Crucial role of TFAP2B in the nervous system for regulating NREM sleep.

The AP-2 transcription factors are crucial for regulating sleep in both vertebrate and invertebrate animals. In mice, loss of function of the transcription factor AP-2ß (TFAP2B) reduces non-rapid eye movement (NREM) sleep. When and where TFAP2B functions, however, is unclear. Here, we used the Cre-loxP system to generate mice in which Tfap2b was specifically deleted in the nervous system during development and mice in which neuronal Tfap2b was specifically deleted postnatally. Both types of mice exhibited reduced NREM sleep, but the nervous system-specific deletion of Tfap2b resulted in more severe sleep phenotypes accompanied by defective light entrainment of the circadian clock and stereotypic jumping behavior. These findings indicate that TFAP2B in postnatal neurons functions at least partly in sleep regulation and imply that TFAP2B also functions either at earlier stages or in additional cell types within the nervous system.

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.

Aripiprazole disrupts cellular synchrony in the suprachiasmatic nucleus and enhances entrainment to environmental light-dark cycles in mice.

Many patients with psychiatric conditions, such as bipolar disorder and major depressive disorder, frequently experience disruptions in their sleep-wake cycles. Several case studies and clinical trials have shown that the administration of aripiprazole, a commonly prescribed antipsychotic drug, alleviates the symptoms of circadian sleep disorders in these patients. This improvement may be attributed to the effects of aripiprazole on the circadian central clock, specifically the hypothalamic suprachiasmatic nucleus (SCN), which regulates various circadian physiological rhythms, including the sleep-wake cycle, in mammals. To examine whether aripiprazole facilitates adaptation to changes in the light-dark cycle, we orally administered aripiprazole to mice and subjected them to jet-lag experiments. Mice receiving aripiprazole were more rapidly entrained to 6 h advanced light-dark cycles. Moreover, we examined the effect of aripiprazole on the cellular rhythms of SCN slice cultures and found that aripiprazole disrupted cellular synchronization in the SCN, thereby accelerating the damping of the SCN rhythm at the population level. Adenosine 3'5' monophosphate (cAMP) assay using a bioluminescence indicator revealed that intracellular cAMP level in the SCN increased following aripiprazole treatment. However, this increase was blocked by pre-treatment with the serotonin 1A receptor (5-HT1AR) antagonist. Based on these findings, we propose that aripiprazole modulates intracellular signaling, including 5-HT1AR-mediated cAMP signaling, and desynchronizes SCN neurons, ultimately leading to enhanced entrainment to phase advanced light-dark cycles in mice. These findings indicate that the improvement in sleep symptoms reported in patients with psychiatric disorders receiving aripiprazole may be due to modulation of the circadian clock. Our study provides novel insights into the potential clinical applications of aripiprazole in patients with various circadian sleep disorders.

GABAergic modulation of sleep-wake states.

Benzodiazepine, a classical medication utilized in the treatment of insomnia, operates by augmenting the activity of the GABAA receptor. This underscores the significance of GABAergic neurotransmission in both the initiation and maintenance of sleep. Nevertheless, an increasing body of evidence substantiates the notion that GABA-mediated neurotransmission also assumes a vital role in promoting wakefulness in specific neuronal circuits. Despite the longstanding belief in the pivotal function of GABA in regulating the sleep-wake cycle, there exists a dearth of comprehensive documentation regarding the specific regions within the central nervous system where GABAergic neurons are crucial for these functions. In this review, we delve into the involvement of GABAergic neurons in the regulation of sleep-wake cycles, with particular focus on those located in the preoptic area (POA) and ventral tegmental area (VTA). Recent research, including our own, has further underscored the importance of GABAergic neurotransmission in these areas for the regulation of sleep-wake cycles.

The present and future of synthetic orexin receptor agonists.

The neuropeptide orexin/hypocretin plays a crucial role in various physiological processes, including the regulation of sleep/wakefulness, appetite, emotion and the reward system. Dysregulation of orexin signaling has been implicated in hypersomnia, especially in narcolepsy, which is a chronic neurological disorder characterized by excessive daytime sleepiness (EDS), sudden loss of muscle tone while awake (cataplexy), sleep paralysis, and hallucinations. Small-molecule orexin receptor agonists have emerged as promising therapeutics for these disorders, and significant progress has been made in this field in the past decade. This review summarizes recent advances in the design and synthesis of orexin receptor agonists, with a focus on peptidic and small-molecule OX2R-selective, dual, and OX1R-selective agonists. The review discusses the key structural features and pharmacological properties of these agonists, as well as their potential therapeutic applications.

