Dorsomedial and preoptic hypothalamic circuits control torpor.

Endotherms can survive low temperatures and food shortage by actively entering a hypometabolic state known as torpor. Although the decrease in metabolic rate and body temperature (Tb) during torpor is controlled by the brain, the specific neural circuits underlying these processes have not been comprehensively elucidated. In this study, we identify the neural circuits involved in torpor regulation by combining whole-brain mapping of torpor-activated neurons, cell-type-specific manipulation of neural activity, and viral tracing-based circuit mapping. We find that Trpm2-positive neurons in the preoptic area and Vgat-positive neurons in the dorsal medial hypothalamus are activated during torpor. Genetic silencing shows that the activity of either cell type is necessary to enter the torpor state. Finally, we show that these cells receive projections from the arcuate and suprachiasmatic nucleus and send projections to brain regions involved in thermoregulation. Our results demonstrate an essential role of hypothalamic neurons in the regulation of Tb and metabolic rate during torpor and identify critical nodes of the torpor regulatory network.

Deficiency of orexin signaling during sleep is involved in abnormal REM sleep architecture in narcolepsy.

Narcolepsy is a sleep disorder caused by deficiency of orexin signaling. However, the neural mechanisms by which deficient orexin signaling causes the abnormal rapid eye movement (REM) sleep characteristics of narcolepsy, such as cataplexy and frequent transitions to REM states, are not fully understood. Here, we determined the activity dynamics of orexin neurons during sleep that suppress the abnormal REM sleep architecture of narcolepsy. Orexin neurons were highly active during wakefulness, showed intermittent synchronous activity during non-REM (NREM) sleep, were quiescent prior to the transition from NREM to REM sleep, and a small subpopulation of these cells was active during REM sleep. Orexin neurons that lacked orexin peptides were less active during REM sleep and were mostly silent during cataplexy. Optogenetic inhibition of orexin neurons established that the activity dynamics of these cells during NREM sleep regulate NREM-REM sleep transitions. Inhibition of orexin neurons during REM sleep increased subsequent REM sleep in "orexin intact" mice and subsequent cataplexy in mice lacking orexin peptides, indicating that the activity of a subpopulation of orexin neurons during the preceding REM sleep suppresses subsequent REM sleep and cataplexy. Thus, these results identify how deficient orexin signaling during sleep results in the abnormal REM sleep architecture characteristic of narcolepsy.

Contrast-enhanced ultrasound imaging for monitoring the efficacy of near-infrared photoimmunotherapy.

BACKGROUND: Near-infrared photoimmunotherapy (NIR-PIT) is a promising cancer therapy combining NIR-light irradiation with an antibody and IR700DX, a light-sensitive substance, to destroy tumours. However, homogeneous irradiation is difficult because the light varies depending on the distance and tissue environment. Therefore, markers that indicate sufficient irradiation are necessary. Nanoparticles sized 10∼200 nm show enhanced permeation and retention within tumours, which is further enhanced via NIR-PIT (super enhanced permeability and retention, SUPR). We aimed to monitor the effectiveness of NIR-PIT by measuring SUPR. METHODS: A xenograft mouse tumour model was established by inoculating human cancer cells in both buttocks of Balb/C-nu/nu mice, and NIR-PIT was performed on only one side. To evaluate SUPR, fluorescent signal examination was performed using QD800-fluorescent nanoparticles and NIR-fluorescent poly (d,l-lactide-co-glycolic acid) (NIR-PLGA) microparticles. Harmonic signals were evaluated using micro-bubbles of the contrast agent Sonazoid and contrast-enhanced ultrasound (CEUS) imaging. The correlation between SUPR immediately after treatment and NIR-PIT effectiveness on the day after treatment was evaluated. FINDINGS: QD800 fluorescent signals persisted only in the treated tumours, and the intensity of remaining signals showed high positive correlation with the therapeutic effect. NIR-PLGA fluorescent signals and Sonazoid-derived harmonic signals remained for a longer time in the treated tumours than in the controls, and the kE value of the two-compartment model correlated with NIR-PIT effectiveness. INTERPRETATION: SUPR measurement using Sonazoid and CEUS imaging could be easily adapted for clinical use as a therapeutic image-based biomarker for monitoring and confirming of NIR-PIT efficacy. FUNDING: This research was supported by ARIM JAPAN of MEXT, the Program for Developing Next-generation Researchers (Japan Science and Technology Agency), KAKEN (18K15923, 21K07217) (JSPS), CREST (JPMJCR19H2, JST), and FOREST-Souhatsu (JST). Mochida Memorial Foundation for Medical and Pharmaceutical Research; Takeda Science Foundation; The Japan Health Foundation; and Princess Takamatsu Cancer Research Fund. Funders only provided financial support and had no role in the study design, data collection, data analysis, interpretation, and writing of the report.

