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Fig. S3. Recovery sleep after 6-h sleep deprivation. (A) Wake, (B) NREMS, (C) REMS during 18-h recovery following 6-h sleep deprivation. (D) NREMS EEG delta during the 18-h recovery. Recovery delta power is normalized by the last hour of the light phase (ZT11) of the baseline. Red-filled box, 6-h sleep deprivation; white box, 6-h recovery sleep; gray-filled box, 12-h dark phase. (E) Change, shown as ratio (recovery/baseline), in time spent in each vigilance state during dark phase (ZT12-ZT24). Male B6J (N = 19), B6N (N = 18) and B10J (N = 20). All data are shown as the mean ± SEM *P < 0.05; ** (blue) P < 0.01; *** (green) P < 0.001; **** (red) P < 0.0001. Two-way ANOVA followed by Tukey's test.
Source publication
Significance
The network of genes and molecules that govern sleep and wakefulness remains largely unknown. Forward genetics is a powerful approach to elucidate important biological phenomena that cannot be predicted from the function of known genes. We established a large-scale screening system using EEG/EMG-based sleep/wake monitoring with reliabl...
Citations
... The forward-genetic approach is based on arhythmic mutants that produce rhythms deviating from 24-h cycles. Although this approach has been instrumental for revealing the detailed molecular mechanisms of circadian rhythms in model systems and various outcrossing species (King and Takahashi 1996;Miyoshi et al. 2019), it has some significant drawbacks. First, its translational relevance for a broad spectrum of species and biological processes is challenging since only a limited number of mutations can be identified. ...
Main conclusion
Molecular mechanisms of biological rhythms provide opportunities to harness functional allelic diversity in core (and trait- or stress-responsive) oscillator networks to develop more climate-resilient and productive germplasm.
Abstract
The circadian clock senses light and temperature in day–night cycles to drive biological rhythms. The clock integrates endogenous signals and exogenous stimuli to coordinate diverse physiological processes. Advances in high-throughput non-invasive assays, use of forward- and inverse-genetic approaches, and powerful algorithms are allowing quantitation of variation and detection of genes associated with circadian dynamics. Circadian rhythms and phytohormone pathways in response to endogenous and exogenous cues have been well documented the model plant Arabidopsis. Novel allelic variation associated with circadian rhythms facilitates adaptation and range expansion, and may provide additional opportunity to tailor climate-resilient crops. The circadian phase and period can determine adaptation to environments, while the robustness in the circadian amplitude can enhance resilience to environmental changes. Circadian rhythms in plants are tightly controlled by multiple and interlocked transcriptional–translational feedback loops involving morning (CCA1, LHY), mid-day (PRR9, PRR7, PRR5), and evening (TOC1, ELF3, ELF4, LUX) genes that maintain the plant circadian clock ticking. Significant progress has been made to unravel the functions of circadian rhythms and clock genes that regulate traits, via interaction with phytohormones and trait-responsive genes, in diverse crops. Altered circadian rhythms and clock genes may contribute to hybrid vigor as shown in Arabidopsis, maize, and rice. Modifying circadian rhythms via transgenesis or genome-editing may provide additional opportunities to develop crops with better buffering capacity to environmental stresses. Models that involve clock gene‒phytohormone‒trait interactions can provide novel insights to orchestrate circadian rhythms and modulate clock genes to facilitate breeding of all season crops.
... EEG/EMG Electrode Implantation Surgery. EEG/EMG electrodes containing four electrode pins and two flexible stainless steel wires were implanted into 8-to 10-wk-old male mice as previously described (42,43). Surgery was performed under stereotaxis control with isoflurane anesthesia (4% for induction and 2% for maintenance). ...
... EEG/EMG Recording and Analysis. EEG/EMG recording and analysis were performed as previously described with some modifications (42,43). EEG/EMG signals were continuously recorded for 48 h, of which the first 24 h was under the 12-h light:12-h dark condition and followed by 24-h constant dark condition. ...
Mammals exhibit circadian cycles of sleep and wakefulness under the control of the suprachiasmatic nucleus (SCN), such as the strong arousal phase-locked to the beginning of the dark phase in laboratory mice. Here, we demonstrate that salt-inducible kinase 3 (SIK3) deficiency in gamma-aminobutyric acid (GABA)-ergic neurons or neuromedin S (NMS)-producing neurons delayed the arousal peak phase and lengthened the behavioral circadian cycle under both 12-h light:12-h dark condition (LD) and constant dark condition (DD) without changing daily sleep amounts. In contrast, the induction of a gain-of-function mutant allele of Sik3 in GABAergic neurons exhibited advanced activity onset and a shorter circadian period. Loss of SIK3 in arginine vasopressin (AVP)-producing neurons lengthened the circadian cycle, but the arousal peak phase was similar to that in control mice. Heterozygous deficiency of histone deacetylase (HDAC) 4, a SIK3 substrate, shortened the circadian cycle, whereas mice with HDAC4 S245A, which is resistant to phosphorylation by SIK3, delayed the arousal peak phase. Phase-delayed core clock gene expressions were detected in the liver of mice lacking SIK3 in GABAergic neurons. These results suggest that the SIK3-HDAC4 pathway regulates the circadian period length and the timing of arousal through NMS-positive neurons in the SCN.
