Regulation of sleep homeostasis mediator adenosine by basal forebrain glutamatergic neurons
Regulation of sleep homeostasis mediator adenosine by basal forebrain glutamatergic neurons
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基底前脑谷氨酸能神经元对睡眠稳态介质腺苷的调节
DOI：
10.1126/science.abb0556
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发表时间：
2020-09-04
期刊：
影响因子：
56.9
通讯作者：
Xu, Min
中科院分区：
文献类型：
--
作者：
Peng, Wanling;Wu, Zhaofa;Xu, Min
大脑中不同的神经活动模式控制着睡眠-觉醒周期。然而，这种活动是如何促进睡眠稳态的，在很大程度上仍然未知。基底前脑中的腺苷是睡眠稳态的重要生理介质。Peng等人使用一种新开发的指标监测小鼠基底前脑中的腺苷浓度。这与清醒状态和快速眼动睡眠有明显的关联。光遗传刺激基底前脑谷氨酸能神经元而非胆碱能神经元后，腺苷的活性依赖性释放也可被诱导。这些发现为清醒时神经元活动如何通过释放睡眠诱导因子来促进睡眠压力提供了新的见解。在小鼠清醒时，基底前脑中的一组神经元介导睡眠压力的增加。睡眠内稳态，即睡眠和清醒时间之间的平衡，是睡眠-觉醒周期的基本特征。在清醒的时候，促进睡眠的促睡因子积累起来，导致睡眠压力或我们对睡眠的需求增加。几十年的研究已经确定了许多基因、分子和参与调节睡眠稳态的生化过程。在涉及睡眠稳态的各种过程中，腺苷是细胞代谢途径的重要组成部分，是睡眠稳态的重要生理介质。基底前脑（basal forebrain， BF）是调节睡眠-觉醒周期的关键脑区，其释放的腺苷可以抑制A1受体介导的神经活动，增加睡眠压力。此外，睡眠-觉醒周期是由大脑中不同的神经活动模式控制的，但这种神经活动如何促进睡眠稳态仍不清楚。在这项研究中，我们通过详细研究BF中腺苷增加的机制来研究睡眠稳态的神经控制。由于传统的微透析测量腺苷浓度的时间分辨率较差，我们首先设计了一种基于遗传编码的G蛋白偶联受体（GPCR）激活（GRAB）的腺苷传感器（GRABAdo），其中细胞外腺苷的数量由绿色荧光蛋白（GFP）产生的荧光强度表示（见图a）。利用GRABAdo，我们首先测量了小鼠BF睡眠-觉醒周期中细胞外腺苷浓度的动态变化。然后，我们使用同时光学记录不同BF神经元中的Ca2+活性和腺苷浓度的变化，以检查腺苷增加与神经活动之间的相关性。我们进一步研究了不同BF神经元控制腺苷释放的能力。最后，我们使用细胞类型特异性病变来证实BF神经元在控制腺苷浓度增加中的作用，并研究其在睡眠稳态调节中的作用。结果我们发现细胞外腺苷的数量在清醒时高，而在非快速眼动（NREM）睡眠时低。得益于GRABAdo的高时间分辨率，我们还发现了快速眼动睡眠期间腺苷的显著增加，并揭示了腺苷浓度在大脑状态转换期间的快速变化。同时纤维光度法记录不同BF神经元内Ca2+活性和胞外腺苷浓度的变化表明，胆碱能神经元和谷氨酸能神经元的活性都与腺苷浓度的变化高度相关（见图A）。在检查这两个信号的时间过程中，我们发现神经活动总是比腺苷动力学的变化早几十秒。当我们利用这两种类型的神经元的生理放电频率测量光遗传激活引起的腺苷释放时，我们发现BF胆碱能神经元的激活仅产生适度的细胞外腺苷增加；相比之下，BF谷氨酸能神经元的激活导致了大量且强劲的增加（见图B）。最后，我们选择性地切除BF谷氨酸能神经元，发现细胞外腺苷的数量明显减少。此外，BF谷氨酸能神经元选择性损伤的小鼠表现出睡眠稳态调节受损，在活动期间显著增加清醒（见图C）。在这里，我们报道了一种具有高灵敏度和特异性、高时间分辨率的遗传编码腺苷传感器的设计和表征；利用该传感器，结合纤维光度法记录、光遗传激活和细胞类型特异性损伤，我们证明了小鼠BF睡眠-觉醒周期中细胞外腺苷浓度的神经活动依赖的快速动态，并揭示了BF谷氨酸能神经元在控制腺苷动力学和睡眠稳态中的关键作用。这些发现表明，清醒时细胞类型特异性的神经活动可以通过刺激促睡因子的释放来促进睡眠压力的增加。快速腺苷动力学和睡眠稳态的神经控制。(A)使用GCaMP和GRABAdo同时光学记录Ca2+活性和腺苷浓度揭示了睡眠-觉醒周期小鼠基底前脑（BF）中神经活动依赖的快速腺苷动力学。(B) BF谷氨酸能神经元的光遗传激活引起细胞外腺苷的强烈增加。(C) BF谷氨酸能神经元细胞类型特异性损伤显著增加清醒。睡眠和清醒受多种因素的动态调节，包括腺苷。然而，睡眠-觉醒周期背后的神经活动如何控制大脑中腺苷的释放仍不清楚。使用新开发的遗传编码腺苷传感器，我们发现小鼠基底前脑（BF）中细胞外腺苷浓度的活动依赖性快速增加，基底前脑是控制睡眠和觉醒的关键区域。虽然BF胆碱能神经元和谷氨酸能神经元的活性都与腺苷浓度的变化有关，但在生理放电频率下，这些神经元的光遗传激活表明，谷氨酸能神经元对腺苷浓度的增加贡献更大。选择性切除BF谷氨酸能神经元的小鼠表现出腺苷增加减少和睡眠稳态调节受损。因此，BF中细胞类型特异性的神经活动动态地控制睡眠的内稳态。
Sleep and basal forebrain activity Different patterns of neural activity in the brain control the sleep-wake cycle. However, how this activity contributes to sleep homeostasis remains largely unknown. Adenosine in the basal forebrain is a prominent physiological mediator of sleep homeostasis. Using a newly developed indicator, Peng et al. monitored adenosine concentration in the mouse basal forebrain. There was a clear correlation with wake state and REM sleep. Activity-dependent release of adenosine could also be elicited after optogenetic stimulation of basal forebrain glutamatergic, but not cholinergic, neurons. These findings offer new insights into how neuronal activity during wakefulness contributes to sleep pressure through the release of sleep-inducing