Regional differences in the ultrastructure of mucosal macrophages in the rat large intestine.

We previously clarified the histological characteristics of macrophages in the rat small intestine using serial block-face scanning electron microscopy (SBF-SEM). However, the regional differences in the characteristics of macrophages throughout the large intestine remain unknown. Here, we performed a pilot study to explore the regional differences in the ultrastructure of mucosal macrophages in the large intestine by using SBF-SEM analysis. SBF-SEM analysis conducted on the luminal side of the cecum and descending colon revealed macrophages as amorphous cells possessing abundant lysosomes and vacuoles. Macrophages in the cecum exhibited a higher abundance of lysosomes and a lower abundance of vacuoles than those in the descending colon. Macrophages with many intraepithelial cellular processes were observed beneath the intestinal superficial epithelium in the descending colon. Moreover, macrophages in contact with nerve fibers were more prevalent in the cecum than in the descending colon, and a subset of them surrounded a nerve bundle only in the cecum. In conclusion, the present pilot study suggested that the quantity of some organelles (lysosomes and vacuoles) in macrophages differed between the cecum and the descending colon and that there were some region-specific subsets of macrophages like nerve-associated macrophages in the cecum.

Fatty Acids Play a Critical Role in Mitochondrial Oxidative Phosphorylation in Effector T Cells in Graft-versus-Host Disease.

Although the role of aerobic glycolysis in activated T cells has been well characterized, whether and how fatty acids (FAs) contribute to donor T cell function in allogeneic hematopoietic stem cell transplantation is unclear. Using xenogeneic graft-versus-host disease (GVHD) models, this study demonstrated that exogenous FAs serve as a crucial source of mitochondrial respiration in donor T cells in humans. By comparing human T cells isolated from wild-type NOD/Shi-scid-IL2rγnull (NOG) mice with those from MHC class I/II-deficient NOG mice, we found that donor T cells increased extracellular FA uptake, the extent of which correlates with their proliferation, and continued to increase FA uptake during effector differentiation. Gene expression analysis showed the upregulation of a wide range of lipid metabolism-related genes, including lipid hydrolysis, mitochondrial FA transport, and FA oxidation. Extracellular flux analysis demonstrated that mitochondrial FA transport was required to fully achieve the mitochondrial maximal respiration rate and spare respiratory capacity, whereas the substantial disruption of glucose supply by either glucose deprivation or mitochondrial pyruvate transport blockade did not impair oxidative phosphorylation. Taken together, FA-driven mitochondrial respiration is a hallmark that differentiates TCR-dependent T cell activation from TCR-independent immune response after hematopoietic stem cell transplant.

Identification of the growth cone as a probe and driver of neuronal migration in the injured brain.

Axonal growth cones mediate axonal guidance and growth regulation. We show that migrating neurons in mice possess a growth cone at the tip of their leading process, similar to that of axons, in terms of the cytoskeletal dynamics and functional responsivity through protein tyrosine phosphatase receptor type sigma (PTPσ). Migrating-neuron growth cones respond to chondroitin sulfate (CS) through PTPσ and collapse, which leads to inhibition of neuronal migration. In the presence of CS, the growth cones can revert to their extended morphology when their leading filopodia interact with heparan sulfate (HS), thus re-enabling neuronal migration. Implantation of an HS-containing biomaterial in the CS-rich injured cortex promotes the extension of the growth cone and improve the migration and regeneration of neurons, thereby enabling functional recovery. Thus, the growth cone of migrating neurons is responsive to extracellular environments and acts as a primary regulator of neuronal migration.

The Mouse Model of Internal Capsule Demyelination: A Novel Tool for Investigating Motor Functional Changes Caused by Demyelination and for Evaluating Drugs That Promote Remyelination.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system, characterized by remyelination failure and axonal dysfunction. Remyelination by oligodendrocytes is critical for improvement of neurological deficits associated with demyelination. Rodent models of demyelination are frequently used to develop and evaluate therapies for MS. However, a suitable mouse model for assessing remyelination-associated recovery of motor functions is currently unavailable. In this review, we describe the development of the mouse model of internal capsule (IC) demyelination by focal injection of lysolecithin into brain and its application in the evaluation of drugs for demyelinating diseases. This mouse model exhibits motor deficits and subsequent functional recovery accompanying IC remyelination. Notably, this model shows enhancement of functional recovery as well as tissue regeneration when treated with clemastine, a drug that promotes remyelination. The IC demyelination mouse model should contribute to the development of novel drugs that promote remyelination and ameliorate neurological deficits in demyelinating diseases.

Apomorphine is a potent inhibitor of ferroptosis independent of dopaminergic receptors.

