Temperature-Dependent Upregulation of Per2 Protein Expression Is Mediated by eIF2α Kinases PERK and PKR through PI3K Activation.

Temperature-dependent translational control of the core clock gene Per2 plays an important role in establishing entrainment of the circadian clock to physiological body temperature cycles. Previously, we found an involvement of the phosphatidylinositol 3-kinase (PI3K) in causing Per2 protein expression in response to a warm temperature shift (WTS) within a physiological range (from 35 to 38.5 °C). However, signaling pathway mediating the Per2 protein expression in response to WTS is only sparsely understood. Additional factor(s) other than PI3K remains unknown. Here we report the identification of eukaryotic initiation factor 2α (eIF2α) kinases, protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK), as a novel mediator of WTS-dependent Per2 protein expression. Canonically, eIF2α has been regarded as a major downstream target of PERK and PKR. However, we found that PERK and PKR mediate WTS response of Per2 in a manner not involving eIF2α. We observed that PERK and PKR serve as an upstream regulator of PI3K rather than eIF2α in the context of WTS-dependent Per2 protein expression. There have been studies reporting PI3K activation occurring depending on PERK and PKR, while its physiological contribution has remained elusive. Our finding therefore not only helps to enrich the knowledge of how WTS affects Per2 protein expression but also extends the region of cellular biology involving the PERK/PKR-mediated PI3K activation to include entrainment-mechanism of the circadian clock.

Regulation of REM sleep in mice: The role of dopamine and serotonin function in the basolateral amygdala.

Animals have a sleep cycle that involves the repetitive occurrence of non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep. In a previous study, we discovered that a transient increase in dopamine (DA) levels in the basolateral amygdala (BLA) during NREM sleep terminates NREM sleep and initiates REM sleep by acting on Drd2-positive neurons (Hasegawa et al., 2022). In this study, we identified the neurons activated by the transient increase of DA in the BLA and found that chemogenetic excitation of these neurons increased REM sleep. Additionally, we demonstrated that acute inhibition of serotonin (5HT) in the BLA elicited a transient increase in DA in the BLA, which triggered REM sleep.

The expression of matrix metalloproteinase-12 in the peritoneum of rats with continuous peritoneal dialysis.

BACKGROUND: Peritoneal dialysis (PD) is an important alternative treatment for end-stage renal disease. Continuous exposure to non-physiological fluids during PD is associated with pathological responses, such as sustained microinflammation, leading to tissue fibrosis and angiogenesis. However, the effect of PD fluid on submesothelial cells has not yet been investigated in detail. METHODS: We investigated the association between macrophages and the expression of matrix metalloproteinase-12 (MMP-12), an elastin proteinase secreted by macrophages, in the peritoneal tissue of rats undergoing continuous PD. RESULTS: Morphological data revealed that the submesothelial layer of the peritoneum in PD model rats was markedly thickened, with fibrosis and angiogenesis. In the fibrillization area, elastin was disorganized and fragmented, and macrophages accumulated, which tended to have M2 characteristics. The expression of MMP-12 was enhanced by continuous exposure to PD fluid, suggesting that MMP-12 expression may be involved in PD fluid-induced peritoneal damage. CONCLUSIONS: The results of this study may lead to a better understanding of the mechanisms underlying fibrosis in PD.

Transcriptome and Methylome Profiling in Rat Skeletal Muscle: Impact of Post-Weaning Protein Restriction.

Skeletal muscle is programmable, and early-life nutritional stimuli may form epigenetic memory in the skeletal muscle, thus impacting adult muscle function, aging, and longevity. In the present study, we designed a one-month protein restriction model using post-weaning rats, followed by a two-month rebound feeding, to investigate how early-life protein restriction affects overall body growth and muscle development and whether these influences could be corrected by rebound feeding. We observed comprehensive alterations immediately after protein restriction, including retarded growth, altered biochemical indices, and disturbed hormone secretion. Transcriptome profiling of the gastrocnemius muscle followed by gene ontology analyses revealed that "myogenic differentiation functions" were upregulated, while "protein catabolism" was downregulated as a compensatory mechanism, with enhanced endoplasmic reticulum stress and undesired apoptosis. Furthermore, methylome profiling of the gastrocnemius muscle showed that protein restriction altered the methylation of apoptotic and hormone secretion-related genes. Although most of the alterations were reversed after rebound feeding, 17 genes, most of which play roles during muscle development, remained altered at the transcriptional level. In summary, early-life protein restriction may undermine muscle function in the long term and affect skeletal muscle development at the both transcriptional and methylation levels, which may hazard future muscle health.

