Background

Sleep is essential for life. Humans spend nearly one third of their lifetime asleep, and sleep quality is closely linked to overall health. Sleep disorders can trigger negative emotions and, in severe cases, lead to major psychiatric illness. Yet fundamental questions remain unresolved: Why do we need sleep? Why do we sleep more deeply after staying up late? How exactly is sleep regulated?

Two systems drive sleepiness in the human body: sleep homeostasis and the circadian rhythm. The circadian rhythm uses an internal biological clock to control the timing of sleep and wakefulness across the day, whereas sleep homeostasis is mainly governed by sleep pressure. The accumulated amount of sleep pressure determines how much an organism sleeps. Previous studies have identified many sleep homeostasis factors and biological processes, among which the release of adenosine is a key physiological regulator of sleep homeostasis.

Many neural nuclei in the brain are involved in sleep and wakefulness, and the basal forebrain (BF) is an important nucleus regulating this cycle. The BF contains cholinergic, glutamatergic and GABAergic neurons. How the activity of these neurons regulates adenosine release has remained an open question.

Recently, the Yu-Long Li lab at Peking University collaborated with the Min Xu lab at the Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences to publish a research paper titled “Glutamatergic neurons in the basal forebrain regulate adenosine dynamics and sleep homeostasis”. Using newly developed genetically encoded sensors with high sensitivity, high specificity and high temporal resolution, combined with fiber photometry, optogenetics and cell-specific ablation, the study revealed the key role of BF glutamatergic neurons in regulating adenosine dynamics and sleep homeostasis, further clarified the neural circuit mechanism underlying sleep homeostasis regulation, and provided an important reference for exploring treatments for sleep disorders.

Selected Results

1. Development and characterization of a novel genetically encoded adenosine probe

To detect dynamic changes in extracellular adenosine concentration in the BF across the sleep-wake cycle with high specificity and high temporal resolution, the Yu-Long Li lab developed an adenosine probe (GRAB_Ado). Its detection principle is based on activation of the adenosine G protein-coupled receptor, so that extracellular adenosine concentration is read out through the fluorescence intensity of a green fluorescent protein. The authors characterized the probe in terms of sensitivity, specificity and coupling to intracellular signaling pathways, and found that the iteratively optimized GRAB_Ado (abbreviated as Ado1.0) has high sensitivity and specificity while showing negligible coupling to intracellular signaling pathways, with no obvious effect on cell physiology (Figure 1).

2. Dynamic changes of extracellular adenosine across the sleep-wake cycle

According to EEG patterns, eye movement and muscle tone during sleep, sleep can be divided into non-rapid eye movement (NREM) sleep and rapid eye movement sleep (REMS) phases. Because REMS episodes are short, conventional microdialysis cannot capture the changes in adenosine concentration; the high temporal resolution of the adenosine probe effectively solved this problem. The authors used AAV2/9-hsyn-GRAB_Ado (Ado1.0) and performed stereotaxic injection into the mouse BF to measure fluorescence signals. To rule out movement artifacts, the authors fused Ado1.0 with the red fluorescent protein mScarlet, which is insensitive to changes in adenosine concentration. The results showed that the order of changes in BF extracellular adenosine concentration in mice was: REMS > wakefulness > NREM sleep (the results for the latter two stages were consistent with conventional microdialysis). Moreover, when mice transitioned between these three states, adenosine concentration changed relatively quickly. Such rapid changes in extracellular adenosine suggest that its release may depend on neural activity (Figure 2).

