Increased spindle-related brain activation in right middle temporal gyrus during N2 than N3 among healthy sleepers: Initial discovery and independent sample replication

•Spindle-related brain activation in the right middle temporal gyrus was significantly increased during N2 compared with N3.•Enhanced right middle temporal spindle-related brain activation in N2 negatively correlated with N2 sleep duration.•Utilized simultaneous EEG-fMRI to compare spindle-related brain activation between sleep stage N2 and N3 in two independent samples. The association between spindle metrics and sleep architecture differs during N2 vs. N3 sleep, the underlying neural mechanism is not clearly illustrated. Here, we tested the discrepancy in spindle-related brain activation between N2 and N3 within healthy college students (dataset 1: n = 27, 59 % females, median age 23 years), using simultaneous electroencephalography-functional magnetic resonance imaging (EEG-fMRI). To assess the replicability of the finding, we repeated the analysis among normal adults (independent dataset 2: n = 30, 50 % females, median age 32 years). The finding from dataset 1 indicated significantly increased blood-oxygen level-dependent signal in the right middle temporal gyrus during N2 compared with N3, which was well replicated in dataset 2. Furthermore, correlation analysis was performed to explore the association between this spindle-related brain activation and N2, N3 sleep duration during EEG-fMRI. We conducted the correlation analysis in N2 and N3, respectively. The negative association between spindle-related brain activation in the right middle temporal gyrus and sleep duration was only observed in N2. Our findings emphasize the unique role of spindle-related brain activation in the right middle temporal gyrus during N2 in shortening N2 sleep duration.