[Rapid Eye Movement Sleep Is Initiated by Dopamine Signaling in the Basolateral Amygdala in Mice].

The sleep cycle alternates between REM and NREM sleep, but the mechanisms by which this cycle is generated are totally unknown. We found that a periodic transient increase of the dopamine level in the amygdala during NREM sleep terminates NREM sleep and initiates REM sleep. This mechanism also plays a role in cataplectic attack, which is a pathological intrusion of REM sleep into wakefulness in narcoleptics.

Lateral habenula glutamatergic neurons projecting to the dorsal raphe nucleus promote aggressive arousal in mice.

The dorsal raphe nucleus (DRN) is known to control aggressive behavior in mice. Here, we found that glutamatergic projections from the lateral habenula (LHb) to the DRN were activated in male mice that experienced pre-exposure to a rival male mouse ("social instigation") resulting in heightened intermale aggression. Both chemogenetic and optogenetic suppression of the LHb-DRN projection blocked heightened aggression after social instigation in male mice. In contrast, inhibition of this pathway did not affect basal levels of aggressive behavior, suggesting that the activity of the LHb-DRN projection is not necessary for the expression of species-typical aggressive behavior, but required for the increase of aggressive behavior resulting from social instigation. Anatomical analysis showed that LHb neurons synapse on non-serotonergic DRN neurons that project to the ventral tegmental area (VTA), and optogenetic activation of the DRN-VTA projection increased aggressive behaviors. Our results demonstrate that the LHb glutamatergic inputs to the DRN promote aggressive arousal induced by social instigation, which contributes to aggressive behavior by activating VTA-projecting non-serotonergic DRN neurons as one of its potential targets.

Discovery of novel orexin receptor antagonists using a 1,3,5-trioxazatriquinane bearing multiple effective residues (TriMER) library.

Structurally diverse small compounds are utilized to obtain hit compounds that have suitable pharmacophores in appropriate three-dimensional conformations for the target drug receptors. We have focused on the 1,3,5-trioxazatriquinane skeleton, which has a rigid bowl-like structure enabling the diverse orientation of side chain units, leading to a novel small-scale focused library based on the skeleton. In the library screening for the orexin receptor, some of the compounds showed orexin receptor antagonistic activity with a high hit rate of 7%. By optimizing the hit compounds, we discovered a potent dual orexin receptor antagonist, 38b, and a selective orexin 1 receptor antagonist, 41b carrying the same plane structure. Both compounds showed reasonable brain permeability and beneficial effects when administered intraperitoneally to wild-type mice. Docking simulations of their eutomers, (-)-38b and (+)-41b, with orexin receptors suggested that the interaction between the 1,3,5-trioxazatriquinane core structure and the hydrophobic subpocket in orexin receptors enables a U-shape structure, which causes tight van der Waals interactions with the receptors similar to SB-334867, a selective orexin 1 receptor antagonist. These results indicate that the library approach utilizing the 1,3,5-trioxazatriquinanes bearing multiple effective residues (TriMERs) might be useful for the hit discovery process targeting not only opioid and orexin receptors but other G-protein coupled receptors.

Rapid eye movement sleep is initiated by basolateral amygdala dopamine signaling in mice.

The sleep cycle is characterized by alternating non-rapid eye movement (NREM) and rapid eye movement (REM) sleeps. The mechanisms by which this cycle is generated are incompletely understood. We found that a transient increase of dopamine (DA) in the basolateral amygdala (BLA) during NREM sleep terminates NREM sleep and initiates REM sleep. DA acts on dopamine receptor D2 (Drd2)-expressing neurons in the BLA to induce the NREM-to-REM transition. This mechanism also plays a role in cataplectic attacks-a pathological intrusion of REM sleep into wakefulness-in narcoleptics. These results show a critical role of DA signaling in the BLA in initiating REM sleep and provide a neuronal basis for sleep cycle generation.

