The Development and Use of Elastic Resonators and Optogenetics to Study Locomotion in Small Soft-bodied Animals

弹性谐振器和光遗传学的开发和使用来研究小型软体动物的运动

• 批准号: 2093560
• 金额: --
• 依托单位: University of St Andrews
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2093560
• 关键词: Development; Use; Elastic; Resonators; Optogenetics

Terrestrial animals with hydrostatic skeletons solve complex motor problems by orchestrating how their soft bodies compress and deform in order to interact with the substrate. Previous studies have made progress in understanding how these animals perform this action flexibly over a wide variety of substrates by researching large animals such as Manduca sexta caterpillars. However, progress has been limited by the insufficient tools to simultaneously record the animal's behaviour from a neuromuscular, behavioural and actuation through forces perspective with high spatiotemporal resolution. Recently, a technique, elastic resonator stress interference microscopy (ERISM), was developed in order to allow for the imaging of migratory forces on a cellular level using the interference of light in a deformable elastic cavity. This technique uses an elastomer between two gold mirrors, the length of which determines the resonant light wavelengths within this cavity. As cells produce forces the cavity deforms, thereby decreasing the overall length, thereby shifting the local resonant wavelengths. Thus, for each given cavity length, the pushing and pulling forces on the substrate can be determined by examining this shift in local resonance. Until recently, this technique did not have the requisite time resolution for resolving beyond the 0.5 Hz region, however, this technique was developed to make use of fewer wavelengths in order to allow for imaging of forces within the 10 Hz timeframe - although this technique is still limited to recording cellular forces. This project aims to develop ERISM to allow for recording forces from small animals by making use of an elastomer with a greater elasticity modulus (20-60 kPa) and to combine it with epifluorescence microscopy to allow for simultaneous force measurement and fluorescence activity measurement. Developing this technique will allow us to observe the forces produced by soft bodied animals. One such animal, Drosophila melanogaster larvae, has a history of almost unrivalled genetic control through use of the Gal-4 UAS system. This animal will allow us to study the simultaneous contraction of muscles via genetic tools such as expressing fluorescent proteins in each of its muscles or by expressing genetically encoded calcium indicators (GECIs) directly in its nervous system to observe neuromuscular activity. This genetic tractability also allows us to express ionophores directly in the specific cells within the nervous system - allowing for optogenetic control of specific aspects of the animal's locomotor behaviour. These larvae also grow within an interesting scale, growing from the size of the largest of cells to the size of the smallest of animals over the course of mere days. This means they have to solve both micro and macro domain problems using the same body. Thus, this study aims to develop two-wavelength ERISM to allow for the utilisation of the full genetic toolkit of Drosophila melanogaster and will be important in understanding both ordinary locomotor behaviour of soft-bodied animals, but also may offer insights into the behavioural phenotypes of Drosophila melanogaster disease models - allowing for a dynamic study of impaired neuromuscular actuation found in Parkinson's models.

具有流体静力骨架的陆生动物通过协调它们柔软的身体如何压缩和变形以与基底相互作用来解决复杂的运动问题。以前的研究已经取得了进展，了解这些动物如何执行这一行动灵活地在各种各样的基板通过研究大型动物，如天蛾毛虫。然而，进展一直受到限制的工具，同时记录动物的行为，从神经肌肉，行为和驱动力的角度与高时空分辨率。最近，一种技术，弹性谐振器应力干涉显微镜（ERISM），被开发，以允许使用光在可变形的弹性腔中的干涉在细胞水平上的迁移力的成像。这种技术在两个金镜之间使用弹性体，其长度决定了该腔内的共振光波长。当细胞产生力时，腔体变形，从而减小总长度，从而使局部谐振波长偏移。因此，对于每个给定的腔长度，可以通过检查局部谐振的这种偏移来确定衬底上的推力和拉力。直到最近，这种技术还没有必要的时间分辨率来分辨超过0.5 Hz的区域，然而，这种技术的发展是为了利用更少的波长，以允许在10 Hz的时间范围内的力的成像-尽管这种技术仍然限于记录细胞力。本项目旨在开发ERISM，以便通过使用具有更大弹性模量（20-60 kPa）的弹性体记录来自小动物的力，并将其与落射荧光显微镜联合收割机结合，以便同时进行力测量和荧光活性测量。发展这项技术将使我们能够观察软体动物产生的力。一种这样的动物，果蝇幼虫，通过使用Gal-4 UAS系统具有几乎无与伦比的遗传控制历史。这种动物将使我们能够通过遗传工具研究肌肉的同时收缩，例如在其每块肌肉中表达荧光蛋白，或者通过直接在其神经系统中表达遗传编码的钙指示剂（GECIs）来观察神经肌肉活动。这种遗传的易处理性也使我们能够直接在神经系统内的特定细胞中表达离子载体-允许对动物运动行为的特定方面进行光遗传学控制。这些幼虫也在一个有趣的规模内生长，从最大的细胞大小到最小的动物大小，仅需几天。这意味着他们必须使用同一个机构来解决微观和宏观领域的问题。因此，本研究旨在开发双波长ERISM，以允许利用果蝇的完整遗传工具包，并且对于理解软体动物的普通运动行为非常重要，而且还可以提供对果蝇疾病模型的行为表型的见解-允许动态研究帕金森模型中发现的神经肌肉驱动受损。