睡眠稳态（sleep homeostasis）是由睡眠压力驱动的维持机体睡醒平衡的生理机制。胞外腺苷（extracellular adenosine，eADO）被视为睡眠稳态的调节因子，其在清醒时积累并推动睡眠发生。随着睡眠时间的延长，睡眠压力和eADO水平均随之下降。腺苷受体对eADO的动态变化作出反应，在抑制唤醒和促进睡眠中起调节作用。然而，在生理条件下，eADO激活的腺苷受体亚型腺苷A3受体（adenosine A3 receptor，A3R）在睡眠稳态中的作用尚不明确。小胶质细胞作为中枢神经系统中的固有免疫细胞，其钙活动的变化或全脑耗竭已被证实可以调节机体的睡醒状态。但小胶质细胞如何感知eADO振荡并以何种机制调节睡眠稳态，仍有待深入研究。去甲肾上腺素（norepinephrine，NE）是与唤醒相关的神经递质，通过β2肾上腺素受体（beta-2-adrenergic receptor，β₂AR）调节小胶质细胞的形态变化。然而，在自然睡眠-觉醒周期中，NE-β₂AR途径如何调节小胶质细胞的动态变化仍需进一步阐释。

    运用微型化双光子显微成像技术，本课题在自由行为小鼠中获取了小胶质细胞形态、小胶质细胞钙活动、NE和eADO的长时程延时图像，以及脑电图、肌电图和行为学视频。在此基础上，本课题探究了自然睡眠-觉醒周期中小胶质细胞形态和钙活动的变化特征，以及这些变化与NE和eADO之间的关系。结合药理学实验和小鼠模型，本课题深入研究了小胶质细胞的β₂AR和A3R在调节细胞动态变化和调制睡眠稳态中的作用。

    研究发现，自然睡眠-觉醒周期中小胶质细胞的形态和钙活动与睡醒状态密切相关，并且受到睡眠压力的调控。小胶质细胞的分支在觉醒状态下相对收缩，在NREM（non-rapid eye movement）睡眠期间相对舒展。小胶质细胞的钙活动则表现为觉醒状态时最高，REM（rapid eye movement）睡眠时次之，NREM睡眠时最低。睡眠剥夺会导致机体睡眠压力上升，这一过程触发小胶质细胞分支的快速收缩和细胞钙活动的增强。随着睡眠压力的降低，小胶质细胞的形态和钙活动可逐渐恢复至正常水平。这些证据表明自然睡眠-觉醒周期中小胶质细胞的动态变化与睡眠稳态有关。

    小胶质细胞的形态和钙活动受到NE和eADO的共同调节。大脑皮层中的NE水平受到蓝斑核（locus coeruleus，LC）中NE能神经元活动的支配，并表现为觉醒状态时最高，NREM睡眠时次之，REM睡眠时最低。在NE能神经元受损小鼠或β₂AR敲除小鼠中，小胶质细胞形态变化的睡醒状态依赖性受到干扰。另一方面，eADO水平和小胶质细胞钙活动均能快速响应睡醒状态转换，并呈现一致的变化趋势。药理学研究表明，小胶质细胞的形态和钙活动受eADO水平与A3R功能的调节。自然睡眠-觉醒周期中，A3R有助于维持睡醒状态依赖的小胶质细胞形态和钙活动变化。在小胶质细胞条件特异性敲除A3R的小鼠中，觉醒状态和NREM睡眠状态之间的转换频率增加，但每种睡醒状态的时间占比不受影响。这提示小胶质细胞A3R可以增强小鼠觉醒状态和NREM睡眠状态的稳定性。

    综上所述，本课题揭示了小胶质细胞调制睡眠稳态的新机制：小胶质细胞通过A3R感知eADO的动态变化，调节细胞形态和钙活动，进而维持觉醒和NREM睡眠的稳定。同时，不同睡醒状态下小胶质细胞的形态变化也受到了LC-NE-β₂AR途径的调控。这些发现为深入研究睡眠稳态中小胶质细胞的功能提供了新的见解。

