Sleep is crucial for our survival, and many diseases are linked to long-term poor sleep quality. Before we can use sleep to enhance our health and performance and alleviate diseases associated with poor sleep, a greater understanding of sleep regulation is necessary. We have identified a mutation in the β 1 -adrenergic receptor gene in humans who require fewer hours of sleep than most. In vitro, this mutation leads to decreased protein stability and dampened signaling in response to agonist treatment. In vivo, the mice carrying the same mutation demonstrated short sleep behavior. We found that this receptor is highly expressed in the dorsal pons and that these ADRB1 + neurons are active during rapid eye movement (REM) sleep and wakefulness. Activating these neurons can lead to wakefulness, and the activity of these neurons is affected by the mutation. These results highlight the important role of β 1 -adrenergic receptors in sleep/wake regulation.

Noradrenergic signaling in the CNS has long been known to regulate sleep (). The network involving the noradrenergic neurons has been extensively studied, and most of the receptor subtypes have been genetically defined. In contrast to α1 and α2 adrenergic receptors (ARs), relatively little is known about the function of β receptors in the CNS (). βARs within the brain were previously suggested to mediate the effect of norepinephrine (NE) for alert waking and rapid eye movement (REM) sleep (). Clinically, β-blockers are widely used and can be associated with difficulty falling asleep and staying asleep, possibly due to reduced production and release of melatonin (). We report here a rare mutation in the βAR gene (ADRB1) found in humans with natural short sleep. Engineering the human mutation into mice resulted in a sleep phenotype similar to that seen in familial natural short sleepers. We show that β1AR is expressed at high levels in the dorsal pons (DP). Neuronal activity measured by calcium imaging in this region demonstrated that ADRB1neurons in DP are wake and REM sleep active. Manipulating the activity of these ADRB1neurons changes sleep/wake patterns. Also, the activity of these neurons was altered in mice harboring the mutation. Together, these results not only support the causative role of this ADRB1 mutation in the human subjects but also provide a mechanism for investigating noradrenaline and β1AR in sleep regulation at the circuit level.

An understanding of the regulatory mechanism for sleep lays at the foundation for healthy living and aging. Sleep behavior has long been thought to be regulated by the interactions of circadian clock and sleep homeostasis pathways (). In humans, variations of genetically inherited sleep features in the population have been recognized for a long time (). Importantly, human sleep has unique features that are different from that of animal models. For example, human sleep is usually consolidated, whereas mice sleep throughout the 24-h day (though more in the light phase than in the dark phase). Drosophila sleep-like behavior is consolidated into one long period, but the level of similarity between the Drosophila and human molecular regulatory mechanisms remains unclear. Previously, we identified a series of genetic variations that influence the timing of sleep in humans, and mouse models of these mutations mostly recapitulate the phenotypes (). Timing of sleep is heavily influenced by the circadian clock, which has been intensely studied, and we now have a large and growing body of knowledge on how the clock is regulated at the molecular level. On the other hand, our understanding of sleep homeostasis regulation for human lags behind. We reported a mutation in the human DEC2 gene that causes mutation carriers to sleep 6 h nightly for their entire lives without apparent negative effects (). Another mutation in DEC2 was later reported in a single individual who is a short sleeper and resistant to sleep deprivation (). Identification of additional genes participating in modulation of human sleep duration provides a unique way to expand our knowledge of genes and pathways critical for human sleep homeostasis regulation.

We analyzed the Adrb1 homozygous mutant mice (Adrb1 m/m) together with the heterozygous mice to clarify the mechanism. Although the protein level in homozygotes is even less than heterozygotes ( Figures S7 A and S7B), homozygous mutant mice have a mobile/sleep phenotype similar to heterozygotes ( Figures S7 C and S7D). In addition, in vivo and in vitro calcium imaging results were also similar between heterozygotes and homozygotes mice ( Figures S7 E and S7F). Together, these data further demonstrate the causative role of ADRB1 A187V mutation and imply that this mutation exerts dominant effects.

