Detection and Quantification of Spreading Depolarization in Drosophila melanogaster Using High‐Throughput, Wide‐Field Brain Imaging

Abstract Spreading depolarization (SD) is a ubiquitous neural phenomenon implicated in mammalian neurological disorders including cerebral ischemia, brain death, stroke, brain injury, and migraine, as well as in insect chill coma. During SD, a breakdown of ion homeostasis and a slowly spreading wave of neural depolarization leads to processing deficits, such as sensory disturbances and paralysis. Our protocols introduce high‐throughput techniques using wide‐field fluorescence brain imaging in Drosophila melanogaster larvae and adults to detect and quantify the spatial and temporal dynamics of SD and its initiation points. Two SD initiation techniques, rapid cooling and pharmacological manipulation, are detailed. The larval protocols describe how to elicit, detect, and quantify SD in up to 16 animals simultaneously in either intact or dissected preparations. The adult SD protocols describe the detailed steps to elicit, detect, and quantify SD in either fully intact animals or after microdissection that enables high resolution imaging of the brain. Detection, quantification, and analysis techniques are described in a separate protocol. Our protocols establish the fly as a high‐throughput model for studying conserved cellular and circuit‐level mechanisms of SD initiation and propagation. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : High‐throughput SD detection in intact Drosophila larvae with fluorescent calcium reporters Alternate Protocol 1 : High‐resolution SD imaging in fillet larvae with fluorescent calcium reporters Support Protocol 1 : SD imaging in larvae with fluorescent membrane voltage reporters Basic Protocol 2 : High‐throughput SD detection in adult Drosophila with fluorescent calcium reporters Alternate Protocol 2 : High‐resolution SD imaging in adult Drosophila with fluorescent calcium reporters Support Protocol 2 : SD imaging in adults with fluorescent membrane voltage reporters Basic Protocol 3 : High‐throughput SD detection analysis in Drosophila with fluorescent calcium reporters Alternate Protocol 3 : High‐resolution SD imaging analysis in Drosophila with fluorescent calcium reporters Support Protocol 3 : SD detection and spread analysis in Drosophila with fluorescent membrane voltage reporters