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0 reviewsDespite the vital role of neuromodulators and neurotransmitters in the neuralsystem, their spatiotemporal correlation with neuronal activities across multiplebrain regions remain unclear. Here, we employed two-photon synthetic aperturemicroscopy (2pSAM) and neurochemical indicators to simultaneously recordcalcium and acetylcholine (ACh)/5-HT dynamics across multiple regions of theDrosophila brain over 2 h. Presenting 3 different odors across multiple trials, ouranalyses 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-relateddynamics. Incorporating both calcium and ACh signals enhanced odor classification accuracy in the global low-dimensional manifold and in specific brainregions where their functional connectivity network features exhibited complementary patterns. Moreover, ACh dynamics demonstrated relatively stabletemporal characteristics compared to calcium and 5-HT. These results suggestthe potential contribution of ACh to consistent odor representations and illustrate the utility of multi-signal imaging in studying neural computation.