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

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.