Integrated single-cell atlases unveil the operation principles of whole-brain 5-HT neuronal subsystems

Investigating 5-hydroxytryptamine (5-HT) neuronal subsystems and the operation principles is crucial for elucidating their complex roles in the regulation of various physiological and behavioral outcomes. Until now, these efforts faced methodological and analytical hurdles. Here, we developed resource datasets in zebrafish that include a single-cell projectomic dataset, two independent scRNA-seq datasets, and a whole-brain 5-HT neuron functional imaging database at cellular resolution. The integrated analysis uncovered seven 5-HT subpopulations, each with unique axon projection patterns, specific soma locations, and distinctive genetic profiles. Within each subsystem, 5-HT neurons were functionally homogeneous, whereas across different subsystems, they were functionally heterogeneous. We further demonstrate that each 5-HT subsystem functions as a distinct module playing a role in multiple tasks, rather than being exclusive to one function. Moreover, the involvement of multiple parallel 5-HT subsystems, rather than a single subsystem, is fundamental to the accomplishment of a specific task. These results provide insights into the operation of 5-HT neuronal system in mammals.