We analyzed males for immunohistochemistry in our manipulations. For functional imaging experiments, we used mixed-sex populations, and did not observe any correlation with sex (not shown). The Y-arena behavior used females due to the size of the arena not being optimal for males. Therefore, the flies dissected post-behavior and used for calyx area quantification in those animals and Brp staining (Figures 1D, 2H, 6F) were also females. Sex differences in the fly are well-documented, including in our own previous work, and anatomic and physiologic sex differences have not been observed in the mushroom body (Brovkina et al., 2021 (link); Clowney et al., 2015 (link)). Any brains that appeared damaged from dissections, or those with the mushroom body region obscured due to insufficient tracheal removal, were not included in the analysis.
Researchers performing quantification could not generally be blinded to experimental condition due to the overt changes in neuron numbers and brain structures induced by our manipulations. However, analysis was performed blind to the goals of the experiment when possible, and quantitation of features on the anterior and posterior sides of the brain were recorded independent of one another and merged after all quantifications were completed. Moreover, many of our analyses make use of variation within an experimental condition or genotype, providing an additional bulwark against observational bias.