Originally, apomorphine was a broad-spectrum dopamine agonist with an affinity for all subtypes of the Dopamine D1 receptor to the D5 receptor. We previously identified apomorphine as a potential therapeutic agent for mitochondrial diseases by screening a chemical library of fibroblasts from patients with mitochondrial diseases. In this study, we showed that apomorphine prevented ferroptosis in fibroblasts from various types of mitochondrial diseases as well as in normal controls. Well-known biomarkers of ferroptosis include protein markers such as prostaglandin endoperoxide synthase 2 (PTGS2), a key gene for ferroptosis-related inflammation PTGS2, lipid peroxidation, and reactive oxygen species. Our findings that apomorphine induced significant downregulation of PTSG2 and suppressed lipid peroxide to the same extent as other inhibitors of ferroptosis also indicate that apomorphine suppresses ferroptosis. To our knowledge, this is the first study to report that the anti-ferroptosis effect of apomorphine is not related to dopamine receptor agonist action and that apomorphine is a potent inhibitor of ferroptotic cell death independent of dopaminergic receptors.

Calcium signals tune AMPK activity and mitochondrial homeostasis in dendrites of developing neurons.

Dendritic outgrowth in immature neurons is enhanced by neuronal activity and is considered one of the mechanisms of neural circuit optimization. It is known that calcium signals affect transcriptional regulation and cytoskeletal remodeling necessary for dendritic outgrowth. Here, we demonstrate that activity-dependent calcium signaling also controls mitochondrial homeostasis via AMP-activated protein kinase (AMPK) in growing dendrites of differentiating mouse hippocampal neurons. We found that the inhibition of neuronal activity induced dendritic hypotrophy with abnormally elongated mitochondria. In growing dendrites, AMPK is activated by neuronal activity and dynamically oscillates in synchrony with calcium spikes, and this AMPK oscillation was inhibited by CaMKK2 knockdown. AMPK activation led to phosphorylation of MFF and ULK1, which initiate mitochondrial fission and mitophagy, respectively. Dendritic mitochondria in AMPK-depleted neurons exhibited impaired fission and mitophagy and displayed multiple signs of dysfunction. Genetic inhibition of fission led to dendritic hypoplasia that was reminiscent of AMPK-deficient neurons. Thus, AMPK activity is finely tuned by the calcium-CaMKK2 pathway and regulates mitochondrial homeostasis by facilitating removal of damaged components of mitochondria in growing neurons during normal brain development.

Shared GABA transmission pathology in dopamine agonist- and antagonist-induced dyskinesia.

Dyskinesia is involuntary movement caused by long-term medication with dopamine-related agents: the dopamine agonist 3,4-dihydroxy-L-phenylalanine (L-DOPA) to treat Parkinson's disease (L-DOPA-induced dyskinesia [LID]) or dopamine antagonists to treat schizophrenia (tardive dyskinesia [TD]). However, it remains unknown why distinct types of medications for distinct neuropsychiatric disorders induce similar involuntary movements. Here, we search for a shared structural footprint using magnetic resonance imaging-based macroscopic screening and super-resolution microscopy-based microscopic identification. We identify the enlarged axon terminals of striatal medium spiny neurons in LID and TD model mice. Striatal overexpression of the vesicular gamma-aminobutyric acid transporter (VGAT) is necessary and sufficient for modeling these structural changes; VGAT levels gate the functional and behavioral alterations in dyskinesia models. Our findings indicate that lowered type 2 dopamine receptor signaling with repetitive dopamine fluctuations is a common cause of VGAT overexpression and late-onset dyskinesia formation and that reducing dopamine fluctuation rescues dyskinesia pathology via VGAT downregulation.

Histological study on the reginal difference in the localization of mucosal enteric glial cells and their sheath structure in the rat intestine.

The present study aimed to histologically clarify the regional specificity of the mucosal enteric glial cells (mEGCs) in the rat intestine with serial block-face scanning electron microscopy (SBF-SEM). SBF-SEM analysis with the ileum, the cecum and the descending colon revealed that mEGC nuclei were more abundant in the data stacks from the apical portion of the villus and the lateral portion of the crypt of the ileum. mEGCs exhibited a high rate of coverage over the nerve bundle around the lateral portion of the ileal crypt, but showed an extremely low level of coverage in the luminal portion of the cecum. These findings evidenced regional differences in the localization of mEGCs and in their sheath structure in the rat intestine.

Unusual nuclear structures in male meiocytes of wild-type rye as revealed by volume microscopy.