[Evaluation on a Simultaneous Analytical Method of Pesticide Residues in Limes].

A LC-MS/MS simultaneous analytical method for screening 191 pesticide residues in limes had been developed and validated. Pesticides were extracted with acetonitrile from samples. Then mixed salts, which were anhydrous magnesium sulfate for dehydration, sodium carbonate for adjusting pH, and sodium chloride for salting out, were added to the sample. After centrifugation, supernatant was transferred to a tube. The sample solution was cleaned up using solid phase extraction (SPE) with C18/GC/PSA for the determination by LC-MS/MS. The developed method was improved the recovery rate of thiabendazole, which had a low recovery rate by the conventional method. Validation study, which was following the guidelines of the Ministry of Health, Labor and Welfare, were carried out at 0.01 and 0.1 µg g-1 to evaluate the method. The results of 175 pesticides out of 191 were with satisfactory. A total of 19 imported lime samples sold in Tokyo was analyzed to evaluate the method, then 18 samples contained pesticide residues below MRLs. The developed method is applicable for detection of pesticide residues in lime.

GABA from vasopressin neurons regulates the time at which suprachiasmatic nucleus molecular clocks enable circadian behavior.

The suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals, is a network structure composed of multiple types of γ-aminobutyric acid (GABA)-ergic neurons and glial cells. However, the roles of GABA-mediated signaling in the SCN network remain controversial. Here, we report noticeable impairment of the circadian rhythm in mice with a specific deletion of the vesicular GABA transporter in arginine vasopressin (AVP)-producing neurons. These mice showed disturbed diurnal rhythms of GABAA receptor-mediated synaptic transmission in SCN neurons and marked lengthening of the activity time in circadian behavioral rhythms due to the extended interval between morning and evening locomotor activities. Synchrony of molecular circadian oscillations among SCN neurons did not significantly change, whereas the phase relationships between SCN molecular clocks and circadian morning/evening locomotor activities were altered significantly, as revealed by PER2::LUC imaging of SCN explants and in vivo recording of intracellular Ca2+ in SCN AVP neurons. In contrast, daily neuronal activity in SCN neurons in vivo clearly showed a bimodal pattern that correlated with dissociated morning/evening locomotor activities. Therefore, GABAergic transmission from AVP neurons regulates the timing of SCN neuronal firing to temporally restrict circadian behavior to appropriate time windows in SCN molecular clocks.

A discrete neuronal circuit induces a hibernation-like state in rodents.

Hibernating mammals actively lower their body temperature to reduce energy expenditure when facing food scarcity1. This ability to induce a hypometabolic state has evoked great interest owing to its potential medical benefits2,3. Here we show that a hypothalamic neuronal circuit in rodents induces a long-lasting hypothermic and hypometabolic state similar to hibernation. In this state, although body temperature and levels of oxygen consumption are kept very low, the ability to regulate metabolism still remains functional, as in hibernation4. There was no obvious damage to tissues and organs or abnormalities in behaviour after recovery from this state. Our findings could enable the development of a method to induce a hibernation-like state, which would have potential applications in non-hibernating mammalian species including humans.

Surveillance of Chlorantraniliprole Residues in Vegetables and Fruits Using LC-MS/MS.

We measured the residual amounts of chlorantraniliprole in various vegetables and fruits. Sample solutions were prepared according to our routine procedure based on the QuEChERS method and analyzed by LC-MS/MS. Performance characteristics were evaluated for 8 kinds of food samples by means of recovery tests of 5 replicates at the concentration of 10 ng/g. Recoveries and RSDs (％) ranged from 50.2 to 93.4％ and from 2.1 to 9.7％, respectively. Application of this method to survey 207 vegetables and 163 fruits gave detection rates of 8.2 and 1.2％, respectively. In vegetables, detection rates were high in okra (4 out of 10 samples), paprika (4 out of 23 samples) and tomato (2 out of 6 samples), and leaf vegetables such as lettuce, mizuna, spinach and wrinkled greens also contained high concentrations of chlorantraniliprole. The highest residual concentration was 571 ng/g in mizuna. The samples containing chlorantraniliprole seemed to be mainly from Asian countries, including samples of domestic Japanese origin. However, none of them contained more than the MRL, which suggests that the use of chlorantraniliprole has been properly controlled.

