One-to-six day old individual female fly brains were dissected in ice-cold saline (in mM NaCl 124, KCl 3, MOPS 20, CaCl2 1.5, MgCl2(H2O)6 4, NaHCO3 5, NaH2PO4(H2O) 1, trehalose 10, sucrose 7, glucose 10). Brains were subsequently transferred and stuck at the bottom of a petri dish filled with saline. Individual MBn cell body was identified using the 488 nm laser of a confocal microscope (Leica). A circular 1-by-1 μm region of interest was defined at the center of a MBn and C3PA-GFP activated using a multi-photon laser (Coherent Inc.) set at 710 nm. The photoactivation stimuli consisted of three stimuli (0.325 ms each) separated by 2 min and for 1 h (90 stimuli total) with a 40x objective. The photoactivation laser power was typically between 4 and 40 mW at the objective. Brains were later flipped and axonal MBn projections imaged using the 63 × objective with the confocal set at 488 nm.
Neuronal projections were manually traced using the Neurolucida software (MBF Bioscience). Total projections length, bifurcations, neuronal projection volume and length of the segments were computed using the ‘Convex Hull Analysis’ and ‘Branched Structure Analysis’ functions of the Neurolucida explorer software (MBF bioscience). α′β′ and γ neurons were discarded as the neurons with no C3PA-GFP diffusion in the calyx or in the lobes or with major gaps or unresolvable structures.
Neuronal projections were manually traced using the Neurolucida software (MBF Bioscience). Total projections length, bifurcations, neuronal projection volume and length of the segments were computed using the ‘Convex Hull Analysis’ and ‘Branched Structure Analysis’ functions of the Neurolucida explorer software (MBF bioscience). α′β′ and γ neurons were discarded as the neurons with no C3PA-GFP diffusion in the calyx or in the lobes or with major gaps or unresolvable structures.