Cre-dependent ACR2-expressing reporter mouse strain for efficient long-lasting inhibition of neuronal activity.

Optogenetics is a powerful tool for manipulating neuronal activity by light illumination with high temporal and spatial resolution. Anion-channelrhodopsins (ACRs) are light-gated anion channels that allow researchers to efficiently inhibit neuronal activity. A blue light-sensitive ACR2 has recently been used in several in vivo studies; however, the reporter mouse strain expressing ACR2 has not yet been reported. Here, we generated a new reporter mouse strain, LSL-ACR2, in which ACR2 is expressed under the control of Cre recombinase. We crossed this strain with a noradrenergic neuron-specific driver mouse (NAT-Cre) to generate NAT-ACR2 mice. We confirmed Cre-dependent expression and function of ACR2 in the targeted neurons by immunohistochemistry and electrophysiological recordings in vitro, and confirmed physiological function using an in vivo behavioral experiment. Our results show that the LSL-ACR2 mouse strain can be applied for optogenetic inhibition of targeted neurons, particularly for long-lasting continuous inhibition, upon crossing with Cre-driver mouse strains. The LSL-ACR2 strain can be used to prepare transgenic mice with homogenous expression of ACR2 in targeted neurons with a high penetration ratio, good reproducibility, and no tissue invasion.

Association of a third vaccination with antibody levels and side reactions in essential workers: A prospective cohort study.

OBJECTIVE: To evaluate the relationship between the change of titer and adverse events after the third vaccination for COVID-19 among healthcare workers. DESIGN AND SETTING: This was a prospective cohort study, and the follow-up period was from December 2021 to November 2023. PARTICIPANTS: A total of 392 healthcare workers aged over 20 years who worked at the facility and wished to have vaccine antibody titers measured participated in this study. EXPOSURES: A third dose of BNT162b2 COVID-19 vaccine was administered to healthcare workers working at the hospital, and we evaluated the changes in antibody titers before and after the vaccine, as well as adverse reactions after vaccination. MAIN OUTCOMES AND MEASURES: The primary endpoints were adverse reactions within 7 days after the third dose of COVID-19 vaccine and the rate of increase in COVID-19 vaccine antibody titer at 4 weeks. RESULTS: A total of 392 people participated in the study, of whom 358 participants had their antibody titers measured before and after the booster vaccination. The overall IgG geometric mean was 609 U/mL (561-663) before booster vaccination and increased to 18,735 U/mL (17,509-20,049) at 4 weeks after vaccination (p < 0.001). Multivariate analysis showed no statistically significant relationship between the primary endpoints, such as a change in antibody titer due to the presence of fever after vaccination or a change in antibody titer due to swelling at the vaccination site. Factors affecting the rate of increase in antibody titer, evaluated as secondary endpoints, were suggested to be age (1.02 (95 % confidence interval (CI): 1.01-1.03)) and hypertension (0.66 (95 % CI: 0.47-0.93)). CONCLUSIONS AND RELEVANCE: In relation to the booster effect of the third dose of COVID-19 vaccination, there was no statistically significant difference in the presence of fever or use of antipyretic or other drugs.

