CARBACHOL INDUCES A TRANSITION FROM SLEEP- TO WAKE-LIKE STATES IN HUMAN WHOLE-BRAIN ORGANOIDS​

Sleep is a fundamental physiological process, yet sleep research has largely relied on animal models, with findings often showing limited translational relevance to the clinic. Whole-brain organoids are three-dimensional constructs derived from human stem cells that recapitulate key structural and functional features of the human brain. It has been already shown that organoids exhibit spontaneous sleep-like electrophysiological activity. The aim of this study was to induce the transition from this intrinsic sleep-like state to a complementary wake-like state through pharmacological intervention. More in detail, we exploited carbachol, a cholinergic agonist known to promote awake-like activity in vitro, together with a custom-developed experimental and analytical pipeline, to induce reproducible changes in network dynamics in whole-brain organoids. Specifically, treatments resulted in quantitative alterations of electrophysiological parameters, including an increase in mean firing rate and a reduction in delta-band energy, consistent with a shift toward an wake-like state observed in vivo. These changes demonstrate that the baseline electrophysiological behavior of whole-brain organoids, which reflects sleep-like features, can be pharmacologically modulated to resemble the wake counterpart. In conclusion, our results establish whole-brain organoids as a versatile in vitro platform for modeling sleep–wake states. In the future, the integration of organoid technology with controlled pharmacological modulation may enable the simulation of full sleep–wake cycles and a more exhaustive modelling of the human wake features, providing a powerful tool to investigate sleep physiology and sleep disorders at the patient-specific level.