Measuring body temperature of freely moving mice under an optogenetics-induced long-term hypothermic state.

We present a protocol for inducing a hibernation-like state in free-moving mice using optogenetics. We have recently developed an optogenetic technique utilizing modified Opsin4, which is activated by weak blue light, resulting in prolonged neuronal excitation. We describe a protocol that includes detailed instructions for virus injection, implantation of optic fibers and temperature transmitters, photostimulation, and real-time recording of body temperature in mice. This method is valuable for investigating the mechanisms underlying torpor and thermoregulation in mice. For complete details on the use and execution of this protocol, please refer to Takahashi et al.1.

Analysis of Odor-Tracking Performance of Silk Moth Using a Sensory-Motor Intervention System.

Animals can adaptively behave in different environmental conditions by converting environmental information obtained from their sensory organs into actions. This sensory-motor integration enables the accomplishment of various tasks and is essential for animal survival. This sensory-motor integration also plays an important role in localization to females, relying on sex pheromones floating in space. In this study, we focused on the localization behavior of the adult male silk moth, Bombyx mori. We investigated sensory-motor integration against time delay using odor plume tracking performance as an index when we set a certain time delay for the sensory and motor responses. Given that it is difficult to directly intervene in the sensory and motor functions of the silk moth, we constructed an intervention system based on a mobile behavior measurement system controlled by them. Using this intervention system, not only can timing the detection of the odor in the environment and timing the presentation of the odor to the silk moth be manipulated, but timing the reflection of the movement of the silk moth can also be manipulated. We analyzed the extent to which the localization strategy of the silk moth could tolerate sensory delays by setting a delay to the odor presentation. We also evaluated behavioral compensation by odor sensory feedback by setting a delay to the motor. The results of the localization experiment have shown that the localization success rate did not decrease when there was a motor delay. However, when there was a sensory delay, the success rate decreased depending on the time delay. Analysis of the change in behavior after detection of the odor stimulus has shown that the movement was more linear when we set a motor delay. However, the movement was accompanied by a large rotational movement when there was a delay in the sensory input. This result has suggested that behavior is compensated for the delay in motor function by feedback control of odor sensation, but not when accompanied by sensory delay. To compensate for this, the silk moth may acquire appropriate information from the environment by making large body movements.

Health profession students' outlooks on the medical profession during the COVID-19 pandemic: a global perspective.

BACKGROUND: This article summarizes a global study of the effect of the COVID-19 pandemic on junior health professions students' outlook on medicine. The pandemic has significantly affected health professions education. There is limited understanding of how students' pandemic experiences will affect them, and what impact these events may have on their career paths or the future of the professions. This information is important as it impacts the future of medicine. METHODS: In the Fall 2020 semester, 219 health professions students at 14 medical universities worldwide responded to the question: 'Has this experience (with COVID-19) changed your outlook on medicine as a profession?'. Short essay responses were semantically coded and organized into themes and subthemes using an inductive approach to thematic analysis. RESULTS: 145 responses were submitted. Themes were identified: (1) students reflected on the interaction between politics and healthcare; (2) reported becoming more aware of the societal expectations placed on healthcare professionals, including undertaking high risks and the sacrifices that healthcare professionals must make; (3) found reassurance from the recognized importance of healthcare professionals and expressed pride to be entering the profession; and (4) reflected on the current state of healthcare, including its limitations and future. CONCLUSION: Most students, independent of the extent of the pandemic in their respective countries, noted a change in their outlook regarding medicine. An overall positive outlook was noted in most junior students. Educators need to work on nurturing these sentiments and attitudes to help young students maintain a healthy relationship towards their chosen profession.

Singularity response reveals entrainment properties in mammalian circadian clock.

Entrainment is characterized by phase response curves (PRCs), which provide a summary of responses to perturbations at each circadian phase. The synchronization of mammalian circadian clocks is accomplished through the receipt of a variety of inputs from both internal and external time cues. A comprehensive comparison of PRCs for various stimuli in each tissue is required. Herein, we demonstrate that PRCs in mammalian cells can be characterized using a recently developed estimation method based on singularity response (SR), which represents the response of desynchronized cellular clocks. We confirmed that PRCs can be reconstructed using single SR measurements and quantified response properties for various stimuli in several cell lines. SR analysis reveals that the phase and amplitude after resetting are distinguishable among stimuli. SRs in tissue slice cultures reveal tissue-specific entrainment properties. These results demonstrate that SRs can be employed to unveil entrainment mechanisms with diverse stimuli in multiscale mammalian clocks.

Analysis of circadian rhythm components in EEG/EMG data of aged mice.