Sleep and Wakefulness Are Controlled by Ventral Medial Midbrain/Pons GABAergic Neurons in Mice.

Sleep-wake behavior is controlled by a wide range of neuronal populations in the mammalian brain. Although the ventral midbrain/pons (VMP) area is suggested to participate in sleep-wake regulation, the neuronal mechanisms have remained unclear. Here, we found that nonspecific cell ablation or selective ablation of GABAergic neurons by expressing diphtheria toxin fragment A in the VMP in male mice induced a large increase in wakefulness that lasted at least 4 weeks. In contrast, selective ablation of dopaminergic neurons in the VMP had little effect on wakefulness. Chemogenetic inhibition of VMP GABAergic neurons also markedly increased wakefulness. The wake-promoting effect of the VMP GABAergic neuron ablation or inhibition was attenuated to varying degrees by the administration of dopamine D1 or D2/3 receptor antagonists and abolished by the administration of both antagonists together. In contrast, chemogenetic activation of VMP GABAergic neurons very strongly increased slow-wave sleep and reduced wakefulness. These findings suggest that VMP GABAergic neurons regulate dopaminergic actions in the sleep-wake behavior of mice.SIGNIFICANCE STATEMENT Current understanding of the neuronal mechanisms and populations that regulate sleep-wake behavior is incomplete. Here, we identified a GABAergic ventral midbrain/pons area that is necessary for controlling the daily amount of sleep and wakefulness in mice. We also found that these inhibitory neurons control wakefulness by suppressing dopaminergic systems. Surprisingly, activation of these neurons strongly induced slow-wave sleep while suppressing wakefulness. Our study reveals a new brain mechanism critical for sleep-wake regulation.

Monoamines Inhibit GABAergic Neurons in Ventrolateral Preoptic Area That Make Direct Synaptic Connections to Hypothalamic Arousal Neurons.

The hypothalamus plays an important role in the regulation of sleep/wakefulness states. While the ventrolateral preoptic nucleus (VLPO) plays a critical role in the initiation and maintenance of sleep, the lateral posterior part of the hypothalamus contains neuronal populations implicated in maintenance of arousal, including orexin-producing neurons (orexin neurons) in the lateral hypothalamic area (LHA) and histaminergic neurons in the tuberomammillary nucleus (TMN). During a search for neurons that make direct synaptic contact with histidine decarboxylase-positive (HDC+), histaminergic neurons (HDC neurons) in the TMN and orexin neurons in the LHA of male mice, we found that these arousal-related neurons are heavily innervated by GABAergic neurons in the preoptic area including the VLPO. We further characterized GABAergic neurons electrophysiologically in the VLPO (GABAVLPO neurons) that make direct synaptic contact with these hypothalamic arousal-related neurons. These neurons (GABAVLPO→HDC or GABAVLPO→orexin neurons) were both potently inhibited by noradrenaline and serotonin, showing typical electrophysiological characteristics of sleep-promoting neurons in the VLPO. This work provides direct evidence of monosynaptic connectivity between GABAVLPO neurons and hypothalamic arousal neurons and identifies the effects of monoamines on these neuronal pathways.SIGNIFICANCE STATEMENT Rabies-virus-mediated tracing of input neurons of two hypothalamic arousal-related neuron populations, histaminergic and orexinergic neurons, showed that they receive similar distributions of input neurons in a variety of brain areas, with rich innervation by GABAergic neurons in the preoptic area, including the ventrolateral preoptic area (VLPO), a region known to play an important role in the initiation and maintenance of sleep. Electrophysiological experiments found that GABAergic neurons in the VLPO (GABAVLPO neurons) that make direct input to orexin or histaminergic neurons are potently inhibited by noradrenaline and serotonin, suggesting that these monoamines disinhibit histamine and orexin neurons. This work demonstrated functional and structural interactions between GABAVLPO neurons and hypothalamic arousal-related neurons.

