Researchers have proposed that wakefulness generates sleep-promoting substances detectable in the cerebrospinal fluid (CSF). However, identities of these molecules, neural circuits that produce and sense them, and how the brain's key sleep-wake regulatory neurons participate in this process, remain mysterious.

In a study published in Nature Neuroscience, Dr. ZHANG Zhe's group from the Center for Excellence in Brain Science and Intelligence Technology of the Chinese Academy of Sciences identified tryptamine as an endogenous sleep-promoting molecule, and found that tryptamine activates GPR139 neurons in the preoptic area, bridging waking neural activity and sleep onset.

Through targeted mass spectrometry and liquid chromatography, researchers profiled CSF neurotransmitters and found that tryptamine was markedly elevated after sleep deprivation. In both nocturnal mice and diurnal miniature pigs, CSF tryptamine accumulated during wakefulness and declined after sleep, reflecting their recent physical activity rather than light-dark cycle. Intracerebroventricular infusion of tryptamine dose-dependently increased non-rapid eye movement sleep and enhanced slow-wave activity.

Researchers engineered a genetically encoded ratiometric fluorescent sensor to trace the source of tryptamine. In vivo fiber-photometry recordings revealed that wake-active monoaminergic neurons in locus coeruleus (LC), dorsal raphe (DR), and ventral tegmental area release tryptamine in an activity-dependent manner during wakefulness. Suppressing tryptamine synthesis in LC and DR eliminated the sleep rebound that normally follows sleep deprivation, demonstrating that tryptamine is a physiological signal linking waking activity to sleep pressure.

To identify the downstream effector, researchers performed single-cell RNA sequencing of hypothalamic preoptic area (POA) in sleep-deprived and recovery-sleep mice. Through covariance analysis of immediate-early genes, they pinpointed an inhibitory neuronal population highly expressing the orphan receptor GPR139.

Structural biology, molecular docking, and calcium-mobilization assays confirmed that tryptamine bound and activated GPR139, which suppressed G protein-gated inwardly rectifying potassium (GIRK) channels via the Gq/11 pathway to raise neuronal excitability. The excitability of POA GPR139-positive neurons was found to rise in proportion to sleep pressure.

Using Gpr139-Cre knock-in mice and CRISPR/Cas9-mediated local GPR139 knockout in the POA, researchers showed that this receptor is essential for tryptamine's sleep-promoting effects. Systemic administration of GPR139 agonists JNJ-63533054 and TAK-041 (the latter currently in clinical trials) significantly prolonged sleep duration and deepened sleep, which are entirely absent in POA-specific knockout mice. Human genetic analyses showed that polymorphisms in GPR139 and AADC genes are associated with an elevated risk of sleep disorders.

The study reveals that the "tryptamine-GPR139" pathway constitutes a molecular substrate of sleep pressure, and provides a new target for developing novel insomnia therapies.