  Sleep homeostasis, driven by sleep pressure, is a fundamental principle that maintains the balance between sleep and wakefulness. Extracellular adenosine (eADO) is regarded as a regulatory factor of sleep homeostasis, accumulating during wakefulness to drive the onset of sleep. As sleep duration lengthens, both sleep pressure and eADO levels are reduced accordingly. Adenosine receptors respond to eADO oscillations, serving a regulatory function in inhibiting arousal and promoting sleep. However, the role of the receptor subtype adenosine A3 receptor (A3R), which is activated by eADO under physiological conditions, remains unclear in the context of sleep homeostasis. Microglia, the intrinsic immune cells of the central nervous system, have been shown to regulate sleep through experiments involving regulated Ca²⁺ activity or whole-brain depletion. Nevertheless, it is still unknown how microglia perceive eADO oscillations and regulate sleep homeostasis, warranting further investigation. Norepinephrine (NE) is a wake-promoting neurotransmitter, regulating microglial morphology through the β2-adrenergic receptor (β₂AR). Nonetheless, the precise mechanisms by which NE-β₂AR axis regulates the dynamic changes of microglia during natural sleep-wake cycles have not yet been fully understood.

  Utilizing miniature two-photon microscopy (mTPM), this research project acquired long-term time-lapse images of microglial morphology and Ca²⁺ activity, NE, and eADO, along with electroencephalography (EEG)/electromyography (EMG) recordings and behavioral video, in freely behaving mice. Based on this, the current study focused on investigating the characteristics of microglial morphology and Ca²⁺ activity during the natural sleep-wake cycle, as well as their relationship with eADO or NE oscillations. Combined with pharmacological experiments and mouse models, the function of microglia β₂AR and A3R in regulating cellular dynamics and modulating sleep homeostasis was investigated in depth. 

  Results indicate that microglial morphology and Ca²⁺ activity are sleep/wake state-dependent and are regulated by sleep pressure during natural sleep-wake cycles. Microglial processes are relatively retracted during wakefulness and extended during NREM (non-rapid eye movement) sleep. Microglia Ca²⁺ activity is highest during wakefulness, followed by REM (rapid eye movement) sleep, and lowest during NREM sleep. Sleep deprivation results in an increase in sleep pressure, which initiates rapid retraction of microglial processes and enhancement of cellular Ca²⁺ activity. As sleep pressure diminishes, the morphology and Ca²⁺ activity of microglia gradually resume. These findings indicate that the dynamic changes in microglia during natural sleep-wake cycles are related to sleep homeostasis. 

  Microglial morphology and Ca²⁺ activity are jointly regulated by NE and eADO. On the one hand, the level of NE in the cerebral cortex is governed by the activity of noradrenergic neurons in the locus coeruleus (LC), which is highest during wakefulness, second highest during NREM sleep, and lowest during REM sleep. The sleep/wake state-dependent microglial morphology is disrupted in LC-axon ablated mice or β₂AR knockout mice. On the other hand, the levels of eADO and microglia Ca²⁺ activity rapidly respond to sleep/wake state transitions, showing a consistent trend of change. Pharmacological experiments demonstrate that microglial morphology and Ca²⁺ activity are regulated by the levels of eADO and A3R function. During natural sleep-wake cycles, A3R promotes the maintenance of sleep/wake state-dependent changes in microglial morphology and Ca²⁺ activity. Furthermore, in microglial A3R conditional knockout mice, the frequency of transitions between wakefulness and NREM sleep increased, but the percentage of time spent in each wake/sleep state was not affected.

  In summary, this study reveals a novel mechanism by which microglia modulate sleep homeostasis. Specifically, microglia A3R responds to eADO oscillations and regulates cellular morphology and Ca²⁺ activity to maintain the stability of wakefulness and NREM sleep. Meanwhile, the morphological changes of microglia are also regulated by the LC-NE-β₂AR axis during natural sleep-wake cycles. These findings provide new insights into the function of microglia in sleep homeostasis.