BACKGROUND AND AIMS: During the analysis of plant male meiocytes coming from destroyed meiocyte columns (united multicellular structures formed by male meiocytes in each anther locule), a considerable amount of information becomes unavailable. Therefore, in this study intact meiocyte columns were studied by volume microscopy in wild-type rye for the most relevant presentation of 3-D structure of rye meiocytes throughout meiosis. METHODS: We used two types of volume light microscopy: confocal laser scanning microscopy and non-confocal bright-field scanning microscopy combined with alcohol and aldehyde fixation, as well as serial block-face scanning electron microscopy. KEY RESULTS: Unusual structures, called nuclear protuberances, were detected. At certain meiotic stages, nuclei formed protuberances that crossed the cell wall through intercellular channels and extended into the cytoplasm of neighbouring cells, while all other aspects of cell structure appeared to be normal. This phenomenon of intercellular nuclear migration (INM) was detected in most meiocytes at leptotene/zygotene. No cases of micronucleus formation or appearance of binucleated meiocytes were noticed. There were instances of direct contact between two nuclei during INM. No influence of fixation or of mechanical impact on the induction of INM was detected. CONCLUSIONS: Intercellular nuclear migration in rye may be a programmed process (a normal part of rye male meiosis) or a tricky artefact that cannot be avoided in any way no matter which approach to meiocyte imaging is used. In both cases, INM seems to be an obligatory phenomenon that has previously been hidden by limitations of common microscopic techniques and by 2-D perception of plant male meiocytes. Intercellular nuclear migration cannot be ignored in any studies involving manipulations of rye anthers.

Three-dimensional structural analysis of papillary thyroid carcinoma nuclei with serial block-face scanning electron microscopy (SBF-SEM).

Nuclear morphology of carcinoma cells is critical for the pathological diagnosis of papillary thyroid carcinoma (PTC). However, three-dimensional architecture of PTC nuclei is still elusive. In this study, we analyzed the three-dimensional ultrastructure of PTC nuclei using serial block-face scanning electron microscopy which takes advantage of the high-throughput acquisition of serial electron microscopic images and three-dimensional reconstruction of subcellular structures. En bloc-stained and resin-embedded specimens were prepared from surgically removed PTCs and normal thyroid tissues. We acquired two-dimensional images from serial block-face scanning electron microscopy and reconstructed three-dimensional nuclear structures. Quantitative comparisons showed that the nuclei of carcinoma cells were larger and more complex than those of normal follicular cells. The three-dimensional reconstruction of carcinoma nuclei divided intranuclear cytoplasmic inclusions into "open intranuclear cytoplasmic inclusions" connecting to cytoplasm outside the nucleus and "closed intranuclear cytoplasmic inclusions" without that connection. Cytoplasm with abundant organelles was observed in open inclusions, but closed inclusions contained fewer organelles with or without degeneration. Granules with a dense core were only observed in closed inclusions. Our observations suggested that open inclusions originate from nuclear invaginations, and disconnection from cytoplasm leads to closed inclusions.

Microglia enable cross-modal plasticity by removing inhibitory synapses.

Cross-modal plasticity is the repurposing of brain regions associated with deprived sensory inputs to improve the capacity of other sensory modalities. The functional mechanisms of cross-modal plasticity can indicate how the brain recovers from various forms of injury and how different sensory modalities are integrated. Here, we demonstrate that rewiring of the microglia-mediated local circuit synapse is crucial for cross-modal plasticity induced by visual deprivation (monocular deprivation [MD]). MD relieves the usual inhibition of functional connectivity between the somatosensory cortex and secondary lateral visual cortex (V2L). This results in enhanced excitatory responses in V2L neurons during whisker stimulation and a greater capacity for vibrissae sensory discrimination. The enhanced cross-modal response is mediated by selective removal of inhibitory synapse terminals on pyramidal neurons by the microglia in the V2L via matrix metalloproteinase 9 signaling. Our results provide insights into how cortical circuits integrate different inputs to functionally compensate for neuronal damage.

Treatment of tubular damage in high-fat-diet-fed obese mice using sodium-glucose co-transporter inhibitors.