3. Cholinergic neurons in the mouse basal forebrain across the sleep-wake cycle

Previous studies have shown that cholinergic neurons (ChAT⁺) and glutamatergic neurons (VGLUT2⁺) are highly active during wakefulness and REMS, and that optogenetic activation of these two neuron types promotes arousal. The authors therefore first investigated the role of BF ChAT⁺ neurons by examining the correlation between ChAT⁺ neuron activity and changes in extracellular adenosine concentration. Using a viral approach, AAV2/9-hsyn-GRAB_Ado and AAV2/9-EF1α-DIO-GCaMP6s were injected into the BF of both hemispheres of ChAT-Cre mice, and fluorescence signals were measured weeks later. ChAT⁺ neuron calcium activity was highly correlated with changes in extracellular adenosine concentration, and the calcium activity usually preceded the change in the adenosine signal, suggesting that BF ChAT⁺ neurons may regulate extracellular adenosine concentration. Next, the authors co-injected AAV2/9-hsyn-GRAB_Ado and AAV2/9-hSyn-FLEX-ChrimsonR-tdTomato into the BF of ChAT-Cre mice, optogenetically activated ChAT⁺ neurons, and used fiber photometry to record adenosine release. Activation of ChAT⁺ neurons caused a small but significant increase in extracellular adenosine, indicating that ChAT⁺ neurons can regulate changes in extracellular adenosine concentration across the sleep-wake cycle. However, the amplitude of the evoked adenosine change varied considerably between trials, suggesting that other types of neural cells in the BF may also regulate extracellular adenosine concentration across the sleep-wake cycle (Figure 3).

4. Glutamatergic neurons in the mouse basal forebrain across the sleep-wake cycle

The authors then used the same viral approach to further investigate the regulatory role of BF VGLUT2⁺ neurons. Calcium activity in VGLUT2⁺ neurons was highly correlated with changes in extracellular adenosine concentration and preceded the adenosine signal. Optogenetic activation of VGLUT2⁺ neurons caused a large increase in extracellular adenosine concentration, larger than that induced by activating ChAT⁺ neurons, indicating that VGLUT2⁺ neurons contribute more to regulating extracellular adenosine concentration across the sleep-wake cycle. Next, the authors used Caspase-3 to specifically ablate BF VGLUT2⁺ neurons and measured changes in adenosine concentration. AAV2/9-hsyn-GRAB_Ado and AAVDJ-flex-taCasp3-TEVp were co-injected into the BF of VGLUT2-Cre mice. Two weeks later, the number of BF VGLUT2⁺ neurons was significantly reduced, and the increase in BF extracellular adenosine concentration during wakefulness and REM stages was significantly attenuated in experimental mice. These results further demonstrate that BF VGLUT2⁺ neurons play an important regulatory role in the increase of extracellular adenosine concentration (Figure 4).

5. Ablation of glutamatergic neurons disrupts sleep homeostasis

Finally, the authors verified through cell-specific ablation that loss of BF VGLUT2⁺ neurons affects sleep homeostasis in mice. Mice lacking VGLUT2⁺ neurons spent significantly longer in the wake state, and this difference was mainly manifested as increased wakefulness at night. The authors then tested whether loss of BF VGLUT2⁺ neurons affects sleep recovery. Mice were kept awake for 6 h under light-on conditions and then allowed recovery sleep over the following 18 h. In experimental mice, NREM sleep was significantly reduced and the rate of decline in NREM sleep time was markedly accelerated. These results indicate that loss of BF VGLUT2⁺ neurons impairs sleep homeostasis in mice (Figure 5).

Summary

In this study, the authors developed a novel adenosine probe (GRAB_Ado) with high sensitivity, high specificity and high temporal resolution. By combining optogenetic manipulation with cell-specific ablation, they demonstrated that glutamatergic neurons in the mouse basal forebrain regulate changes in extracellular adenosine concentration across the sleep-wake cycle, accumulate sleep pressure, and thereby play an important regulatory role in sleep homeostasis. This study provides new insight into the mechanism by which neural activity during wakefulness increases sleep pressure by stimulating the release of sleep-promoting factors.

Original Article

Original article link: https://www.nature.com/

Viral Tools and Usage in This Study

Other Neurotransmitter Probes from the Yu-Long Li Lab (Partial List)