... Consequently, genome-wide association studies and systems genetics approaches have detected hundreds of genes affecting different aspects of sleep and circadian behavior [5-7, 9-17, 19-26]. Furthermore, mutational screens in mice and flies revealed that a striking 14%-16% of mutations tested have quantitative effects on some aspect of sleep [42,43] and 0.1%-0.2% of those tested have Mendelian (2-3 standard deviations) effects on sleep [29,44]. As sleep is polygenic, it is likely that a single mutation does not act in isolation. ...
Selective breeding is a classic technique that enables an experimenter to modify a heritable target trait as desired. Direct selective breeding for extreme sleep and circadian phenotypes in flies successfully alters these behaviors, and sleep and circadian perturbations emerge as correlated responses to selection for other traits in mice, rats, and dogs. The application of sequencing technologies to the process of selective breeding identifies the genetic network impacting the selected trait in a holistic way. Breeding techniques preserve the extreme phenotypes generated during selective breeding, generating community resources for further functional testing. Selective breeding is thus a unique strategy that can explore the phenotypic limits of sleep and circadian behavior, discover correlated responses of traits having shared genetic architecture with the target trait, identify naturally-occurring genomic variants and gene expression changes that affect trait variability, and pinpoint genes with conserved roles.
... However, in general, a heterozygous loss-of-function mutation results in mild changes in sleep/wakefulness and, thus, dominant screening usually leads to heterozygous gain-of-function gene mutations. In fact, this method also detected a loss-of-function mutation of the calcium voltage-gated channel subunit α1A (Cacna 1a) gene in the long sleeper pedigree Drowsy [25]. ...
... Previous studies showed that Intraperitoneal administration of a chemical mutagen, ethyl nitrosourea (ENU), to male B6J mice causes numerous point mutations in spermatogonia, and subsequently in the sperm, at a rate of approximately one mutation per million bases [27,28] To obtain the next generation, the ENUtreated B6J mice or their sperm were subjected to natural mating or in vitro fertilization with wild-type B6N females. The offspring (F1 generation) were evaluated for sleep abnormalities using EEG/EMG-based sleep staging [25,29]. ...
... Based on our examination of the reproducibility of sleep parameters, we used simple parameters, such as total wake time, as an indicator to detect mice with sleep abnormalities [25]. When the abnormalities were shown in F1 mice, these were crossed with wild-type female B6N mice to obtain the next generation, namely, N2 mice. ...
Genetics is one of the various approaches adopted to understand and control mammalian sleep. Reverse genetics, which is usually applied to analyze sleep in gene-deficient mice, has been the mainstream field of genetic studies on sleep for the past three decades and has revealed that various molecules, including orexin, are involved in sleep regulation. Recently, forward genetic studies in humans and mice have identified gene mutations responsible for heritable sleep abnormalities, such as SIK3, NALCN, DEC2, the neuropeptide S receptor, and β1 adrenergic receptor. Furthermore, the protein kinase A-SIK3 pathway was shown to represent the intracellular neural signaling for sleep need. Large-scale genome-wide analyses of human sleep have been conducted, and many gene loci associated with individual differences in sleep have been found. The development of genome-editing technology and gene transfer by an adeno-associated virus has updated and expanded the genetic studies on mammals. These efforts are expected to elucidate the mechanisms of sleep–wake regulation and develop new therapeutic interventions for sleep disorders.
... While spike-wave seizures that interrupt active wakefulness can cause paroxysmal arrests of movement, these events are typically much shorter in duration (2-6 s on average 22,37,38 ) and would not have been registered by our algorithm's cutoff. Average NREM or REM sleep bouts in mice are significantly more prolonged (180-360s or 60-120s [39][40][41][42] , respectively). Further, in patients with genetic or symptomatic generalized epilepsies, spike-wave discharges commonly occur during NREM sleep 43 , where they often persist in patients who are clinically seizure-free. ...