A long-term high-fat diet (HFD) causes obesity and changes in renal lipid metabolism and lysosomal dysfunction in mice, causing renal damage. Sodium-glucose co-transporter inhibitors, including phlorizin, exert nephroprotective effects in patients with chronic kidney disease, but the underlying mechanism remains unclear. A HFD or standard diet was fed to adult C57BL/6J male mice, and phlorizin was administered. Lamellar body components of the proximal tubular epithelial cells (PTECs) were investigated. After phlorizin administration in HFD-fed mice, sphingomyelin and ceramide in urine and tissues were assessed and label-free quantitative proteomics was performed using kidney tissue samples. Mitochondrial elongation by fusion was effective in the PTECs of HFD-fed obese mice under phlorizin administration, and many lamellar bodies were found in the apical portion of the S2 segment of the proximal tubule. Phlorizin functioned as a diuretic, releasing lamellar bodies from the apical membrane of PTECs and clearing the obstruction in nephrons. The main component of the lamellar bodies was sphingomyelin. On the first day of phlorizin administration in HFD-fed obese mice, the diuretic effect was increased, and more sphingomyelin was excreted through urine than in vehicle-treated mice. The expressions of three peroxisomal ß-oxidation proteins involved in fatty acid metabolism were downregulated after phlorizin administration in the kidneys of HFD-fed mice. Fatty acid elongation protein levels increased with phlorizin administration, indicating an increase in long-chain fatty acids. Lamellar bodies accumulated in the proximal renal tubule of the S2 segment of the HFD-fed mice, indicating that the urinary excretion of lamellar bodies has nephroprotective effects.

Pharmacological treatment promoting remyelination enhances motor function after internal capsule demyelination in mice.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system characterized by remyelination failure, axonal degeneration, and progressive worsening of motor functions. Animal models of demyelination are frequently used to develop and evaluate therapies for MS. We recently reported that focal internal capsule (IC) demyelination in mice with lysophosphatidylcholine injection induced acute motor deficits followed by recovery through remyelination. However, it remains unknown whether the IC demyelination mouse model can be used to evaluate changes in motor functions caused by pharmacological treatments that promote remyelination using behavioral testing and histological analysis. In this study, we examined the effect of clemastine, an anti-muscarinic drug that promotes remyelination, in the mouse IC demyelination model. Clemastine administration improved motor function and changed forepaw preference in the IC demyelinated mice. Moreover, clemastine-treated mice showed increased mature oligodendrocyte density, reduced axonal injury, an increased number of myelinated axons and thicker myelin in the IC lesions compared with control (PBS-treated) mice. These results suggest that the lysophosphatidylcholine-induced IC demyelination model is useful for evaluating changes in motor functions following pharmacological treatments that promote remyelination.

Cerebrospinal fluid-contacting neuron tracing reveals structural and functional connectivity for locomotion in the mouse spinal cord.

Cerebrospinal fluid-contacting neurons (CSF-cNs) are enigmatic mechano- or chemosensory cells lying along the central canal of the spinal cord. Recent studies in zebrafish larvae and lampreys have shown that CSF-cNs control postures and movements via spinal connections. However, the structures, connectivity, and functions in mammals remain largely unknown. Here we developed a method to genetically target mouse CSF-cNs that highlighted structural connections and functions. We first found that intracerebroventricular injection of adeno-associated virus with a neuron-specific promoter and Pkd2l1-Cre mice specifically labeled CSF-cNs. Single-cell labeling of 71 CSF-cNs revealed rostral axon extensions of over 1800 µm in unmyelinated bundles in the ventral funiculus and terminated on CSF-cNs to form a recurrent circuitry, which was further determined by serial electron microscopy and electrophysiology. CSF-cNs were also found to connect with axial motor neurons and premotor interneurons around the central canal and within the axon bundles. Chemogenetic CSF-cNs inactivation reduced speed and step frequency during treadmill locomotion. Our data revealed the basic structures and connections of mouse CSF-cNs to control spinal motor circuits for proper locomotion. The versatile methods developed in this study will contribute to further understanding of CSF-cN functions in mammals.

Correlative light and volume electron microscopy to study brain development.

Recent advances in volume electron microscopy (EM) have been driving our thorough understanding of the brain architecture. Volume EM becomes increasingly powerful when cells and their subcellular structures that are imaged in light microscopy are correlated to those in ultramicrographs obtained with EM. This correlative approach, called correlative light and volume electron microscopy (vCLEM), is used to link three-dimensional ultrastructural information with physiological data such as intracellular Ca2+ dynamics. Genetic tools to express fluorescent proteins and/or an engineered form of a soybean ascorbate peroxidase allow us to perform vCLEM using natural landmarks including blood vessels without immunohistochemical staining. This immunostaining-free vCLEM has been successfully employed in two-photon Ca2+ imaging in vivo as well as in studying complex synaptic connections in thalamic neurons that receive a variety of specialized inputs from the cerebral cortex. In this mini-review, we overview how volume EM and vCLEM have contributed to studying the developmental processes of the brain. We also discuss potential applications of genetic manipulation of target cells using clustered regularly interspaced short palindromic repeats-associated protein 9 and subsequent volume EM to the analysis of protein localization as well as to loss-of-function studies of genes regulating brain development. We give examples for the combinatorial usage of genetic tools with vCLEM that will further enhance our understanding of regulatory mechanisms underlying brain development.

