Prefrontal synaptic regulation of homeostatic sleep pressure revealed through synaptic chemogenetics.

Sleep is regulated by homeostatic processes, yet the biological basis of sleep pressure that accumulates during wakefulness, triggers sleep, and dissipates during sleep remains elusive. We explored a causal relationship between cellular synaptic strength and electroencephalography delta power indicating macro-level sleep pressure by developing a theoretical framework and a molecular tool to manipulate synaptic strength. The mathematical model predicted that increased synaptic strength promotes the neuronal "down state" and raises the delta power. Our molecular tool (synapse-targeted chemically induced translocation of Kalirin-7, SYNCit-K), which induces dendritic spine enlargement and synaptic potentiation through chemically induced translocation of protein Kalirin-7, demonstrated that synaptic potentiation of excitatory neurons in the prefrontal cortex (PFC) increases nonrapid eye movement sleep amounts and delta power. Thus, synaptic strength of PFC excitatory neurons dictates sleep pressure in mammals.

Cortical parvalbumin neurons are responsible for homeostatic sleep rebound through CaMKII activation.

The homeostatic regulation of sleep is characterized by rebound sleep after prolonged wakefulness, but the molecular and cellular mechanisms underlying this regulation are still unknown. In this study, we show that Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent activity control of parvalbumin (PV)-expressing cortical neurons is involved in homeostatic regulation of sleep in male mice. Prolonged wakefulness enhances cortical PV-neuron activity. Chemogenetic suppression or activation of cortical PV neurons inhibits or induces rebound sleep, implying that rebound sleep is dependent on increased activity of cortical PV neurons. Furthermore, we discovered that CaMKII kinase activity boosts the activity of cortical PV neurons, and that kinase activity is important for homeostatic sleep rebound. Here, we propose that CaMKII-dependent PV-neuron activity represents negative feedback inhibition of cortical neural excitability, which serves as the distributive cortical circuits for sleep homeostatic regulation.

Bioorthogonal chemical labeling of endogenous neurotransmitter receptors in living mouse brains.

Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent labeling by chemical reaction (chemical labeling) of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labeling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labeling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-color receptor imaging; and pulse-chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions.

CUBIC-Cloud provides an integrative computational framework toward community-driven whole-mouse-brain mapping.

Recent advancements in tissue clearing technologies have offered unparalleled opportunities for researchers to explore the whole mouse brain at cellular resolution. With the expansion of this experimental technique, however, a scalable and easy-to-use computational tool is in demand to effectively analyze and integrate whole-brain mapping datasets. To that end, here we present CUBIC-Cloud, a cloud-based framework to quantify, visualize, and integrate mouse brain data. CUBIC-Cloud is a fully automated system where users can upload their whole-brain data, run analyses, and publish the results. We demonstrate the generality of CUBIC-Cloud by a variety of applications. First, we investigated the brain-wide distribution of five cell types. Second, we quantified Aß plaque deposition in Alzheimer's disease model mouse brains. Third, we reconstructed a neuronal activity profile under LPS-induced inflammation by c-Fos immunostaining. Last, we show brain-wide connectivity mapping by pseudotyped rabies virus. Together, CUBIC-Cloud provides an integrative platform to advance scalable and collaborative whole-brain mapping.

Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues.

Whole-organ/body three-dimensional (3D) staining and imaging have been enduring challenges in histology. By dissecting the complex physicochemical environment of the staining system, we developed a highly optimized 3D staining imaging pipeline based on CUBIC. Based on our precise characterization of biological tissues as an electrolyte gel, we experimentally evaluated broad 3D staining conditions by using an artificial tissue-mimicking material. The combination of optimized conditions allows a bottom-up design of a superior 3D staining protocol that can uniformly label whole adult mouse brains, an adult marmoset brain hemisphere, an ~1 cm3 tissue block of a postmortem adult human cerebellum, and an entire infant marmoset body with dozens of antibodies and cell-impermeant nuclear stains. The whole-organ 3D images collected by light-sheet microscopy are used for computational analyses and whole-organ comparison analysis between species. This pipeline, named CUBIC-HistoVIsion, thus offers advanced opportunities for organ- and organism-scale histological analysis of multicellular systems.

Chemical Landscape for Tissue Clearing Based on Hydrophilic Reagents.

We describe a strategy for developing hydrophilic chemical cocktails for tissue delipidation, decoloring, refractive index (RI) matching, and decalcification, based on comprehensive chemical profiling. More than 1,600 chemicals were screened by a high-throughput evaluation system for each chemical process. The chemical profiling revealed important chemical factors: salt-free amine with high octanol/water partition-coefficient (logP) for delipidation, N-alkylimidazole for decoloring, aromatic amide for RI matching, and protonation of phosphate ion for decalcification. The strategic integration of optimal chemical cocktails provided a series of CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis) protocols, which efficiently clear mouse organs, mouse body including bone, and even large primate and human tissues. The updated CUBIC protocols are scalable and reproducible, and they enable three-dimensional imaging of the mammalian body and large primate and human tissues. This strategy represents a future paradigm for the rational design of hydrophilic clearing cocktails that can be used for large tissues.