factors. Science, this issue p. eabb0556 A group of neurons in the basal forebrain mediates increased sleep pressure during wakefulness in mice. INTRODUCTION Sleep homeostasis, the balance between the duration of sleep and wakefulness, is a fundamental feature of the sleep-wake cycle. During wakefulness, sleep-promoting somnogenic factors accumulate and cause an increase in sleep pressure or our need for sleep. Decades of research have identified many genes, molecules, and biochemical processes involved in the regulation of sleep homeostasis. Among various processes implicated in sleep homeostasis, adenosine—a critical component of the cell metabolic pathway—is a prominent physiological mediator of sleep homeostasis. Adenosine released in the basal forebrain (BF), a brain region that plays a critical role in regulating the sleep-wake cycle, can suppress neural activity mediated by the A1 receptor and increase sleep pressure. In addition, the sleep-wake cycle is controlled by different patterns of neural activity in the brain, but how this neural activity contributes to sleep homeostasis remains mostly unclear. In this study, we examine the neural control of sleep homeostasis by investigating in detail the mechanisms underlying adenosine increase in the BF. RATIONALE Because the traditional microdialysis measurement of adenosine concentration has a poor temporal resolution, we first designed a genetically encoded G protein–coupled receptor (GPCR)–activation-based (GRAB) sensor for adenosine (GRABAdo), in which the amount of extracellular adenosine is indicated by the intensity of fluorescence produced by green fluorescent protein (GFP) (see the figure, panel A). Using the GRABAdo, we first measured the dynamics of extracellular adenosine concentrations during the sleep-wake cycle in the mouse BF. We then used a simultaneous optical recording of the Ca2+ activity in different BF neurons and the change in adenosine concentrations to examine the correlation between adenosine increase and neural activity. We further studied the ability of different BF neurons in controlling the adenosine release using optogenetic activation. Finally, we used cell type–specific lesion to confirm the contribution of BF neurons in controlling the increase in adenosine concentrations and examine its contribution to the sleep homeostasis regulation. RESULTS We found that the amount of extracellular adenosine was high during wakefulness and low during non–rapid eye movement (NREM) sleep. Benefiting from the high temporal resolution of the GRABAdo, we also found a prominent increase in adenosine during REM sleep and revealed rapid changes in adenosine concentrations during brain state transitions. Simultaneous fiber photometry recording of the Ca2+ activity in different BF neurons and the change in extracellular adenosine concentrations showed that both cholinergic neurons and glutamatergic neurons had highly correlated activity with changes in the adenosine concentration (see the figure, panel A). In examining the time course