Swell application reveals and quantifies wavefront propagation of halorhodopsin-induced spreading depolarization

Abstract Background Spreading depolarization (SD) is the cellular correlate of migraine aura and often occurs during brain injury and seizures. SD can be experimentally induced by multiple methods, including elevated extracellular K + and emerging optogenetic paradigms employing either channelrhodopsin or halorhodopsin (HaloR). Interestingly, HaloR-induced SD can arise while neurons are hyperpolarized and extracellular K + is below baseline, conditions opposite to conventional induction; however, consistent propagation of these events has not been established. Intrinsic optical signal (IOS) imaging has long been used to track SD propagation, but no dedicated user-friendly open-source tools exist for SD event characterization validated across traditional and optogenetic induction. Methods Here, we present Swell, a desktop application for identifying and quantifying SD-associated IOS. Experiments used ex vivo brain slices from male and female C57BL/6 and HaloR × EMXCre mice. Swell was first validated with SD events induced by elevated extracellular K + , comparing its output with electrophysiology data and manual SD segmentation. Swell was then used to characterize HaloR-induced SD, and event metrics were compared to those of elevated extracellular K + -induced events. Analyses used nonparametric methods with percentile cluster-bootstrap confidence intervals. Results Swell-derived IOS arrival was coincident with direct current electrophysiology deflection (bias − 0.2 s, 10/10 events within 3 s); its generated masks matched those produced by trained human annotators (Dice 0.92 vs. inter-annotator 0.92); and exported propagation speed fell within published values. Swell was used to demonstrate that HaloR-induced SD events propagate beyond the induction site both during and following illumination, at speeds similar to those triggered by elevated extracellular K + . Conclusions Swell is a validated open-source desktop application for quantifying SD. Applied across paradigms, Swell revealed that SD initiated during HaloR illumination propagate at speeds that do not detectably differ from those induced by elevated extracellular K⁺, demonstrating that HaloR-induced SD propagate consistently following their induction and that optogenetically triggered SD can be reliably tracked with IOS. These findings further validate HaloR as an SD induction model whose unique extracellular conditions offer promising potential to decouple the mechanisms of SD induction from propagation.