Robinson_2026_Genetics__

In Drosophila melanogaster, Neuroligin 3 (Nlg3) is a postsynaptic membrane protein important for synapse development and social spacing behaviour, and its human orthologs are associated with autism. We localized Nlg3 to three brain structures: the mushroom bodies (MB) calyx, the optic lobes (OL), and the protocerebral bridge (PB). RNA knockdown of nlg3 in each structure independently replicated the effect of knocking down in all nlg3-expressing neurons. In contrast, hyperactivating and silencing all nlg3-expressing neurons, including those within the MB, PB, and OL, affected social spacing by sex: females were further apart after either manipulation, whereas males' social space was increased, only after hyperactivation. Decreasing acetylcholine release in the MB via choline acetyltransferase (ChAT)-RNAi caused decreased social space in both sexes. In contrast, RNAi targeting dopamine receptors in the MB increased social space mainly in males. Lastly, we also tested fruitless-expressing (fruP1) neurons, which regulate sexually dimorphic behaviours. Hyperactivating fruP1 neurons decreased social space in both sexes, while silencing increased social space only in males. These results highlight sex-specific neural substrates for social space and implicate the OL, MB, nlg3, and fruP1 neurons, in this behaviour.