Experimental modulation of physiological force application on leg joint neurons in intact Drosophila melanogaster.

Abstract

The study of how mechanical forces affect biological events in living tissue is important for the understanding of a multitude of physiogical and pathophysiological phenomena. However, these investigations are often impeded by insufficient knowledge about force parameters, inadequate experimental administration of force stimuli and lack of noninvasive means to record their molecular and cellular effects. We therefore introduced a procedure to study the impact of force stimulation on adhesion G-protein-coupled receptor dissociation in mechanosensory neurons. Here, we detail a procedure to harness the mechanical force spectrum that emerges during the natural flexion-extension cycle of the femorotibial joint of adult fruit flies (Drosophila melanogaster). Mechanical load generated during the joint's motion is transmitted to specialized mechanosensory neurons residing close to the joint axis, which serve as proprioceptive sensors in the peripheral nervous system of the animal. Temporary immobilization of the joint by a restraint made of a human hair allows for the observation of transgenic mechanosensitive reporters by using fluorescent readout in the neurons before, during and after cessation of mechanical stimulation. The assay harnesses physiologically adequate stimuli for joint flexion and extension, can be conducted noninvasively in live specimens and is compatible with various transgenic reporter systems beyond the initially conceived strategy and mechanobiological hypotheses tested. The application of the protocol requires knowledge in Drosophila genetics, husbandry and fluorescence imaging and micromanipulation skills. The experimental procedure can be completed in 10 h and requires an additional 30 min in advance for fly fixation and leg immobilization. The apple agar cooking and heptane glue preparation requires a maximum of 30 min on the day before the experiment is conducted.