An analysis modality for vascular structures combining tissue-clearing technology and topological data analysis.

The blood and lymphatic vasculature networks are not yet fully understood even in mouse because of the inherent limitations of imaging systems and quantification methods. This study aims to evaluate the usefulness of the tissue-clearing technology for visualizing blood and lymphatic vessels in adult mouse. Clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) enables us to capture the high-resolution 3D images of organ- or area-specific vascular structures. To evaluate these 3D structural images, signals are first classified from the original captured images by machine learning at pixel base. Then, these classified target signals are subjected to topological data analysis and non-homogeneous Poisson process model to extract geometric features. Consequently, the structural difference of vasculatures is successfully evaluated in mouse disease models. In conclusion, this study demonstrates the utility of CUBIC for analysis of vascular structures and presents its feasibility as an analysis modality in combination with 3D images and mathematical frameworks.

Prevention of droplet dispersal with 'e-mask': A new daily use endoscopic mask during bronchoscopy.

BACKGROUND AND OBJECTIVE: Bronchoscopy is an airborne particle-generating procedure. However, few methods for safe bronchoscopy have been developed. To reduce airborne particles during bronchoscopy, we created an 'e-mask', which is a simple, disposable mask for patients. Our objective was to evaluate the e-mask's protective ability against airborne particles and to assess respiratory adverse events and complications. METHODS: Patients with stage 2-4 chronic obstructive pulmonary disease were excluded. We performed visualization and quantifying experiments on airborne particles with and without the e-mask. We prospectively evaluated whether wearing the e-mask during bronchoscopy was associated with the incidence of patients requiring >5 L/min oxygen to maintain >90% oxygen saturation, and patients with >45 mm Hg end-tidal carbon dioxide (EtCO2 ) elevation, in addition to complications, compared to historical controls. RESULTS: In the visualization experiment, more than ten thousand times of airborne particles were generated without the e-mask than with the e-mask. The volume of airborne particles was significantly reduced with the e-mask, compared to that without the e-mask (p = 0.011). Multivariate logistic regression analysis revealed that wearing the e-mask had no significant effect on the incidence of patients requiring >5 L/min oxygen to maintain >90% oxygen saturation, (p = 0.959); however, wearing the e-mask was a significant factor in >45 mm Hg EtCO2 elevation (p = 0.026). No significant differences in complications were observed between the e-mask and control groups (5.8% vs. 2.5%, p = 0.395). CONCLUSION: Wearing the e-mask during bronchoscopy significantly reduced the generation of airborne particles during bronchoscopy without increasing complications.

GABA neurons in the ventral tegmental area regulate non-rapid eye movement sleep in mice.

Sleep/wakefulness cycle is regulated by coordinated interactions between sleep- and wakefulness-regulating neural circuitry. However, the detailed mechanism is far from understood. Here, we found that glutamic acid decarboxylase 67-positive GABAergic neurons in the ventral tegmental area (VTAGad67+) are a key regulator of non-rapid eye movement (NREM) sleep in mice. VTAGad67+ project to multiple brain areas implicated in sleep/wakefulness regulation such as the lateral hypothalamus (LH). Chemogenetic activation of VTAGad67+ promoted NREM sleep with higher delta power whereas optogenetic inhibition of these induced prompt arousal from NREM sleep, even under highly somnolescent conditions, but not from REM sleep. VTAGad67+ showed the highest activity in NREM sleep and the lowest activity in REM sleep. Moreover, VTAGad67+ directly innervated and inhibited wake-promoting orexin/hypocretin neurons by releasing GABA. As such, optogenetic activation of VTAGad67+ terminals in the LH promoted NREM sleep. Taken together, we revealed that VTAGad67+ play an important role in the regulation of NREM sleep.