Aging disrupts circadian clocks, as evidenced by a reduction in the amplitude of circadian rhythms. Because the circadian clock strongly influences sleep-wake behavior in mammals, age-related alterations in sleep-wake patterns may be attributable, at least partly, to functional changes in the circadian clock. However, the effect of aging on the circadian characteristics of sleep architecture has not been well assessed, as circadian behaviors are usually evaluated through long-term behavioral recording with wheel-running or infrared sensors. In this study, we examined age-related changes in circadian sleep-wake behavior using circadian components extracted from electroencephalography (EEG) and electromyography (EMG) data. EEG and EMG were recorded from 12 to 17-week-old and 78 to 83-week-old mice for 3 days under light/dark and constant dark conditions. We analyzed time-dependent changes in the duration of sleep. Rapid eye movement (REM) and non-REM (NREM) sleep significantly increased during the night phase in old mice, whereas no significant change was observed during the light phase. The circadian components were then extracted from the EEG data for each sleep-wake stage, revealing that the circadian rhythm in the power of delta waves during NREM sleep was attenuated and delayed in old mice. Furthermore, we used machine learning to evaluate the phase of the circadian rhythm, with EEG data serving as the input and the phase of the sleep-wake rhythm (environmental time) as the output. The results indicated that the output time for the old mice data tended to be delayed, specifically at night. These results indicate that the aging process significantly impacts the circadian rhythm in the EEG power spectrum despite the circadian rhythm in the amounts of sleep and wake attenuated but still remaining in old mice. Moreover, EEG/EMG analysis is useful not only for evaluating sleep-wake stages but also for circadian rhythms in the brain.

Structure and Function of Neuronal Circuits Linking Ventrolateral Preoptic Nucleus and Lateral Hypothalamic Area.

To understand how sleep-wakefulness cycles are regulated, it is essential to disentangle structural and functional relationships between the preoptic area (POA) and lateral hypothalamic area (LHA), since these regions play important yet opposing roles in the sleep-wakefulness regulation. GABA- and galanin (GAL)-producing neurons in the ventrolateral preoptic nucleus (VLPO) of the POA (VLPOGABA and VLPOGAL neurons) are responsible for the maintenance of sleep, while the LHA contains orexin-producing neurons (orexin neurons) that are crucial for maintenance of wakefulness. Through the use of rabies virus-mediated neural tracing combined with in situ hybridization (ISH) in male and female orexin-iCre mice, we revealed that the vesicular GABA transporter (Vgat, Slc32a1)- and galanin (Gal)-expressing neurons in the VLPO directly synapse with orexin neurons in the LHA. A majority (56.3 ± 8.1%) of all VLPO input neurons connecting to orexin neurons were double-positive for Vgat and Gal Using projection-specific rabies virus-mediated tracing in male and female Vgat-ires-Cre and Gal-Cre mice, we discovered that VLPOGABA and VLPOGAL neurons that send projections to the LHA received innervations from similarly distributed input neurons in many brain regions, with the POA and LHA being among the main upstream areas. Additionally, we found that acute optogenetic excitation of axons of VLPOGABA neurons, but not VLPOGAL neurons, in the LHA of male Vgat-ires-Cre mice induced wakefulness. This study deciphers the connectivity between the VLPO and LHA, provides a large-scale map of upstream neuronal populations of VLPO→LHA neurons, and reveals a previously uncovered function of the VLPOGABA→LHA pathway in the regulation of sleep and wakefulness.SIGNIFICANCE STATEMENT We identified neurons in the ventrolateral preoptic nucleus (VLPO) that are positive for vesicular GABA transporter (Vgat) and/or galanin (Gal) and serve as presynaptic partners of orexin-producing neurons in the lateral hypothalamic area (LHA). We depicted monosynaptic input neurons of GABA- and galanin-producing neurons in the VLPO that send projections to the LHA throughout the entire brain. Their input neurons largely overlap, suggesting that they comprise a common neuronal population. However, acute excitatory optogenetic manipulation of the VLPOGABA→LHA pathway, but not the VLPOGAL→LHA pathway, evoked wakefulness. This study shows the connectivity of major components of the sleep/wake circuitry in the hypothalamus and unveils a previously unrecognized function of the VLPOGABA→LHA pathway in sleep-wakefulness regulation. Furthermore, we suggest the existence of subpopulations of VLPOGABA neurons that innervate LHA.

Shank3a/b isoforms regulate the susceptibility to seizures and thalamocortical development in the early postnatal period of mice.

