoclave. Label “Solution A” and store at 4°C.

Coating Coverslips: Put autoclaved coverslips in a 60 mm petri dish. Pipet 5 ul of Con A/Laminin mix onto center of each coverslip. Place in 37 C incubator for 2 hours. Rinse 3x coverslips with 100 ul of autoclaved water each. Use vacuum attached to a sterile Pasteur pipet. During the last rinse, pick up the coverslip with forceps and dry both sides. Transfer the coverslip to a 35mm Petri dish. Store at room temperature for up to a month.