We also recorded spontaneous excitatory postsynaptic currents (sEPSCs) in slices from ADRB1-Cre; Adrb1+/+ or ADRB1-Cre; Adrb1+/m mice in the presence of the GABAergic antagonist bicuculline (BIC; 50 μM). Because cells in the DP region are heterogeneous (), we analyzed sEPSCs that were blocked by post hoc treatment of the glutamatergic antagonists 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) and DL-2-amino-5-phosphonopentanoic acid (DAP5) (6/15 cells for Adrb1+/+, 40%; 7/22 cells for Adrb1+/m, 32%) ( Figures 7 E and 7F). Basal sEPSC frequency was slightly higher in Adrb1+/m than Adrb1+/+ neurons (p = 0.057) ( Figure 7 G). Interestingly, dobutamine treatment increased sEPSC frequency in Adrb1+/m neurons, but not Adrb1+/+ neurons ( Figure 7 G). Moreover, no significant change in sEPSC amplitude was found for either the genotype or treatment of dobutamine ( Figure 7 H). We further analyzed the miniature EPSCs (mEPSCs) in the presence of TTX from Adrb1+/m slices ( Figure 7 I). We found that the increase of EPSC frequencies were preserved in the presence of TTX, indicating that the increase in spontaneous glutamate release from the presynaptic terminals was independent of action potential generation ( Figure 7 J). This significant increase of the sEPSC (mEPSC) frequency suggests that presynaptic neurons (of the patched ADRB1neuron) are more excitable as a population in response to dobutamine and that glutamatergic transmission is more active in the ADRB1neurons in mutant brain slices.

The increased population activity of ADRB1neurons from Adrb1 mutant mice could also be caused by enhanced excitability. To test this, we injected the Cre-dependent AAV encoding EGFP (AAV2/EF1a-DIO-EGFP-L10a) into the DP area to label the ADRB1cells with GFP. Using current clamp recordings of GFP- labeled ADRB1neurons from brain slices from either ADRB1-Cre; Adrb1+/+ (n = 6, N = 6 out of 23 brain slices from 11 animals) or ADRB1-Cre; Adrb1+/m mice (n = 6, N = 6 out of 15 brain slices from 15 animals), we obtained action potentials ( Figures 7 A and 7B ). These action potentials were blocked by 500 nM tetrodotoxin (TTX) (data not shown). We found that mutant neurons had a significantly lower rheobase current compared with that of neurons from Adrb1+/+ mice ( Figure 7 C) without any significant differences in the resting membrane potential (Adrb1 +/+, −60.14 ± 1.28 mV; Adrb1 +/m, −58.11 ± 1.32 mV), voltage threshold (Adrb1 +/+, −43.83 ± 3.00 mV; Adrb1 +/m, −45.50 ± 2.48 mV) or action potential overshoot (Adrb1 +/+, 21 ± 0.85 mV; Adrb1 +/m, 20.17 ± 0.14 mV). Also, a significant increase in firing frequencies was noted in Adrb1+/m neurons compared with those from Adrb1+/+ neurons ( Figure 7 D). These electrophysiological properties imply that Adrb1+/m neurons are more excitable.

To further explore whether the changes of GCaMP signals are neuron-autonomous effects and not secondary to circuitry or behavior changes, we performed single cell imaging experiments on acutely isolated DP explants from ADRB1-Cre; Adrb1+/+ and ADRB1-Cre; Adrb1+/m mice ( Figure 6 C). We measured the percentage of ADRB1neurons responding with changes in GCaMP signal during treatment with dobutamine (a selective βagonist). Neurons were categorized into three groups based on their response to the dobutamine treatment: inhibition, no change, and activation ( Figures S6 A–S6C). This is consistent with the previous observations that β-ARs in CNS could be either excitatory or inhibitory (). As shown in Figure 6 D, the percentage of DP ADRB1neurons that were inhibited by dobutamine was significantly lower (56.7%) in the Adrb1+/m slice. Meanwhile, the percentages of DP ADRB1neurons that were activated by dobutamine were comparable between the two groups. These results indicate that the mutation affects ADRB1neurons to various extents. However, as a whole, the portion of neurons inhibited is much smaller in mutant than in WT, consistent with an overall increase in ADRB1neuron population activity in mutant mice ( Figures 6 A and 6B).

