Blinded Electrophysiology of Drosophila Motoneurons

The experimenter was blinded to genotype during both recordings and subsequent data analysis. L3 larvae were dissected under extracellular saline as described^{59 (link)} with the only modification being a red filter applied to both the dissecting light and compound microscope to minimize DmCRY degradation before and during recordings. Thick-walled borosilicate glass electrodes (GC100F-10; Harvard Apparatus) were fire-polished to resistances of 10–15 MΩ. Recordings were made using the Multiclamp 700B amplifier controlled by pCLAMP (v.10.4) and the Digidata 1440A analogue-to-digital converter (Molecular Devices). Only cells with an input resistance of ≥500 MΩ were used. Traces were filtered at 10 kHz and sampled at 20 kHz. The extracellular saline solution contained the following: 135 mM NaCl, 5 mM KCl, 4 mM MgCl₂.6H₂O, 2 mM CaCl₂.2H₂O, 5 mM TES and 36 mM sucrose, pH 7.15. The intracellular patch solution contained the following (in mM): 140 mM potassium-d-gluconate, 2 mM MgCl₂.6H₂O, 2 mM EGTA, 5 mM KCl and 20 mM HEPES, pH 7.4. KCl and CaCl₂ were from Thermo Fisher Scientific; sucrose was from BDH; all of the remaining chemicals were from Sigma-Aldrich. Mecamylamine (1 mM) was applied to all preparations to isolate the aCC motoneurons from excitatory cholinergic synaptic input. For recordings supplemented with additional FAD or riboflavin (Sigma-Aldrich), dilutions were made up in intracellular saline and kept in the dark.