Loss of GPRC5B impairs synapse formation of Purkinje cells with cerebellar nuclear neurons and disrupts cerebellar synaptic plasticity and motor learning.

GPRC5B is a membrane glycoprotein robustly expressed in mouse cerebellar Purkinje cells (PCs). Its function is unknown. In Gprc5b-/- mice that lack GPRC5B, PCs develop distal axonal swellings in deep cerebellar nuclei (DCN). Numerous misshapen mitochondria, which generated excessive amounts of reactive oxygen species (ROS), accumulated in these distal axonal swellings. In primary cell cultures of Gprc5b-/- PCs, pharmacological reduction of ROS prevented the appearance of such swellings. To examine the physiological role of GPRC5B in PCs, we analyzed cerebellar synaptic transmission and cerebellum-dependent motor learning in Gprc5b-/- mice. Patch-clamp recordings in cerebellum slices in vitro revealed that the induction of long-term depression (LTD) at parallel fiber-PC synapses was normal in adult Gprc5b-/- mice, whereas the induction of long-term potentiation (LTP) at mossy fiber-DCN neuron synapses was attenuated in juvenile Gprc5b-/- mice. In Gprc5b-/- mice, long-term motor learning was impaired in both the rotarod test and the horizontal optokinetic response eye movement (HOKR) test. These observations suggest that GPRC5B plays not only an important role in the development of distal axons of PCs and formation of synapses with DCN neurons, but also in the synaptic plasticity that underlies long-term motor learning.

Comparative characterization of GPRC5B and GPRC5CLacZ knockin mice; behavioral abnormalities in GPRC5B-deficient mice.

Although GPRC5B and GPRC5C are categorized into the G protein-coupled receptor family C, including glutamate receptors, GABA receptors, and taste receptors, their physiological functions remain unknown. Since both receptors are expressed in the brain and evolutionarily conserved from fly to human, it is conceivable that they have significant biological roles particularly in the central nervous system (CNS). We generated GPRC5B- and GPRC5C-deficient mice to examine their roles in the CNS. Both homozygous mice were viable, fertile, and showed no apparent histological abnormalities, though GPRC5B-deficient mice resulted in partial perinatal lethality. We demonstrated that the expressions of GPRC5B and GPRC5C are developmentally regulated and differentially distributed in the brain. GPRC5B-deficient mice exhibited altered spontaneous activity pattern and decreased response to a new environment, while GPRC5C-deficient mice have no apparent behavioral deficits. Thus, GPRC5B has important roles for animal behavior controlled by the CNS. In contrast, GPRC5C does not affect behavior, though it has a high sequence similarity to GPRC5B. These findings suggest that family C, group 5 (GPRC5) receptors in mammals are functionally segregated from their common ancestor.

Association of oral yeast carriage with specific host factors and altered mouth sensation.

OBJECTIVES: The aim of this study was to determine if there was a significant association between the presence of altered mouth and taste sensations with oral carriage of yeasts and to assess the factors that influence the yeast carriage. STUDY DESIGN: The oral and dental status including unstimulated (USFR) and stimulated (SSFR) whole salivary flow rates of a total of 509 subjects was recorded. Saliva specimens were collected for microbiologic examination. Multiple logistic regression analysis was performed to identify any factors that were significantly associated with the prevalence of oral yeasts. RESULTS: Old age, clinical signs of oral dryness, denture wearing, and a reduction in USFR increased the prevalence of yeasts, whereas patient gender, levels of dentition, the sensation of dry or burning mouth, taste disorders, and SSFR were not associated with increased prevalence of oral yeasts. CONCLUSIONS: An increased prevalence of oral yeasts was not found to relate to changes in mouth sensation alone. Other factors, most notably patient age, the wearing of dentures, clinical signs of oral dryness, and salivary flow rate under rest conditions, were, however, found to be closely associated with oral yeast carriage.

A comparison of lower lip hypoesthesia measured by trigeminal somatosensory-evoked potential between different types of mandibular osteotomies and fixation.