In many childhood-onset genetic epilepsies, seizures are accompanied by neurobehavioral impairments and motor disability. In the Stargazer mutant mouse, genetic disruptions of Cacng2 result in absence-like spike-wave seizures, cerebellar gait ataxia and vestibular dysfunction, which limit traditional approaches to behavioral phenotyping. Here, we combine videotracking and instrumented home-cage monitoring to resolve the neurobehavioral facets of the murine Stargazer syndrome. We find that despite their gait ataxia, stargazer mutants display horizontal hyperactivity and variable rates of repetitive circling behavior. While feeding rhythms, circadian or ultradian oscillations in activity are unchanged, mutants exhibit fragmented bouts of behaviorally defined “sleep”, atypical licking dynamics and lowered sucrose preference. Mutants also display an attenuated response to visual and auditory home-cage perturbations, together with profound reductions in voluntary wheel-running. Our results reveal that the seizures and ataxia of Stargazer mutants occur in the context of a more pervasive behavioral syndrome with elements of encephalopathy, repetitive behavior and anhedonia. These findings expand our understanding of the function of Cacng2.
... At 3-4 months of age, male mice were implanted with electroencephalography/electromygography (EEG/EMG) electrodes with four EEG electrode pins and 2 flexible stainless EMG wires under anesthesia using isoflurane (4% for induction, 2.5% for maintenance) as described previously (Miyoshi et al., 2019). An EEG/EMG electrode insulator was attached to the skull using dental cement. ...
... EEG/EMG recordings were analyzed as described (Miyoshi et al., 2019). The recording room was kept under a 12:12 h light/dark cycle and a constant temperature (24-25 • C). ...
In addition to the well-known motor control, the cerebellum has recently been implicated in memory, cognition, addiction, and social behavior. Given that the cerebellum contains more neurons than the cerebral cortex and has tight connections to the thalamus and brainstem nuclei, it is possible that the cerebellum also regulates sleep/wakefulness. However, the role of the cerebellum in sleep was unclear, since cerebellar lesion studies inevitably involved massive inflammation in the adjacent brainstem, and sleep changes in lesion studies were not consistent with each other. Here, we examine the role of the cerebellum in sleep and wakefulness using mesencephalon- and rhombomere 1-specific Ptf1a conditional knockout (Ptf1a cKO) mice, which lack the cerebellar cortex and its related structures, and exhibit ataxic gait. Ptf1a cKO mice had similar wake and non-rapid eye movement sleep (NREMS) time as control mice and showed reduced slow wave activity during wakefulness, NREMS and REMS. Ptf1a cKO mice showed a decrease in REMS time during the light phase and had increased NREMS delta power in response to 6 h of sleep deprivation, as did control mice. Ptf1a cKO mice also had similar numbers of sleep spindles and fear memories as control mice. Thus, the cerebellum does not appear to play a major role in sleep-wake control, but may be involved in the generation of slow waves.
... MGD: mouse genome database, zfin.org). These initiatives contributed not only to speed-up the identification of both disease targets and models (Miyoshi et al. 2019) but also to 'popularize' the exploitation of the mutants by research laboratories. ...
Although any macromolecule linked to a pathophysiological phenomenon is a
natural candidate for therapeutic intervention, not all of them fulfil the criteria of
drug target candidates. The selection of molecular targets with the highest potential
for developing selective, effective and safe drugs depends, to a significant
extent, on the correct assessment of their structural, biochemical and biological
properties. The validation of a molecular target is the first experimental approach
to be conducted in a target-based drug discovery campaign and is critical to diminish the high attrition rate and costs associated with the subsequent phases.
In medicinal chemistry, target validation embraces the determination of the
indispensability or physiological relevance of the molecular target, its druggability
and assayability in vitro or in vivo. Different computational, genetic-, biochemical-
and chemical-based approaches can be used complementary to address
this goal. In this chapter, we provide an overview of the diverse strategies available
to (bio)chemically and biologically validate a molecular target and discuss
important concepts in this area
... Timing: 2-3 weeks EEG/EMG electrode implantation surgery, EEG/EMG recording and analysis is performed (Iwasaki et al., 2021;Miyoshi et al., 2019). Here, we show the procedure of EEG/EMG implantation surgery. ...
Elucidating the molecular pathways that regulate animal behavior such as sleep is essential for understanding how the brain works. However, to examine how a certain functional domain of protein is involved in animal behavior is challenging. Here, we present a protocol for inducing endogenous protein that lacks a specific functional domain using Cre-mediated allele modification in neurons followed by electroencephalogram/electromyogram (EEG/EMG) recording to study the role of kinases in sleep. This strategy is applicable to other gene targets or behaviors.
For complete details on the use and execution of this protocol, please refer to Iwasaki et al. (2021).
