Impact of NPAS2 on mPFC dopamine synthesis and nap behavior

The biological basis of afternoon nap, a widespread yet poorly understood phenomenon, has remained elusive. Here we identify NPAS2, among core circadian regulators, as a sex-independent determinant of the nap behavior in mice. Specifically, medial prefrontal cortex (mPFC)-expressed NPAS2 orchestrates nap regulation through circadian modulation of local dopaminergic activity. We demonstrate that tyrosine hydroxylase-positive (TH+) neurons in mPFC exhibit time-of-day dependent wake-promoting activity, showing minimal excitation precisely during nap hours. Mechanistically, NPAS2 achieves this circadian suppression through a POU2F2-TH regulatory pathway: 1) transcriptional activation of the transcription repressor POU2F2, and 2) consequent downregulation of TH expression (a rate-limiting enzyme for dopamine synthesis) and dopamine production in mPFC TH+ neurons. These findings establish an endogenous circadian mechanism where mPFC NPAS2 periodically inhibits wake-promoting dopaminergic activity to drive nap behavior, providing fundamental insights into the neural and molecular regulation of nap biology. Overall design: To elucidate the molecular mechanisms by which mPFC NPAS2 regulates sleep-wake cycles and nap behavior, we performed a comparative single-nucleus transcriptomic analysis using Npas2 KO mice and wild-type littermate controls. Medial prefrontal cortex (mPFC) tissues were harvested from four mice per genotype at circadian time ZT20 (the peak of nap behavior) and pooled by genotype to ensure robust biological representation. Nuclei were isolated using the Shbio Cell Nuclear Isolation Kit (52009-10) in combination with NP-40 lysis buffer to obtain high-quality single-nucleus suspensions. Single-nucleus libraries were then constructed using the 10x Genomics Chromium Next GEM Single Cell 3' Reagent Kit v3.1, followed by high-depth sequencing on the Illumina NovaSeq 6000 platform with a paired-end 150 bp (PE150) configuration.