Robinson_2025_bioRxiv__

In Drosophila melanogaster , the autism-related Neuroligin 3 (Nlg3) protein is a postsynaptic membrane protein important for synapse development and regulation, which plays a role in social spacing behaviour. Here, we report the localization of Nlg3 to the calyx of the mushroom bodies (MB), optic lobes (OL), and protocerebral bridge (PB). Using RNA interference, nlg3 knockdown in the OL increased fly social space, especially in the males, while knocking down nlg3 independently in the MB or the PB did not change social spacing. However, hyperactivation and silencing of these neurons in the MB, but not the PB, controls social space in males and females, while hyperactivating and silencing of all nlg3 -expressing neurons, including within the MB, PB, and OL, regulates male and female social space. Knocking down neurotransmitter biosynthesis enzymes, which decreases the amount of neurotransmitter release, showed that reducing acetylcholine release from the MB decreased female social space, whereas knocking down any dopamine receptor in the MB increased male social space. Lastly, to investigate the sexually dimorphic effects on social spacing previously seen in nlg3 mutants, we examined a subset of sexually dimorphic fruitless- expressing ( fru)P1 neurons known to regulate sexually dimorphic behaviours. Hyperactivation of those fruP1 neurons decreased social space in both sexes, while silencing those fruP1 neurons specifically increased male social space without affecting females. Our findings highlight a sex-specific social space neural circuitry that includes the OL, MB, and fruP1 neurons, while uncovering the underlying basis of some of the sex differences in this behaviour. ARTICLE SUMMARY: In vinegar flies ( Drosophila melanogaster ), the autism-related Neuroligin 3 protein (Nlg3) controls neuronal development and regulation but also affects fly social behaviour. Nlg3 is localized to the mushroom bodies (MB), protocerebral bridge, and the optic lobes. Genetically reducing Nlg3 in the optic lobes made male flies more socially distant, while hyperactivating or silencing neurons in the MB affected social behavior in both sexes. Reducing acetylcholine release from the MB affects female social space, while reducing dopamine receptors of the MB only affect male behaviour. Finally, the fruitless sexually dimorphic neurons control social behavior differently in males and females.