Epileptic seizures are distinct but frequent comorbidities in children with autism spectrum disorder (ASD). The hyperexcitability of cortical and subcortical neurons appears to be involved in both phenotypes. However, little information is available concerning which genes are involved and how they regulate the excitability of the thalamocortical network. In this study, we investigate whether an ASD-associated gene, SH3 and multiple ankyrin repeat domains 3 (Shank3), plays a unique role in the postnatal development of thalamocortical neurons. We herein report that Shank3a/b, the splicing isoforms of mouse Shank3, were uniquely expressed in the thalamic nuclei, peaking from two to four weeks after birth. Shank3a/b-knockout mice showed lower parvalbumin signals in the thalamic nuclei. Consistently, Shank3a/b-knockout mice were more susceptible to generalized seizures than wild-type mice after kainic acid treatments. Together, these data indicate that NT-Ank domain of Shank3a/b regulates molecular pathways that protect thalamocortical neurons from hyperexcitability during the early postnatal period of mice.

A thematic analysis of students' discussions on death and body donation in international online focus groups.

Historically, Anatomy education is an in-person discipline involving exposure to human body donors that facilitates personal and professional growth through, in part, the initiation of reflection on the topic of death. However, during the COVID-19 pandemic the decreased exposure to cadaveric anatomy for many health professions students may have influenced the depth of their individual reflections on this topic. Accordingly, this study aimed to investigate the effect of an alternate approach-focus group discussions between peers with varying degrees of exposure to cadaveric material-that may offer one strategy to stimulate deep reflection on the topic of death. A programmatic intervention was introduced, wherein students (n = 221) from 13 international universities discussed differences in their anatomy courses during small focus group sessions as part of an online exchange program. An inductive semantic thematic analysis was conducted on responses to an open-ended text-response question on how the activity influenced students' reflections about death. Resulting themes were organized into categories that described the content and topics of the students' discussions as they grappled with this sensitive topic. The students reportedly engaged in deep reflection and expressed an increased sense of connectedness with their peers, despite their disparate exposure levels to cadaveric anatomy and being physically distanced. This demonstrates that focus groups with students experiencing different laboratory contexts can be used to help all students reflect on the topic of death and that interchanges between dissecting and non-dissecting students can initiate thoughts about death and body donation among non-dissecting students.

Optogenetic induction of hibernation-like state with modified human Opsin4 in mice.

We recently determined that the excitatory manipulation of Qrfp-expressing neurons in the preoptic area of the hypothalamus (quiescence-inducing neurons [Q neurons]) induced a hibernation-like hypothermic/hypometabolic state (QIH) in mice. To control the QIH with a higher time resolution, we develop an optogenetic method using modified human opsin4 (OPN4; also known as melanopsin), a G protein-coupled-receptor-type blue-light photoreceptor. C-terminally truncated OPN4 (OPN4dC) stably and reproducibly induces QIH for at least 24 h by illumination with low-power light (3 µW, 473 nm laser) with high temporal resolution. The high sensitivity of OPN4dC allows us to transcranially stimulate Q neurons with blue-light-emitting diodes and non-invasively induce the QIH. OPN4dC-mediated QIH recapitulates the kinetics of the physiological changes observed in natural hibernation, revealing that Q neurons concurrently contribute to thermoregulation and cardiovascular function. This optogenetic method may facilitate identification of the neural mechanisms underlying long-term dormancy states such as sleep, daily torpor, and hibernation.

Hypothalamic orexin prevents non-alcoholic steatohepatitis and hepatocellular carcinoma in obesity.

Non-alcoholic steatohepatitis (NASH) occasionally occurs under obesity; however, factors modulating the natural history of fatty liver disease remain unknown. Since hypothalamic orexin that regulates physical activity and autonomic balance prevents obesity, we investigate its role in NASH development. Male orexin-deficient mice fed a high-fat diet (HFD) show severe obesity and progression of NASH with fibrosis in the liver. Hepatic fibrosis also develops in ovariectomized orexin-deficient females fed an HFD but not ovariectomized wild-type controls. Moreover, long-term HFD feeding causes hepatocellular carcinoma (HCC) in orexin-deficient mice. Intracerebroventricular injection of orexin A or pharmacogenetic activation of orexin neurons acutely activates hepatic mTOR-sXbp1 pathway to prevent endoplasmic reticulum (ER) stress, a NASH-causing factor. Daily supplementation of orexin A attenuates hepatic ER stress and inflammation in orexin-deficient mice fed an HFD, and autonomic ganglionic blocker suppresses the orexin actions. These results suggest that hypothalamic orexin is an essential factor for preventing NASH and associated HCC under obesity.

