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bioRxiv · 10.1101/2025.10.09.681455

Whole brain fluorescence imaging in Drosophila reveals spreading depression and its initiation, propagation, and resilience dynamics.

Harris, A. L.·Crowe, G.·Nelson, E.·Al Mokbil, M.·Stein, W.
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Abstract

Spreading depression (SD) is a wave of neuronal hyperactivity followed by depolarization block that propagates through large brain regions and is associated with disorders such as migraine, stroke, and brain injury. The mechanisms that initiate SD and alter susceptibility to it remain incompletely understood. Here, we use whole-brain fluorescence imaging with genetically encoded pan-neuronal calcium and voltage sensors to observe SD in Drosophila melanogaster. We show that rapid cooling, a naturally occurring environmental condition, as well as elevated extracellular potassium reliably elicit SD in both adult and larval flies. SD was characterized by a rapid and large rise in intracellular calcium that was accompanied by neuronal depolarization and stark changes in the transperineuronal potential. In adults, SD occurred at 6.7 {+/-} 0.6{degrees}C (N=15) and in larvae at 6.0 {+/-} 0.3{degrees}C (N=30). SD initiation was not restricted to specific sites, but initiated at multiple, variable sites across and within individuals, with an average of 3.0 {+/-} 0.7 (N=8) initiation points per brain. In all cases, SD spread throughout large areas of the nervous system. In a high-throughput larval assays that allows the simultaneous monitoring of up to 16 animals with repeated cooling cycles, we demonstrate that single SD events are followed by a transient refractory period lasting up to 45 minutes, during which the threshold for subsequent SD was significantly elevated. This was the case in adult and larval brains of all developmental stages. The refractory effect was independent of neuronal depolarization, suggesting that homeostatic processes alter SD susceptibility following an initial SD event. Taken together, our findings demonstrate that SD initiation and propagation are not restricted to specific regions, neuronal populations, or developmental stages, and they reveal fundamental properties of adaptive changes to SD susceptibility in a genetically tractable model. Building upon the extensive genetic toolkit available in Drosophila, this work establishes the fly as a complementary model for understanding conserved cellular and circuit-level mechanisms of SD relevant to human neurological disorders.

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Harris, A. L., Crowe, G., Nelson, E., Al Mokbil, M., Stein, W.. 2025-10-10. Whole brain fluorescence imaging in Drosophila reveals spreading depression and its initiation, propagation, and resilience dynamics.. https://doi.org/10.1101/2025.10.09.681455

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