Prominent involvement of acetylcholine dynamics in stable olfactory representation across the Drosophila brain

Despite the vital role of neuromodulators and neurotransmitters in the neural system, their spatiotemporal correlation with neuronal activities across multiple brain regions remain unclear. Here, we employed two-photon synthetic aperture microscopy (2pSAM) and neurochemical indicators to simultaneously record calcium and acetylcholine (ACh)/5-HT dynamics across multiple regions of the Drosophila brain over 2 h. Presenting 3 different odors across multiple trials, our analyses revealed signal-specific differences in responsiveness, functional connectivity, and odor classification accuracy across the brain. We further constructed low-dimensional manifolds to characterize the global odor-related dynamics. Incorporating both calcium and ACh signals enhanced odor classification accuracy in the global low-dimensional manifold and in specific brain regions where their functional connectivity network features exhibited complementary patterns. Moreover, ACh dynamics demonstrated relatively stable temporal characteristics compared to calcium and 5-HT. These results suggest the potential contribution of ACh to consistent odor representations and illustrate the utility of multi-signal imaging in studying neural computation.