Large-scale forward genetics screening identifies Trpa1 as a chemosensor for predator odor-evoked innate fear behaviors.

Innate behaviors are genetically encoded, but their underlying molecular mechanisms remain largely unknown. Predator odor 2,4,5-trimethyl-3-thiazoline (TMT) and its potent analog 2-methyl-2-thiazoline (2MT) are believed to activate specific odorant receptors to elicit innate fear/defensive behaviors in naive mice. Here, we conduct a large-scale recessive genetics screen of ethylnitrosourea (ENU)-mutagenized mice. We find that loss of Trpa1, a pungency/irritancy receptor, diminishes TMT/2MT and snake skin-evoked innate fear/defensive responses. Accordingly, Trpa1 -/- mice fail to effectively activate known fear/stress brain centers upon 2MT exposure, despite their apparent ability to smell and learn to fear 2MT. Moreover, Trpa1 acts as a chemosensor for 2MT/TMT and Trpa1-expressing trigeminal ganglion neurons contribute critically to 2MT-evoked freezing. Our results indicate that Trpa1-mediated nociception plays a crucial role in predator odor-evoked innate fear/defensive behaviors. The work establishes the first forward genetics screen to uncover the molecular mechanism of innate fear, a basic emotion and evolutionarily conserved survival mechanism.

Serotonin neurons in the dorsal raphe mediate the anticataplectic action of orexin neurons by reducing amygdala activity.

Narcolepsy is a sleep disorder caused by the loss of orexin (hypocretin)-producing neurons and marked by excessive daytime sleepiness and a sudden weakening of muscle tone, or cataplexy, often triggered by strong emotions. In a mouse model for narcolepsy, we previously demonstrated that serotonin neurons of the dorsal raphe nucleus (DRN) mediate the suppression of cataplexy-like episodes (CLEs) by orexin neurons. Using an optogenetic tool, in this paper we show that the acute activation of DRN serotonin neuron terminals in the amygdala, but not in nuclei involved in regulating rapid eye-movement sleep and atonia, suppressed CLEs. Not only did stimulating serotonin nerve terminals reduce amygdala activity, but the chemogenetic inhibition of the amygdala using designer receptors exclusively activated by designer drugs also drastically decreased CLEs, whereas chemogenetic activation increased them. Moreover, the optogenetic inhibition of serotonin nerve terminals in the amygdala blocked the anticataplectic effects of orexin signaling in DRN serotonin neurons. Taken together, the results suggest that DRN serotonin neurons, as a downstream target of orexin neurons, inhibit cataplexy by reducing the activity of amygdala as a center for emotional processing.

Fine-Tuning Circadian Rhythms: The Importance of Bmal1 Expression in the Ventral Forebrain.

Although, the suprachiasmatic nucleus (SCN) of the hypothalamus acts as the central clock in mammals, the circadian expression of clock genes has been demonstrated not only in the SCN, but also in peripheral tissues and brain regions outside the SCN. However, the physiological roles of extra-SCN circadian clocks in the brain remain largely elusive. In response, we generated Nkx2.1-Bmal1-/- mice in which Bmal1, an essential clock component, was genetically deleted specifically in the ventral forebrain, including the preoptic area, nucleus of the diagonal band, and most of the hypothalamus except the SCN. In these mice, as expected, PER2::LUC oscillation was drastically attenuated in the explants of mediobasal hypothalamus, whereas it was maintained in those of the SCN. Although, Nkx2.1-Bmal1-/- mice were rhythmic and nocturnal, they showed altered patterns of locomotor activity during the night in a 12:12-h light:dark cycle and during subjective night in constant darkness. Control mice were more active during the first half than the second half of the dark phase or subjective night, whereas Nkx2.1-Bmal1-/- mice showed the opposite pattern of locomotor activity. Temporal patterns of sleep-wakefulness and feeding also changed accordingly. Such results suggest that along with mechanisms in the SCN, local Bmal1-dependent clocks in the ventral forebrain are critical for generating precise temporal patterns of circadian behaviors.

Corrigendum to "High glucose concentration-induced expression of pentraxin-3 in a rat model of continuous peritoneal dialysis".

This correct the article DOI: 10.14670/HH-11-756.