PURPOSE: The purpose of this study was to compare objectively, the recovery of hypoestheia of the lower lip following orthognathic surgery using different procedures (sagittal split ramus osteotomy [SSRO]) and intra-oral vertical ramus osteotomy (IVRO)) and fixation methods (monocortical plate fixation and bi-cortical plate fixation). Hypoesthesia was evaluated using the trigeminal somatosensory-evoked potential (TSEP). PATIENTS AND METHODS: The subjects consisted of 174 patients (348 sides) with mandibular prognathism with or without asymmetry, who underwent mandibular ramus osteotomies using different fixation types. The patients were divided into 4 groups. The OAM group consisted of 128 sides who had SSRO using the Obwegeser method with mono-cortical absorbable plate fixation, the ODTM group consisted of 84 sides who had the Obwegeser-Dal Pont method with mono-cortical titanium plate fixation, the OTB group consisted of 32 sides who had the Obwegeser method with bi-cortical titanium plate fixation and the VO group consisted of 104 sides who underwent IVRO according to the Bell method without fixation. Trigeminal nerve hypoestheia at the region of the lower lip was assessed bilaterally by the TSEP method. An electroencephalograph recording system (Neuropack Sigma; Nion Koden Corp., Tokyo, Japan) was used to analyze the potentials. Each patient was evaluated pre-operatively and then post-operatively at 1 and 2 weeks, 1, 3, and 6 months, and 1 year. RESULTS: The mean measurable period and standard deviation of TSEP of the lower lip in the OAM group was 5.2 +/- 9.9 weeks, 10.9 +/- 13.1 weeks in the ODTM group, 7.8 +/- 4.5 weeks in the OTB group, and 2.5 +/- 6.3 weeks in the VO group. There were significant differences between the OAM and ODTM groups (P < .0001), the ODTM and OTB groups (P = .0001), the OTB and VO groups (P = .0221), the OAM and VO groups (P < .0001), and the ODTM and VO groups (P < .0001). CONCLUSION: This study proved using objective measurements that the recovery period from hypoesthesia of the lower lip following orthognathic surgery was dependent on the surgical procedure. Recovery in lower lip hypoesthesia after IVRO was significantly earlier than SSRO.

Comparison of maxillary stability after Le Fort I osteotomy for occlusal cant correction surgery and maxillary advanced surgery.

OBJECTIVE: To compare postoperative maxillary stability following Le Fort I osteotomy for the correction of occlusal cant as compared with conventional Le Fort I osteotomy for maxillary advancement. STUDY DESIGN: The subjects were 40 Japanese adults with jaw deformities. Of these, 20 underwent a Le Fort I osteotomy and intraoral vertical ramus osteotomy (IVRO) to correct asymmetric skeletal morphology and inclined occlusal cant. The other 20 patients underwent a Le Fort I osteotomy and sagittal split ramus osteotomy (SSRO) to advance the maxilla. Lateral and posteroanterior cephalograms were taken postoperatively and assessed statistically. Thereafter, the 2 groups were followed for time-course changes. RESULTS: There was no significant difference between the 2 groups with regard to time-course changes during the immediate postoperative period. CONCLUSION: This suggests that maxillary stability after Le Fort I osteotomy for cant correction does not differ from that after Le Fort I osteotomy for maxillary advancement.

Evaluation of upper lip hypoesthesia with a trigeminal somatosensory-evoked potential following Le Fort I osteotomy in combination with mandibular osteotomy.

PURPOSE: The purpose of this study was to objectively evaluate hypoesthesia of the upper lip following Le Fort I osteotomy in combination with mandibular osteotomy with trigeminal somatosensory evoked potential (TSEP). SUBJECTS AND METHODS: The subjects consisted of 25 patients with mandibular prognathism with maxillary retrognathism mandibular prognathism with or without asymmetry, who underwent Le Fort I osteotomy in combination with sagittal split ramus osteotomy (SSRO) or intraoral vertical ramus osteotomy (IVRO). Trigeminal nerve hypoesthesia at the region of the upper lip was assessed bilaterally by the TSEP method. The electrodes were placed exactly above the highest point of the vermilion border and on the mucosa of the upper lip. An electroencephalograph recording system (Neuropack Sigma; Nihon Koden Corp., Tokyo, Japan) was used to analyze the potentials. Each patient was evaluated preoperatively and then postoperatively at 1 week, 2 weeks, 1 month, 3 months, 6 months, and 1 year. RESULTS: The average measurable period and standard deviation of TSEP of the upper lip was 7.8 +/- 10.7 weeks following Le Fort I osteotomy, TSEP of the lower lip was 4.6 +/- 9.2 weeks in the patients who underwent SSRO with Le Fort I osteotomy, and 1.2 +/- 0.4 weeks in the patients who underwent IVRO with Le Fort I osteotomy. CONCLUSION: This study objectively proved that hypoesthesia could appear in the upper lips following Le Fort I osteotomy with TSEP. The measurable period for the upper lip following Le Fort I osteotomy tended to be longer than that for the lower lip in the patients who underwent SSRO and IVRO with Le Fort I osteotomy.