Probing pathways by which rhynchophylline modifies sleep using spatial transcriptomics

Rhynchophylline (RHY) is an alkaloid component of Uncaria, which are plants extensively used in traditional Asian medicines. Uncaria treatments increase sleep time and quality in humans, and RHY induces sleep in rats. However, like many traditional natural treatments, the mechanisms of action of RHY and Uncaria remain evasive. Moreover, it is unknown whether RHY modifies key brain oscillations during sleep. We thus aimed at defining the effects of RHY on sleep architecture and oscillations throughout a 24-h cycle, as well as identifying the underlying molecular mechanisms. Mice received systemic RHY injections at two times of the day (beginning and end of the light period), and vigilance states were studied by electrocorticographic recordings. RHY enhanced slow wave sleep (SWS) after both injections, suppressed paradoxical sleep (PS) in the light but enhanced PS in the dark period. Furthermore, RHY modified brain oscillations during both wakefulness and SWS (including delta activity dynamics) in a time-dependent manner. Interestingly, most effects were larger in females. A brain spatial transcriptomic analysis showed that RHY modifies the expression of genes linked to cell movement, apoptosis/necrosis, and transcription/translation in a brain region-independent manner, and changes those linked to sleep regulation (e.g., Hcrt, Pmch) in a brain region-specific manner (e.g., in the hypothalamus). The findings provide support to the sleep-inducing effect of RHY, expose the relevance to shape wake/sleep oscillations, and highlight its effects on the transcriptome with a high spatial resolution. The exposed molecular mechanisms underlying the effect of a natural compound should benefit sleep- and brain-related medicine.