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Neuroscience

离体 癫痫 果蝇 模型的钙成像

Published: October 13, 2023
doi:
10.3791/65825
, , , , ,
1Department of Neurology, Institute of Neuroscience, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China,the Second Affiliated Hospital, Guangzhou Medical University, 2The Second Clinical Medicine School of Guangzhou Medical University, 3Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Guangdong Provincial Key Laboratory of Major Obstetric Diseases,The Third Affiliated Hospital of Guangzhou Medical University, 4Key Laboratory for Reproductive Medicine of Guangdong Province,The Third Affiliated Hospital of Guangzhou Medical University

Summary

在这里，我们提出了一种在表达 GCaMP6 的成年果蝇中进行离体钙成像以监测癫痫样活动的方案。该协议为通过离体钙成像研究成年果蝇的发作事件提供了有价值的工具，从而可以在细胞水平上探索癫痫的潜在机制。

Abstract

癫痫是一种以反复发作为特征的神经系统疾病，部分与遗传起源相关，影响全球超过7000万人。尽管癫痫具有临床重要性，但中枢神经系统神经活动的功能分析仍有待开发。成像技术的最新进展，结合遗传编码钙指示剂（如GCaMP6）的稳定表达，在全脑和单细胞分辨率水平上彻底改变了癫痫的研究。黑腹果蝇因其复杂的分子遗传学和行为分析而成为研究癫痫分子和细胞机制的工具。在这项研究中，我们提出了一种新颖有效的方案，用于在表达 GCaMP6 的成年果蝇中进行离体钙成像以监测癫痫样活动。整个大脑由众所周知的癫痫基因 cac 制备，敲低苍蝇用共聚焦显微镜进行钙成像，以识别神经活动，作为爆炸敏感癫痫样行为测定的后续行动。与野生型苍蝇相比，cac敲除果蝇表现出更高的癫痫样行为和异常钙活性，包括更多的大尖峰和更少的小尖峰。钙活性与癫痫样行为相关。该方法在细胞水平上筛选癫痫致病基因和探索癫痫的潜在机制方面是一种有效的方法。

Introduction

癫痫是一种复杂的慢性神经系统疾病，其特征是自发性和无诱因性癫痫发作的复发以及神经元网络活动异常，已影响全球超过7000万人，使其成为最常见的神经系统疾病之一1，并导致家庭和社会的沉重负担。考虑到癫痫的影响，已经进行了许多研究以确定癫痫发作的病因，其中遗传学已被批准为许多类型的癫痫或癫痫综合征的主要原因2。在过去的几十年里，基因组技术的进步导致新的癫痫相关基因的发现迅速增加，这些基因在癫痫发作中起着至关重要的作用，包括离子通道和非离子通道基因3,4。然而，基因和癫痫表型之间的潜在机制和功能分析尚不完全清楚。鉴定癫痫相关基因和机制为有效管理患者提供了可能性5,6。

胞质钙信号是神经元活动和突触传递的关键元件。自 1970 年代以来，钙成像（包括脑切片7、体内8、9 和 ex 体内10）已被用于监测神经元活动11 作为神经元兴奋性的标志物12,13。成像技术的最新进展，结合基因编码的钙指示剂（GECI），如GCaMP6，在全脑和单细胞分辨率水平14,15,16上彻底改变了癫痫的研究，具有高水平的时空精度。分别在动作电位和突触传递中观察到钙浓度和瞬态的变化14，表明细胞内钙水平的改变与神经元的电兴奋性表现出严格的相关性17,18。钙成像也已被用作发育性癫痫发作模型9，并在果蝇中用于筛选抗惊厥化合物19。

黑腹果蝇因其复杂的分子遗传学和行为测定而成为科学研究中强大的模式生物，例如癫痫20,21,22。此外，果蝇中先进的遗传工具有助于遗传编码的钙指示剂GCaMP6的表达。例如，基于 Gal4 和 UAS 的二元转录系统能够以空间和时间控制的方式特异性表达 GCaMP6。由于果蝇是一种微小的生物，体内钙成像需要熟练的操作技能来执行手术干预，其中只有一小部分大脑背侧通过小窗口暴露14,23。同时，果蝇完整大脑中的离体钙成像可用于监测整个大脑的感兴趣区域（ROI）。

在这项研究中，我们在表达 GCaMP6 的成年果蝇中进行了离体钙成像，以监测癫痫样活动。CACNA1A是众所周知的癫痫基因，cac属于Cav2通道，是CACNA1A的同源物。我们首先解剖了cac敲低果蝇tub-Gal4>GCaMP6m/cac-RNAi的大脑，并使用具有xyt扫描模式的共聚焦显微镜对它们进行成像。然后，我们通过计算量化自发性癫痫样事件的指标来分析ROIs的钙信号变化，例如GCaMP6荧光的%ΔF/F值和钙事件。此外，我们通过涡旋机进行机械刺激，以诱导对 cac 敲除苍蝇的癫痫发作行为测试，以验证钙成像的结果。总体而言，该协议为通过离体钙成像研究成年果蝇的发作事件提供了有价值的工具，从而可以在细胞水平上探索癫痫的潜在机制。

