Department of Biomedical Engineering, Washington University in St. Louis.
Department of Biomedical Engineering, Washington University in St. Louis; Department of Computer Science and Engineering, Washington University in St. Louis.
J Vis Exp. 2022 Aug 25(186). doi: 10.3791/63939.
Using Drosophila melanogaster (fruit fly) as a model organism has ensured significant progress in many areas of biological science, from cellular organization and genomic investigations to behavioral studies. Due to the accumulated scientific knowledge, in recent years, Drosophila was brought to the field of modeling human diseases, including heart disorders. The presented work describes the experimental system for monitoring and manipulating the heart function in the context of a whole live organism using red light (617 nm) and without invasive procedures. Control over the heart was achieved using optogenetic tools. Optogenetics combines the expression of light-sensitive transgenic opsins and their optical activation to regulate the biological tissue of interest. In this work, a custom integrated optical coherence tomography (OCT) imaging and optogenetic stimulation system was used to visualize and modulate the functioning D. melanogaster heart at the 3rd instar larval and early pupal developmental stages. The UAS/GAL4 dual genetic system was employed to express halorhodopsin (eNpHR2.0) and red-shifted channelrhodopsin (ReaChR), specifically in the fly heart. Details on preparing D. melanogaster for live OCT imaging and optogenetic pacing are provided. A lab-developed integration software processed the imaging data to create visual presentations and quantitative characteristics of Drosophila heart function. The results demonstrate the feasibility of initiating cardiac arrest and bradycardia caused by eNpHR2.0 activation and performing heart pacing upon ReaChR activation.
利用黑腹果蝇（果蝇）作为模式生物，确保了在从细胞组织和基因组研究到行为研究的许多生物学科学领域的重大进展。由于科学知识的积累，近年来，果蝇被引入到人类疾病建模领域，包括心脏疾病。本工作描述了一种实验系统，用于在整个活体生物的背景下使用红光（617nm）监测和操纵心脏功能，而无需进行侵入性操作。通过光遗传学工具实现对心脏的控制。光遗传学将光敏感转基因 opsin 的表达与其光学激活相结合，以调节感兴趣的生物组织。在这项工作中，使用定制的集成光学相干断层扫描（OCT）成像和光遗传学刺激系统，可视化和调节 3 龄幼虫和早期蛹发育阶段的 D. melanogaster 心脏的功能。使用 UAS/GAL4 双重遗传系统表达 halorhodopsin（eNpHR2.0）和红移通道蛋白（ReaChR），特别是在果蝇心脏中。提供了用于活体 OCT 成像和光遗传学起搏的 D. melanogaster 准备的详细信息。一个实验室开发的集成软件处理成像数据，以创建果蝇心脏功能的视觉表示和定量特征。结果表明，可以通过 eNpHR2.0 的激活来引发心脏骤停和心动过缓，并在 ReaChR 的激活下进行心脏起搏。