Given that the activity of ADRB1neurons of the DP region modulates the wakeful state and that the Adrb1-A187V mutant mice showed altered sleep/wake behavior, we next tested whether the Adrb1-A187V mutation affects the activity of ADRB1neurons in the DP. We crossed the transgenic ADRB1-Cre mice with the Adrb1-A187V mice, and a photometry strategy similar to that described above was applied to ADRB1-Cre; Adrb1+/+ and ADRB1-Cre; Adrb1+/m mice. The absolute photometry signals depend on the expression level of the virus around the tip of the optical fibers, which makes it difficult to analyze the neural activity between different mice directly. Thus, we compared the relative GCaMP signal amplitude from the active phase (ZT13–16) to the sleep phase (ZT1–4) in the same mouse. The relative amplitude was significantly greater in the Adrb1+/m mice than in the Adrb1+/+ mice ( Figures 6 A and 6B ). These results suggest that activity of ADRB1neurons in the DP is altered by the Adrb1 A187V mutation. Notably, these neurons are wake-active and the Adrb1+/m mice spend 9% more time in wakefulness at ZT13–16. However, mutant mice exhibited 34% increase in the fluorescence signal shown in Figure 6 B, suggesting the change of fluorescence here could not be solely attributed to the altered behavior.

(D) Percentage of ADRB1cells that respond differentially to dobutamine treatment in both Adrb1+/+ (N = 7) and Adrb1+/m (N = 5) brain slices. The bottom table shows the original cell numbers in different categories. See Figure S6 for the representative fluorescence traces of the cells in different categories.

We next applied an optogenetic approach to activate the neurons in the DP. We unilaterally injected a Cre-inducible AAV expressing channel rhodopsin 2 fused with EYFP (AAV5-DIO-hChR2(H134R)-EYFP, ChR2 hereafter) or the control virus (AAV5-EF1α-DIO-EYFP, EYFP hereafter) into the DP of ADRB1-Cre mice and implanted the optical fiber and EEG-EMG electrodes ( Figures 5 A and 5B ). The 10-s trains of light trials were randomly applied during EEG/EMG recording in the light phase. Based on the post hoc analysis of EEG/EMG, the trials were then categorized to be starting from NREM, REM, and wakefulness. Interestingly, light stimulation during NREM sleep elicited immediate NREM-to-wake transitions in the ChR2-infected mice, but not in the EYFP-infected mice ( Figures 5 C–5E). However, similar stimulation could not trigger REM-to-wake transitions ( Figures 5 C, 5D, and 5F). Stimulation during the wake phase produced no significant effect on state transitions, although it may play a role in maintaining the wakefulness afterward ( Figures 5 C, 5D, and 5G). This is consistent with the fact that these neurons are wake and REM active under physiological conditions ( Figures 4 D and 4E). During wakefulness and REM sleep states, additional activation of these neurons cannot change the states, because the neurons are already active. Nonetheless, the strong effect of NREM-to-wake transition demonstrates that ADRB1cells in the DP region are primarily wake promoting.

To assess the population activity of the ADRB1cells in DP across spontaneous sleep-wake states, we used fiber photometry to record calcium signals from ADRB1neurons in freely moving animals ( Figure S5 A) (). We injected a Cre-dependent AAV encoding the fluorescent calcium indicator GCaMP6s (AAV1/Syn-Flex-GCaMP6s-WPRE-SV40) into the DP of ADRB1-Cre mice. We implanted both a fiber-optic probe (for subsequent delivery of excitation light and collection of fluorescent emission) and EEG/EMG electrodes (for simultaneous sleep-wake recordings) ( Figures S5 B and S5C) (). When aligning the fluorescence signals of calcium activity across different sleep states, robust alterations in the population activity of the ADRB1neurons revealed that these cells are active during wakefulness and REM sleep and remain quiescent during NREM sleep ( Figures 4 D and 4E). Notably, these neurons began to increase their activity before NREM-to-REM and NREM-to-wake transitions and decrease their activity before wake-to-NREM and REM-to-wake transitions ( Figure S5 D). These findings demonstrate that ADRB1neurons in DP change their population activity across sleep-wake states and offer a mechanistic framework for their participation in the regulation of sleep and wakefulness.

