Reviewer #1 (Public review): In vivo autofluorescence lifetime imaging of spatial metabolic heterogeneities and learning-induced changes in the Drosophila mushroom body

This valuable study uses NAD(P)H fluorescence lifetime imaging (FLIM) to map metabolic states in the Drosophila brain.The authors reveal subtype-specific metabolic profiles in Kenyon cells and report learning-related changes, supported by solid evidence and careful methodology.However, the FLIM shifts observed after memory formation in α/β neurons are small and only weakly significant, so the ability of FLIM to detect subtle physiological changes still requires further validation.Nevertheless, this work provides a strong starting point and demonstrates the promising potential of FLIM for probing neural metabolism in vivo.