A high-performance fluorescent sensor spatiotemporally reveals cell-type specific regulation of intracellular adenosine in vivo

Adenosine (Ado), a nucleoside bridging intracellular metabolism with intercellular communication, plays an essential role in regulating processes such as sleep and seizure. While the functions of extracellular Ado ("eAdo") are well documented, our knowledge about the distribution and regulatory functions of intracellular Ado ("iAdo") is limited by a lack of methods for detecting iAdo in vivo. Here, we develop HypnoS, a genetically encoded fluorescent sensor for iAdo characterized by its high sensitivity, specificity, spatiotemporal resolution, and rapid response (sub-seconds). HypnoS enables real-time visualization of iAdo dynamics in live cultures, acute brain slices, flies, and freely moving mice. Using HypnoS for dual-color mesoscopic imaging in mice, we show that seizure-induced iAdo waves propagated across the cortex, following calcium signals. Additionally, two-photon imaging reveals that iAdo decays more rapidly in astrocytes than in neurons during seizures. Moreover, by recording iAdo dynamics in the basal forebrain during the sleep-wake cycle, we observe that iAdo signals are present during wakefulness and rapid eye movement (REM) sleep, regulated by equilibrative nucleoside transporters (ENT1/2). Thus, HypnoS is a versatile and powerful tool for investigating the biological functions of iAdo across a range of physiological and pathological states.