Since DP contains different populations of neurons (), we then labeled the DP ADRB1neurons by injecting the virus AAV8-hSyn-DIO-mCherry (mCherry hereafter). Co-staining revealed that the ADRB1cells in the DP were glutamatergic (∼37%, 221/600) or GABAergic (∼25%, 150/600) based on the expression of vesicular glutamate transporter 2 (Vglut2) or glutamate decarboxylase 1 (Gad1), respectively ( Figures S4 A–S4F). There were very few cholinergic or noradrenergic cells based on the expression of choline acetyltransferase (ChAT) or tyrosine hydroxylase (TH) ( Figures S4 G–S4L).

We next generated ADRB1-Cre bacterial artificial chromosome/clone (BAC) transgenic mice in order to study neural activity of ADRB1cells and their possible role in sleep regulation. A large BAC construct (150 kb) containing the entire ADRB1 locus was modified so that the coding sequence of ADRB1 was replaced by that of Cre recombinase ( Figure 4 A) (). The transgenic mice were crossed with two different reporter lines, ROSA mT/mG () and Ai32 (loxP-flanked-ChR2-EYFP). We sectioned the whole brain to identify cells positive for the reporters. There was good agreement between the two reporter lines, and several regions were found to have high CRE activity, including hippocampus, lateral septal nucleus, medial prefrontal cortex, DP ( Figures 4 B and 4C), and various structures in the medulla ( Figure S2 ). The DP region contains the major portions of the laterodorsal tegmental nucleus (LDTg), laterodorsal tegmental nucleus ventral (LDTgV), and part of parabrachial nucleus (PB), regions that are known to be involved in regulation of sleep behaviors ( Figure S3 A) (). We thus focused our attention on the DP. We injected the Cre-dependent adeno-associated virus (AAV) encoding EGFP (AAV2/EF1a-DIO-EGFP-L10a) into this area and stained the brain sections with probes against endogenous Adrb1 and gfp. Approximately 80% of the GFP-positive cells also stained positively for Adrb1 ( Figure S3 B). Translating ribosome affinity purification (TRAP) of mRNA populations in CRE-expressing cells in DP also showed ∼3- to 4-fold enrichment of endogenous Adrb1 ( Figures S3 C and S3D) compared to control genes. Taken together, these results indicate that the Cre activity largely represents Adrb1 expression in this region.

Since sleep pressure positively correlates with delta power (the 1–4.0 Hz frequency range of the EEG during NREM sleep) (), we examined changes in the NREM EEG delta power during the light phase (see STAR Methods ). The delta power of the mutant mice was significantly higher at the beginning (ZT1–2) and decreased rapidly throughout the light phase when compared to WT mice ( Figure 3 M). These data suggested that the Adrb1 mutant mice accumulate more sleep pressure, probably due to the shorter sleep in the dark phase.

To determine whether this mutation has any influence on sleep-related behaviors, we measured the sleep/wake behavior by two independent methods: ANY-maze (locomotor activity monitored by infrared cameras) and electroencephalogram/electromyogram (EEG/EMG). The mutant (Adrb1 +/m) mice showed increased activity by ANY-maze as reflected by more mobile time during the 24 h day ( Figure 3 A). The increased mobile time in the mutant mice was observed in both the light and dark phases ( Figures 3 B and 3C). In parallel, total sleep time within 24 h was ∼55 min shorter in the mutant mice when measured by EEG/EMG ( Figure 3 D), and this decrease was only seen in the dark phase ( Figures 3 E, 3F, and S1 A). The short-sleep phenotype of these mice is also demonstrated by the significant shortening of both non-REM (NREM) and REM sleep in the dark phase for Adrb1 +/m mice when compared to WT mice. NREM sleep was ∼53 min less and REM sleep was ∼7 min less in the Adrb1 +/m versus WT mice during the dark phase ( Figures 3 G–3L, S1 B, and S1C). The decreased NREM and REM sleep was due to the reduction of sleep bouts rather than episode duration ( Figures S1 D–S1G). These results indicated that the Adrb1 A187V mutation can lead to short sleep and increased mobile time, similar to what we observe in the human subjects.

