The glymphatic-lymphatic system is increasingly recognized as fundamental for the homeostasis of the brain milieu since it defines cerebral spinal fluid flow in the brain parenchyma and eliminates metabolic waste. Animal and human studies have uncovered several important physiological factors regulating the glymphatic system including sleep, aquaporin-4, and hemodynamic factors. Yet, our understanding of the modulation of the glymphatic system is limited, which has hindered the development of glymphatic-based treatment for aging and neurodegenerative disorders. Here, we present the evidence from fluorescence tracing, two-photon recording, and dynamic contrast-enhanced magnetic resonance imaging analyses that 40 Hz light flickering enhanced glymphatic influx and efflux independently of anesthesia and sleep, an effect attributed to increased astrocytic aquaporin-4 polarization and enhanced vasomotion. Adenosine-A _2A receptor (A _2A R) signaling emerged as the neurochemical underpinning of 40 Hz flickering-induced enhancement of glymphatic flow, based on increased cerebrofluid adenosine levels, the abolishment of enhanced glymphatic flow by pharmacological or genetic inactivation of equilibrative nucleotide transporters-2 or of A _2A R, and by the physical and functional A _2A R–aquaporin-4 interaction in astrocytes. These findings establish 40 Hz light flickering as a novel non-invasive strategy of enhanced glymphatic flow, with translational potential to relieve brain disorders.

Sleep–wake behavior is controlled by a wide range of neuronal populations in the mammalian brain. Although the ventral midbrain/pons (VMP) area is suggested to participate in sleep–wake regulation, the neuronal mechanisms have remained unclear. Here, we found that nonspecific cell ablation or selective ablation of GABAergic neurons by expressing diphtheria toxin fragment A in the VMP in male mice induced a large increase in wakefulness that lasted at least 4 weeks. In contrast, selective ablation of dopaminergic neurons in the VMP had little effect on wakefulness. Chemogenetic inhibition of VMP GABAergic neurons also markedly increased wakefulness. The wake-promoting effect of the VMP GABAergic neuron ablation or inhibition was attenuated to varying degrees by the administration of dopamine D1 or D2/3 receptor antagonists and abolished by the administration of both antagonists together. In contrast, chemogenetic activation of VMP GABAergic neurons very strongly increased slow-wave sleep and reduced wakefulness. These findings suggest that VMP GABAergic neurons regulate dopaminergic actions in the sleep–wake behavior of mice. SIGNIFICANCE STATEMENT Current understanding of the neuronal mechanisms and populations that regulate sleep–wake behavior is incomplete. Here, we identified a GABAergic ventral midbrain/pons area that is necessary for controlling the daily amount of sleep and wakefulness in mice. We also found that these inhibitory neurons control wakefulness by suppressing dopaminergic systems. Surprisingly, activation of these neurons strongly induced slow-wave sleep while suppressing wakefulness. Our study reveals a new brain mechanism critical for sleep–wake regulation.

Optochemistry, an emerging pharmacologic approach in which light is used to selectively activate or deactivate molecules, has the potential to alleviate symptoms, cure diseases, and improve quality of life while preventing uncontrolled drug effects. The development of in-vivo applications for optochemistry to render brain cells photoresponsive without relying on genetic engineering has been progressing slowly. The nucleus accumbens (NAc) is a region for the regulation of slow-wave sleep (SWS) through the integration of motivational stimuli. Adenosine emerges as a promising candidate molecule for activating indirect pathway neurons of the NAc expressing adenosine A_2A receptors (A_2ARs) to induce SWS. Here, we developed a brain-permeable positive allosteric modulator of A_2ARs (A_2AR PAM) that can be rapidly photoactivated with visible light (λ > 400 nm) and used it optoallosterically to induce SWS in the NAc of freely behaving male mice by increasing the activity of extracellular adenosine derived from astrocytic and neuronal activity.

An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma. The rat plasma was treated with liquid-liquid extraction using ethyl acetate. The determination was performed on the UPLC HSS T3 column (50mm×2.1mm, 1.8μm) with a mobile phase consisting of acetonitrile-water (containing 0.1 formic acid) and gradient elution at a flow rate of 0.4mL/min. Electrospray (ESI) positive ion mode detection and multireaction monitoring (MRM) quantitative analysis were performed. A total of 36 rats were given tenacissoside G, tenacissoside H, and tenacissoside I, respectively, orally (5mg/kg) and intravenously (1mg/kg), with 6 rats in each group, to evaluate the pharmacokinetic difference of tenacissoside G, tenacissoside H, and tenacissoside I in rats. The calibration curves showed good linearity in the range of 52000ng/mL, where r was greater than 0.99. The results of precision, accuracy, recovery, matrix effect, and stability met the requirements of biological sample detection methods. The established UPLC-MS/MS method was successfully applied to pharmacokinetic studies of tenacissoside G, tenacissoside H, and tenacissoside I, and the bioavailability was 22.9, 89.8, and 9.4, respectively.

Chronic intermittent hypoxia (CIH), a defining feature of obstructive sleep apnea, is strongly associated with cognitive impairment and increased risk of neurodegenerative disease, yet the underlying mechanisms linking hypoxic stress to disrupted brain homeostasis remain poorly defined. Impaired glymphatic clearance has been reported in patients with obstructive sleep apnea, but whether and how intermittent hypoxia directly alters glymphatic function is unknown. Here, we investigated the effects of acute and chronic intermittent hypoxia on cerebrospinal fluid–interstitial fluid exchange in male mice and examined the molecular mechanisms governing these effects. Using tracer-based influx and efflux assays, in vivo two-photon imaging, behavioral testing, and genetic and pharmacological manipulation, we show that intermittent hypoxia exerts a duration-dependent, biphasic effect on glymphatic function. Acute exposure transiently enhanced glymphatic influx and efflux, whereas prolonged CIH progressively impaired glymphatic transport, disrupted perivascular aquaporin-4 (AQP4) polarization, reduced vascular pulsatility, and impaired spatial working memory. CIH was associated with reduced extracellular adenosine levels, suppression of cerebral energy metabolism, and altered expression of equilibrative nucleoside transporters (ENTs). Genetic ablation of AQP4 abolished CIH-induced glymphatic impairment, confirming its essential role in hypoxia-induced glymphatic dysfunction. Importantly, pharmacological inhibition or genetic deletion of ENT1 and deletion of ENT2 restored adenosine availability, normalized AQP4 polarization and vascular dynamics, and rescued glymphatic dysfunction and cognitive deficits under CIH. These findings identify ENT-dependent dysregulation of adenosine signaling as a key mechanism by which chronic intermittent hypoxia compromises glymphatic clearance, providing mechanistic insight into how sleep-disordered breathing disrupts brain waste removal and cognitive function.