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bioRxiv · 10.1101/2025.08.18.670936

An optogenetic assay of Drosophila larval motor neuron performance in vivo

Arab, Y.·Bonassi, G. G.·Macleod, G. T.
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Abstract

BackgroundOver fifty million people worldwide currently live with neurodegenerative diseases, many of which are the result of pathogenic gene variants. Genetically malleable model organisms provide an avenue for research into the genetic bases of these diseases, and the large motor neurons of fruit fly larvae provide a test bed for investigating neuronal mechanisms impacted by pathogenic gene variants. However, it is challenging to collect information from these neurons under physiological conditions as they terminate on muscle fibers that are perpetually contracting - driven by motor neuron burst-firing. New MethodAs a test of in vivo neuronal performance, we expressed light-activated opsins in motor-neurons of unrestrained intact Drosophila larva and used light pulses to drive cyclical body-wall contractions that were captured on camera and analyzed offline. ResultsWe describe the assembly of an apparatus to systematically activate motor-neurons in Drosophila larvae and an image acquisition system to capture the resulting body-wall contractions. To test the utility of the assay we performed a motor-neuron specific knock-down of dMiro, an adaptor for mitochondrial transport into motor-neuron terminals. As predicted, contractions were poorly sustained in larvae with impaired axonal transport of mitochondria. Comparison with Existing MethodsThis in vivo assay allows for a test of sustained neuronal performance while sidestepping the shortcomings of electrophysiological assays of neurotransmission in situ where hemolymph-like solutions may not recapitulate hemolymph properties, axons are severed and where recordings are mechanically disrupted at endogenous firing rates. Secondly, unlike adult climbing assays and larval locomotion assays, performance is assayed independently of the organisms motivation to perform or ability to detect stimuli. ConclusionsWe demonstrated that Drosophila 3rd instar larvae cannot sustain body-wall contractions if mitochondria are not delivered to motor nerve terminals - validating a motor neuron performance assay in a model organism suited for molecular genetic analysis.

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Arab, Y., Bonassi, G. G., Macleod, G. T.. 2025-08-21. An optogenetic assay of Drosophila larval motor neuron performance in vivo. https://doi.org/10.1101/2025.08.18.670936

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