Characterization of Seizure Induction Methods in Drosophila

Comparison of seizure induction methods in Drosophila adults and larvae. We tested BS pk-sple, eas, jus, and bss lines and heat-sensitive DS and GEFS+ lines. A, Vortex-induced seizures in pk-sple 15.22 s [95% CI: 11.37, 20.69], eas 107.54 s [95% CI: 88.71, 123.84], jus 73.35 s [95% CI: 64.60, 79.88], and bss 168.35 s [95CI: 157.31, 179.39]. No SLA was observed in CS, DS-C, DS, GEFS-C, and GEFS+ flies. B, Seizure duration induced by the heat assay. DS showed seizures of average 71.82 s and GEFS+ of 431.03 s, with no seizures recorded for the respective matched controls (DS-C and GEFS-C). pk-sple showed seizures of 136.67 s [95% CI: 87.42, 191.84]. By comparison, CS, OR, jus, bss, or eas did not show heat-induced seizures. C, Cumulative fraction of flies seizing throughout the 120 s of heat assay. Only 100% of DS flies seized during the 2-min period. Other genotypes reached a maximum of: 90% for GEFS+, 83% for pk-sple, and 20% for eas. D, Electroshock induced seizures in all genotypes tested. Results of OR, jus, eas, bss, and pk-sple are reported as a ratio to CS seizure duration which was measured at 97.0 s [95% CI: 95.4, 101.6]. There were no differences between OR and CS. Pk-sple exhibited the weakest phenotype with seizure duration increased by 57.47%, whereas bss showed an increase of 244.11%. eas and jus had 112.62% and 104.95% longer seizures. DS and GEFS+ seizure durations are reported as a ratio to their respective controls (DS-C and GEFS-C). DS showed 119.78% and GEFS+ 70.67% increase in seizures as compared with controls. E, Seizure induction in larva and adult flies have similar effectiveness (R² = 0.85 [95% CI: 0.03, 1.0], shaded area indicates 95% CI for the linear regression model fit). Effect sizes for adults were derived from vortex assay for BS and heat assay for temperature-sensitive lines.

Adult seizure mutants do not exhibit obvious changes in locomotor activity. A, Schematic showing the locomotor assay. B, During the 12-h period of lights on, jus (1298.14 mm/h), eas (724.40 mm/h), pk-sple (1148.98 mm/h), and GEFS+ (1007.61 mm/h) show reduced locomotor activity (compared with appropriate controls), with eas and GEFS+ having the most prominent phenotypes; slower by 1012.09 mm/h [95% CI: −1211.17, −818.82] and 654.49 mm/h [95% CI: −1017.36, −267.84], respectively. DS showed increased activity by 361 mm/h [95% CI: 154.58, 592.13] over its control (DS-C) with an average speed of 727.60 mm/h. C, During lights off, several mutants showed a small increase in activity levels. Of these, eas had the most prominent phenotype at 667.02 mm/h [95% CI: 493.03, 870.27]. jus and bss showed similar activity levels with an increase of 261.51 mm/h [95% CI: 128.67, 404.60] and 285.95 mm/h [95% CI: 138.50, 458.49], respectively. GEFS+ is the only genotype remaining slower than its respective control (GEFS-C) by 313.14 mm/h [95% CI: −489.98, −154.34]. Other genotypes showed no meaningful differences. Fly activity was tracked for 3 d at 25°C at 12/12 h light/dark cycle. All genotypes were recorded at least twice. D, Schematic showing the set up for larval tracking. E, Total distances crawled by BS mutants pk-sple (49.27 mm), eas (60.51 mm), jus (52.29 mm), and bss (32.43 mm) were greatly lower in comparison to CS (161.47 mm). Heat-sensitive mutants did not show any meaningful reduction in the total distance crawled from their matched controls.

Seizure mutants show elongated motor neuron bursting. A, B, Examples of cell-attached recordings from larval RP2 and aCC motor neuron bursting patterns in CS wild-type (A) and the bss seizure mutant (B). C, The fraction of elongated bursts (i.e., activity shown in B) observed in wild-type and the BS genotypes: CS, OR (0%), pk-sple (31.8%), eas (14.29%), jus (41.67%), and bss (15.38%). D, Fraction of elongated bursts recorded in heat-sensitive mutants and their respective controls: GEFS-C (16.67%), GEFS+ (41.67%), DS-C (50.00%), and DS (58.33%). E, Fraction of elongated bursts detected in CS larvae fed with either vehicle (–PTX) or PTX. Vehicle showed only normal bursting, but recordings from PTX-fed larvae showed 83.33% elongated bursts. F, PTX induces larval seizures with recovery time 86% longer in the experimental group than control.