Protocol

1. 爆炸敏感测定方案 通过Gal4/UAS系统21将tub-Gal4驱动线与UAS-cac-RNAi线交叉，建立实验果蝇。收集tub-Gal4系的处女果蝇和UAS-cac-RNAi系的雄性果蝇。然后，将处女苍蝇和雄性苍蝇转移到同一个小瓶中以收获后代。注意： tub-Gal4 驱动系将允许实现 cac 基因的全局敲低。使用 UAS-cac-RNAi 系作为对照组。 闭合3-5天?…

Representative Results

使用该方案，我们发现 cac 敲低果蝇表现出明显高于WT果蝇的癫痫样行为率（17.00±2.99 [n = 6] vs 4.50 ± 2.03 [n = 6]; P = 0.0061;学生的 t 检验， 图 1A）。大多数tub-Gal4>UAS-cac-RNAi 果蝇在1-5秒内恢复，而 UAS-cac-RNAi 果蝇在2秒内恢复。1 s内 cac 敲除果蝇的回收率显著低于WT果蝇（88.08±1.89 [n = 6] vs 96.50 ± 1.82 [n = 6]）; P = 0.009…

Discussion

钙离子是关键的第二信使，在对化学和电扰动的一系列生理和病理生理反应中起着关键作用。此外，由人CACNA1A基因编码的突触前 P/Q 通道的拓扑元件已被确定为负责介导各种神经递质（包括谷氨酸30,31,32）的放电，并且与癫痫密切相关 33,34。以前，Cac 敲除果蝇的行为没?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了广东省基础与应用基础研究基金会（批准号：2022A15151111123）的支持，并计划加强GMU（京大桥）的科学研究。这项工作还得到了广州医科大学学生创新能力项目（资助编号：02-408-2304-02038XM）的支持。

Materials

Brushes Panera AAhc022-2 for handling flies
Calcium chloride (CaCl2) Sigma-Aldrich C4901
Confocal microscope SP8; Zeiss, Jena, Germany. N/A for calcium imaging
CO2 anesthesia machine N/A N/A for Anesthetizing the flies.
C-sharp holder N/A N/A handmade, for mounting the brain
Culture vials Biologix 51-0500 2.5 cm diameter, 9.5 cm height
Fiji software National Institutes of Health, Bethesda, MD, USA version: 2.14.0 for analysis
Fly morgue N/A N/A handmade, for handling flies
Fly stocks cac-RNAi 27244 from Bloomington Drosophila Stock Center
Fly stocks GCaMP6m 42750 from Bloomington Drosophila Stock Center
Fly stocks tub-Gal4 N/A from the Sion-Frech Hoffmann Institute, Guangzhou Medical University
Glucose Sigma-Aldrich G8270
High-resolution camera N/A N/A for recording the seizure-like behavior assay
L-lysine Sigma-Aldrich L5626
Magnesium chloride solution (MgCl2) Sigma-Aldrich M1028
Papain suspension Worthington Biochemical LS003126
Petri dishes Sigma-Aldrich SLW1480/02D for dissection
Pipette Thermo Scientific 4640010, 4640030, 4640050, 4640060 for transporting a measured volume of liquid and diseccected brain
Potassium chloride (KCl) Sigma-Aldrich P4504
Recording dish Thermo Scientific 150682- Glass Based Dish for holding the brain and calcium imaging
Sodium bicarbonate (NaHCO3) Sigma-Aldrich S5761
Sodium chloride (NaCl) Sigma-Aldrich S5886
Sodium hydroxide (NaOH) Fisher Scientific S25550
Sodium phosphate monobasic (NaH2PO4) Sigma-Aldrich S8282
Stereo-binocular microscope SHANG GUANG XTZ-D for handling flies and dissection
Syringe needles pythonbio HCL0693 for dissection
Tripod WEIFENG 45634732523 for recording the seizure-like behavior assay
Vortex mixer Lab dancer, IKA, Germany/Sigma-Aldrich Z653438 for performing the seizure-like behavior assay
Whiteboard N/A N/A handmade, foam pad or paper for background
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Tags

Keywords: Ex VivoCalcium ImagingDrosophilaEpilepsyNeural ActivityGCaMP6CacSeizure-like BehaviorImaging Technique
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He, M., Liu, C., Zhang, X., Lin, Y., Mao, Y., Qiao, J. Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy. J. Vis. Exp. (200), e65825, doi:10.3791/65825 (2023).
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