... Sleep analysis was performed as described previously [28]. Eight-to ten-week-old mice were subjected to EEG/ EMG electrode implantation surgery. ...
22q11.2 deletion syndrome (22q11.2DS) is a disorder caused by the segmental deletion of human chromosome 22. This chromosomal deletion is known as high genetic risk factors for various psychiatric disorders. The different deletion types are identified in 22q11.2DS patients, including the most common 3.0-Mb deletion, and the less-frequent 1.5-Mb and 1.4-Mb deletions. In previous animal studies of psychiatric disorders associated with 22q11.2DS mainly focused on the 1.5-Mb deletion and model mice mimicking the human 1.5-Mb deletion have been established with diverse genetic backgrounds, which resulted in the contradictory phenotypes. On the other hand, the contribution of the genes in 1.4-Mb region to psychiatric disorders is poorly understood. In this study, we generated two mouse lines that reproduced the 1.4-Mb and 1.5-Mb deletions of 22q11.2DS [ Del(1.4 Mb)/ + and Del(1.5 Mb)/ +] on the pure C57BL/6N genetic background. These mutant mice were analyzed comprehensively by behavioral tests, such as measurement of locomotor activity, sociability, prepulse inhibition and fear-conditioning memory. Del(1.4 Mb)/ + mice displayed decreased locomotor activity, but no abnormalities were observed in all other behavioral tests. Del(1.5 Mb)/ + mice showed reduction of prepulse inhibition and impairment of contextual- and cued-dependent fear memory, which is consistent with previous reports. Furthermore, apparently intact social recognition in Del(1.4 Mb)/ + and Del(1.5 Mb)/ + mice suggests that the impaired social recognition observed in Del(3.0 Mb)/ + mice mimicking the human 3.0-Mb deletion requires mutations both in 1.4-Mb and 1.5 Mb regions. Our previous study has shown that Del(3.0 Mb)/ + mice presented disturbance of behavioral circadian rhythm. Therefore, we further evaluated sleep/wakefulness cycles in Del(3.0 Mb)/ + mice by electroencephalogram (EEG) and electromyogram (EMG) recording. EEG/EMG analysis revealed the disturbed wakefulness and non-rapid eye moving sleep (NREMS) cycles in Del(3.0 Mb)/ + mice, suggesting that Del(3.0 Mb)/ + mice may be unable to maintain their wakefulness. Together, our mouse models deepen our understanding of genetic contributions to schizophrenic phenotypes related to 22q11.2DS.
... For intact, gonadectomized, and hormone-supplemented groups, mice were implanted electroencephalogram (EEG)/electromyography (EMG) electrode at 7 weeks of age as described previously (Miyoshi et al., 2019), under anesthesia with isoflurane (4% for induction and 1% for maintenance). Each mouse was implanted with an EEG/EMG electrode containing four electrode pins and two flexible stainless steel wires. ...
... Recording environment was kept under a 12-h light/12-h dark cycle and a constant temperature (24-25 • C). EEG/EMG signaling was obtained and analyzed as previously described with some modifications (Miyoshi et al., 2019). EEG/EMG signals were amplified, filtered (EEG: 0.3-300 Hz; EMG: 30-300 Hz) with a multichannel amplifier (NIHON KODEN, #AB-611J), digitized at 250-Hz sampling rate using an analog-to-digital converter (National Instruments #PCI-6220) and LabView (National Instruments)-based custom-made software. ...
There are various sex differences in sleep/wake behaviors in mice. However, it is unclear whether there are sex differences in sleep homeostasis and arousal responses and whether gonadal hormones are involved in these sex differences. Here, we examined sleep/wake behaviors under baseline condition, after sleep deprivation by gentle handling, and arousal responses to repeated cage changes in male and female C57BL/6 mice that are hormonally intact, gonadectomized, or gonadectomized with hormone supplementation. Compared to males, females had longer wake time, shorter non-rapid eye movement sleep (NREMS) time, and longer rapid eye movement sleep (REMS) episodes. After sleep deprivation, males showed an increase in NREMS delta power, NREMS time, and REMS time, but females showed a smaller increase. Females and males showed similar arousal responses. Gonadectomy had only a modest effect on homeostatic sleep regulation in males but enhanced it in females. Gonadectomy weakened arousal response in males and females. With hormone replacement, baseline sleep in gonadectomized females was similar to that of intact females, and baseline sleep in gonadectomized males was close to that of intact males. Gonadal hormone supplementation restored arousal response in males but not in females. These results indicate that male and female mice differ in their baseline sleep–wake behavior, homeostatic sleep regulation, and arousal responses to external stimuli, which are differentially affected by reproductive hormones.