Lipid droplets in the pheromone glands of bombycids: Effects of larval diet on their size and pheromone titer.

In addition to the blend ratio, the quantity of sex pheromone components secreted by female moths may affect the efficient attraction of conspecific males. The present study using the silkmoth Bombyx mori, which has bombykol as its pheromone component, demonstrated that pheromone titer, body weight, and lipid droplet (LD) diameter in the pheromone gland were affected by the larval diet. Although the artificial diet contained approximately 11-fold more total fatty acids than mulberry leaf, the pheromone titer in the group fed the artificial diet (group AD) was approximately 2-fold higher than that of the group fed mulberry (group M). The diameter of LDs, which store the pheromone-precursor fatty acyl, E10,Z12-16:Acyl, was also larger in the AD group. The relatively small increase in sex pheromone titer by feeding on a fatty-acid-rich diet may be partly attributable to the storage of excess precursors in the LDs. We detected LDs in the pheromone glands of Trilocha varians, the closest non-congener of B. mori available in Bombycidae. T. varians uses bombykal and bombykyl acetate as sex pheromone components, which are biosynthesized via the same precursor fatty acyl as that of B. mori. The presence of LDs in T. varians suggests that the storage and mobilization mechanisms of the pheromone precursor fatty acyl via LDs may be conserved in bombycids.

The Anatomy Course During COVID-19: The Impact of Cadaver-Based Learning on the Initiation of Reflection on Death.

Background: During the COVID-19 pandemic, in-person cadaveric dissection laboratories for teaching anatomy were omitted by many schools around the world. While knowledge domains can be easily evaluated via remote exams, non-traditional discipline-independent skills such as those encouraged through reflection on the topic of death are often overlooked. This study investigated how different anatomy course formats played a role in initiating students' reflections on death during the COVID-19 pandemic. Method: In fall 2020, 217 medical, dental, premedical, and health sciences students from 13 international universities discussed differences in their anatomy courses online. Formats of anatomy courses ranged from dissection-based, prosection-based, hybrid (combination of dissection and prosection) to no laboratory exposure at all. Students' responses to the question, "Did/does your anatomy course initiate your thinking about life's passing?" were collected, and they self-reported themes that were present in their reflections on death using a multiple-choice prompt. Statistical analyses to detect differences between students with and without exposure to cadavers were performed using the chi-squared test. Results: When comparing students who had exposure to human anatomical specimens to those who had no exposure, the majority of students with exposure thought that the course did initiate thoughts about life's passing, compared to students without exposure (P < 0.05). Reflection themes were consistent across groups. Discussion: These findings indicate that anatomy dissection courses are important for the initiation of students' feelings about the topic of death. Omission of cadaveric dissection- or prosection-based laboratories will decrease the likelihood that students initiate reflection on this topic and gain important transferable skills.

Immune-Triggered Forms of Plasticity Across Brain Regions.

Immune cells play numerous roles in the host defense against the invasion of microorganisms and pathogens, which induces the release of inflammatory mediators (e.g., cytokines and chemokines). In the CNS, microglia is the major resident immune cell. Recent efforts have revealed the diversity of the cell types and the heterogeneity of their functions. The refinement of the synapse structure was a hallmark feature of the microglia, while they are also involved in the myelination and capillary dynamics. Another promising feature is the modulation of the synaptic transmission as synaptic plasticity and the intrinsic excitability of neurons as non-synaptic plasticity. Those modulations of physiological properties of neurons are considered induced by both transient and chronic exposures to inflammatory mediators, which cause behavioral disorders seen in mental illness. It is plausible for astrocytes and pericytes other than microglia and macrophage to induce the immune-triggered plasticity of neurons. However, current understanding has yet achieved to unveil what inflammatory mediators from what immune cells or glia induce a form of plasticity modulating pre-, post-synaptic functions and intrinsic excitability of neurons. It is still unclear what ion channels and intracellular signaling of what types of neurons in which brain regions of the CNS are involved. In this review, we introduce the ubiquitous modulation of the synaptic efficacy and the intrinsic excitability across the brain by immune cells and related inflammatory cytokines with the mechanism for induction. Specifically, we compare neuro-modulation mechanisms by microglia of the intrinsic excitability of cerebellar Purkinje neurons with cerebral pyramidal neurons, stressing the inverted directionality of the plasticity. We also discuss the suppression and augmentation of the extent of plasticity by inflammatory mediators, as the meta-plasticity by immunity. Lastly, we sum up forms of immune-triggered plasticity in the different brain regions with disease relevance. Together, brain immunity influences our cognition, sense, memory, and behavior via immune-triggered plasticity.

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.