To gain insight into whether this mutation has functional consequences, we first compared the mutant versus wild-type (WT) protein in cultured cells. As shown in Figure 2 A, the mutant protein was less stable than the WT protein in a cycloheximide (CHX) assay. Because βAR mediates the catecholamine-induced activation of adenylatecyclase (thus cyclic AMP [cAMP]-mediated signaling), we next examined the effect of the mutation on the synthesis of cAMP. To mimic the situation of human carriers, we transfected the cells with a mixture (1:1) of WT and mutant βAR. We found that heterozygous expression of the receptors led to decreased cAMP production in response to treatment with the nonselective agonist isoproterenol compared to expression of WT receptor alone ( Figure 2 B). This result indicates that the mutant protein likely has altered function. We thus generated an Adrb1-A187V knockin mouse model using CRISPR/Cas9. Endogenous βAR levels were decreased significantly in the mutant (Adrb1 +/m) mice ( Figures 2 C and 2D), whereas mRNA levels remained unchanged ( Figures 2 E and 2F), suggesting the decreased protein levels were caused by post-transcriptional events. This is consistent with the finding that the mutant protein is less stable than the WT protein in the CHX assay in cultured cells ( Figure 2 A).

(C and D) Western blotting results of endogenous β 1 AR protein from the heart (C) and brain (D) lysates of Adrb1+/+ and Adrb1+/m animals. N = 4 mice per group. NS, nonspecific band. Quantified results are shown on the right.

(A) Degradation assay of β 1 AR in transfected HEK293 cells. 24 h after transfection, cells were treated with 100 μg/mL CHX and harvested at indicated time points. Bands inside the two red boxes indicate β 1 AR protein of different sizes in the SDS gel. Quantified results are shown on the right. β 1 AR protein levels at the starting point (t = 0 h) were normalized to 1.

We searched for genes that are important in sleep regulation by screening for mutations in human individuals who exhibit unusual sleep patterns. Natural short sleep (NSS) refers to individuals who have a lifelong tendency to sleep only 4–6 h per night and feel well rested (). Kindred 50025 is a family segregating an autosomal dominant allele for familial natural short sleep (FNSS) ( Figure 1 A; Table S1 ). SNP-based linkage analysis followed by whole-exome sequencing identified a very rare variant in the ADRB1 gene, located on chromosome 10q25.3. The mutation co-segregates with FNSS in the family and involves a C→T change in the coding sequence that is predicted to cause an alanine→valine alteration at amino acid position 187 of the βAR. This change was not found in the unaffected members of the same family. In the human population, this is a rare mutation, with an incidence of 4.028/100,000 according to the Exome Aggregation Consortium database. The alanine at position 187 of βAR (A187) is highly conserved in vertebrates and invertebrates ( Figure 1 B). Notably, βAR is a G-protein-coupled receptor (GPCR) that has seven transmembrane domains. The A187 residue is located in the 4transmembrane domain.

Discussion

He et al., 2009 He Y.

Jones C.R.

Fujiki N.

Xu Y.

Guo B.

Holder J.L.

Rossner M.J.

Nishino S.

Fu Y.H. The transcriptional repressor DEC2 regulates sleep length in mammals. While NSS has long been recognized in sporadic cases from the general population, familial NSS was only first reported in 2009 (), thus enabling the use of human genetics to identify novel sleep genes. The initial report was in a small nuclear family, and thus, when a candidate mutation was identified in DEC2, in vitro and in vivo experiments were necessary to prove causality. We have continued to collect small- and moderate-sized families (2- and 3-generation) segregating FNSS alleles. It is likely that “required sleep time” is a genetic trait resulting from contributions of many variants in many genes. The >50 FNSS families identified to date have a sufficiently strong phenotype (lifelong requirement of <6.5 h per night) so that we have enriched for single mutations of large effect. Still, there is variability in the expression of FNSS among affected individuals, even within the same family. Taken together, these observations suggest that FNSS is caused by alleles of strong effect and that the phenotype is modified by the genetic background of each mutation carrier. In the moderate-sized family reported here, there is clear transmission of an autosomal dominant allele in affected members of the family. We used a staged approach based on whole-exome sequencing but using the genetic linkage data to take advantage of the information contained in the family structure to interpret the rare variants identified. This led to identification of only one coding variant from this locus that co-segregated with the FNSS phenotype. The ADRB1 allele is present in all “affected” individuals but also in a carrier (#100784) with a total sleep time of 7.5 h. This is still 1 h shorter than the population mean but does not meet our strict criteria to be classified as affected (we classified this individual as “unknown” and an obligate carrier, since he has a daughter who is definitely affected). Collectively, these data suggest that the ADRB1 mutation is likely to be causative for FNSS in this family. Variable expressivity is likely due to the genetic background of each mutation carrier.