of the two signals, we found that neural activity always preceded changes in adenosine dynamics by tens of seconds. When we measured the evoked adenosine release by optogenetic activation of these two types of neurons using their physiological firing frequencies, we found that the activation of BF cholinergic neurons only produced a moderate increase in extracellular adenosine; by contrast, the activation of BF glutamatergic neurons caused a large and robust increase (see the figure, panel B). Finally, we selectively ablated BF glutamatergic neurons and found a significantly reduced increase in the amounts of extracellular adenosine. Also, mice with a selective lesion of BF glutamatergic neurons showed impaired sleep homeostasis regulation, with significantly increased wakefulness during the active period (see the figure, panel C). CONCLUSION Here, we report the design and characterization of a genetically encoded adenosine sensor with high sensitivity and specificity, and high temporal resolution; using the sensor, in combination with fiber photometry recording, optogenetic activation, and cell type–specific lesion, we demonstrate a neural activity–dependent rapid dynamics of the extracellular adenosine concentration during the sleep-wake cycle in the mouse BF and uncover a critical role of the BF glutamatergic neurons in controlling adenosine dynamics and sleep homeostasis. These findings suggest that cell type–specific neural activity during wakefulness can contribute to the increase in sleep pressure by stimulating the release of somnogenic factors. Neural control of rapid adenosine dynamics and sleep homeostasis. (A) Simultaneous optical recording of the Ca2+ activity and adenosine concentration using GCaMP and GRABAdo reveals neural activity–dependent rapid adenosine dynamics in the mouse basal forebrain (BF) during the sleep-wake cycle. (B) Optogenetic activation of BF glutamatergic neurons evokes a robust increase of extracellular adenosine. (C) Cell type–specific lesion of BF glutamatergic neurons significantly increases wakefulness. Sleep and wakefulness are homeostatically regulated by a variety of factors, including adenosine. However, how neural activity underlying the sleep-wake cycle controls adenosine release in the brain remains unclear. Using a newly developed genetically encoded adenosine sensor, we found an activity-dependent rapid increase in the concentration of extracellular adenosine in mouse basal forebrain (BF), a critical region controlling sleep and wakefulness. Although the activity of both BF cholinergic and glutamatergic neurons correlated with changes in the concentration of adenosine, optogenetic activation of these neurons at physiological firing frequencies showed that glutamatergic neurons contributed much more to the adenosine increase. Mice with selective ablation of BF glutamatergic neurons exhibited a reduced adenosine increase and impaired sleep homeostasis regulation. Thus, cell type–specific neural activity in the BF dynamically controls sleep homeostasis.