Bonassi, G. G. | Search bioRxiv

bioRxiv2025

An optogenetic assay of Drosophila larval motor neuron performance in vivo

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.

neuroscience↗

bioRxiv2025

Optimal Neuromuscular Performance Requires Motor Neuron Phosphagen Kinases

Phosphagen systems are crucial for muscle bioenergetics - rapidly regenerating ATP to support the high metabolic demands of intense musculoskeletal activity. However, their roles in motor neurons that drive muscle contraction have received little attention. Here, we knocked down expression of the primary phosphagen kinase [Arginine Kinase 1; ArgK1] in Drosophila larval motor neurons and assessed the impact on presynaptic energy metabolism and neurotransmission in situ. Fluorescent metabolic probes showed a deficit in presynaptic energy metabolism and some glycolytic compensation. Glycolytic compensation was revealed through a faster elevation in lactate at high firing frequencies, and the accumulation of pyruvate subsequent to firing. Our performance assays included two tests of endurance: enforced cycles of presynaptic calcium pumping, and, separately, enforced body-wall contractions for extended periods. Neither test of endurance revealed deficits when ArgK1 was knocked down. The only performance deficits were detected at firing frequencies that approached, or exceeded, twice the firing frequencies recorded during fictive locomotion, where both electrophysiology and SynaptopHluorin imaging showed an inability to sustain neurotransmitter release. Our computational modeling of presynaptic bioenergetics indicates that the phosphagen systems contribution to motor neuron performance is likely through the removal of ADP in microdomains close to sites of ATP hydrolysis, rather than the provision of a deeper reservoir of ATP. Taken together, these data demonstrate that, as in muscle fibers, motor neurons rely on phosphagen systems during activity that imposes intense energetic demands.

neuroscience↗