Our mutant mice showed increased mobile time and a decreased sleep time of 55 min every 24 h, whereas the human mutation carriers, on average, sleep 2 h/day less than non-mutation carriers. It is not uncommon that animal models only partially recapitulate a human phenotype. This may be due to differences in physiology of mice versus humans. Mice are nocturnal and have much more fragmented sleep than humans. Thus, it is possible that mice are less dependent on sleep duration, consolidation, timing, and other variables for sleep than humans. Also, the phenotype of mice modeling human mutations is often more subtle than those obtained in forward mutagenesis screens. Mutations identified in FNSS families exist in humans who have survived on this planet. It is not surprising that the phenotypes are not as strong as those seen in mutagenized flies and mice under laboratory conditions. Our mouse model resembles features of human FNSS, further strengthening the genetic data in support of a causative role of this ADRB1 mutation in sleep. Interestingly, the delta power of Adrb1 mutant mice is higher at the beginning of the sleep phase, indicating a higher sleep pressure accumulated at the end of the active phase than the control mice. Further, the delta power decreased to the basal level during the light phase, suggesting these mice can sustain a higher sleep pressure than control mice. More work is needed to test whether human mutation carriers have a similar alteration in delta power.

+ neurons are either wake or REM sleep active under physiological conditions (+ neurons that are potentially inhibited by agonist was significantly dampened, while ADRB1+ neurons that are potentially excited by agonist remained unchanged ( 1 AR is more sensitive to the reduction of its protein abundance while the excitatory function is more tolerant to decreased protein levels. With fewer neurons being inhibited in the presence of natural ligand, the DP ADRB1+ neurons (as a population) are more active. This is consistent with the increased neuron activity in vivo in the mutant mice (+ neurons may contribute to the short sleep phenotype. Of note, although we chose to study ADRB1+ neurons in the DP, it is possible that ADRB1+ neurons (or glia) in other brain regions ( Berridge et al., 2012 Berridge C.W.

Schmeichel B.E.

España R.A. Noradrenergic modulation of wakefulness/arousal. Paschalis et al., 2009 Paschalis A.

Churchill L.

Marina N.

Kasymov V.

Gourine A.

Ackland G. beta1-Adrenoceptor distribution in the rat brain: an immunohistochemical study. We found a heterogeneous group of neurons in DP that express ADRB1 at high levels. Using fiber photometry, we showed that these DP ADRB1neurons are either wake or REM sleep active under physiological conditions ( Figure 4 ). Consistently, optogenetic studies indicated that these neurons are primarily wake promoting ( Figure 5 ). Thus, these sleep-relevant neurons provided an opportunity to investigate the neural behavior that might be affected by the Adrb1 A187V mutation. In mutant brain slices, the portion of ADRB1neurons that are potentially inhibited by agonist was significantly dampened, while ADRB1neurons that are potentially excited by agonist remained unchanged ( Figure 6 D). Based on our finding in Figure 2 , we speculated that the inhibitory function of βAR is more sensitive to the reduction of its protein abundance while the excitatory function is more tolerant to decreased protein levels. With fewer neurons being inhibited in the presence of natural ligand, the DP ADRB1neurons (as a population) are more active. This is consistent with the increased neuron activity in vivo in the mutant mice ( Figure 6 B) and the increased excitability and spontaneous excitatory glutamatergic neurotransmission in the mutant slices ( Figure 7 ). Together, these results imply that the increased activity of the wake-promoting DP ADRB1neurons may contribute to the short sleep phenotype. Of note, although we chose to study ADRB1neurons in the DP, it is possible that ADRB1neurons (or glia) in other brain regions () ( Figure S2 ) may also play important roles in sleep regulation and contribute to the short sleep phenotype. Further investigation is needed to address this possibility.

In summary, we present human genetic data in FNSS, mouse modeling, in vitro, and in vivo functional data. Collectively, these diverse approaches support the role of the Adrb1-A187V as a causative mutation in FNSS and of DP βARs in the regulation of sleep/wake behavior. This, in turn, provides an opportunity to further explore the mechanisms and potential drug targets of β1-AR for the treatment of sleep-related disorders. Much more work is needed to dissect the complex circuitry underlying sleep/wake regulation.