Correlative light and electron microscopic observation of calcium phosphate particles in a mouse kidney formed under a high-phosphate diet.

Calcium phosphate forms particles under excessive urinary excretion of phosphate in the kidney. While the formation of calcium phosphate particles (CaPs) has been implicated in the damage to renal tubular cells and renal dysfunction, clarifying the ultrastructural information and the elemental composition of the small CaPs in the wide areas of kidney tissue has been technically difficult. This study introduces correlative and sequential light as well as electron microscopic CaP observation in the kidney tissue by combining fluorescent staining for CaPs and energy-dispersive X-ray spectroscopy (EDS) in scanning electron microscopy (SEM) on resin sections prepared using high-pressure freezing and freeze substitution. CaPs formed in mouse kidneys under long-term feeding of a high-phosphate diet were clearly visualized on resin sections by fluorescence-conjugated alendronate derivatives and toluidine blue metachromasia. These CaPs were verified by correlative observation with EDS. Furthermore, small CaPs formed in the kidney under short-term feeding were detected using fluorescent probes. The elemental composition of the particles, including calcium and magnesium, was identified following EDS analyses. These results suggest that the correlative microscopy approach is helpful for observing in situ distribution and elemental composition of CaPs in the kidney and contributing to studies regarding CaP formation-associated pathophysiology.

Histological study on regional specificity of the mucosal nerve network in the rat large intestine.

Our previous studies and others have revealed detailed characteristics of the mucosal nerve network in the small intestine, but much remains unknown about the corresponding network in the large intestine. We herein investigated regional differences in the expression of neurochemical markers, the nerve network structure, and the cells in contact with nerve fibers by histological analysis using both immunohistochemistry and serial block-face scanning electron microscopy (SBF-SEM). Immunohistochemistry revealed that immunopositive structures for protein gene product 9.5, vasoactive intestinal peptide (VIP), calretinin and vesicular acetylcholine transporter were more prevalent in the lamina propria of the ascending colon than the cecum and descending colon (DC). There was no significant difference in the frequency of most neurochemical markers between the cecum and DC, but the frequencies of VIP+ structures were higher in the cecum than in the DC. SBF-SEM analysis showed that the nerve network structure was more developed on the luminal side of the DC than the cecum. The cells that nerve fibers abundantly contacted were subepithelial and lamina propria fibroblast-like cells and macrophages. In addition, nerve fibers in the cecum were in more frequent contact with immune cells such as macrophages and plasma cells than nerve fibers in the DC. Thus, the present histological analysis suggested that the mucosal nerve network in the large intestine possessed both regional universality and various specificities, and revealed the intimate relationship between the nerve network and immune cells, especially in the cecum.

Ultrastructure of the lamprey head mesoderm reveals evolution of the vertebrate head.

The cranial muscle is a critical component in the vertebrate head for a predatory lifestyle. However, its evolutionary origin and possible segmental nature during embryogenesis have been controversial. In jawed vertebrates, the presence of pre-otic segments similar to trunk somites has been claimed based on developmental observations. However, evaluating such arguments has been hampered by the paucity of research on jawless vertebrates. Here, we discovered different cellular arrangements in the head mesoderm in lamprey embryos (Lethenteron camtschaticum) using serial block-face scanning electron and laser scanning microscopies. These cell populations were morphologically and molecularly different from somites. Furthermore, genetic comparison among deuterostomes revealed that mesodermal gene expression domains were segregated antero-posteriorly in vertebrates, whereas such segregation was not recognized in invertebrate deuterostome embryos. These findings indicate that the vertebrate head mesoderm evolved from the anteroposterior repatterning of an ancient mesoderm and developmentally diversified before the split of jawless and jawed vertebrates.

Ultrastructural characteristics of oligodendrocyte precursor cells in the early postnatal mouse optic nerve observed by serial block-face scanning electron microscopy.

Oligodendrocyte precursor cells (OPC) arise from restricted regions of the central nervous system (CNS) and differentiate into myelin-forming cells after migration, but their ultrastructural characteristics have not been fully elucidated. This study examined the three-dimensional ultrastructure of OPCs in comparison with other glial cells in the early postnatal optic nerve by serial block-face scanning electron microscopy. We examined 70 putative OPCs (pOPC) that were distinct from other glial cells according to established morphological criteria. The pOPCs were unipolar in shape with relatively few processes, and their Golgi apparatus were localized in the perinuclear region with a single cisterna. Astrocytes abundant in the optic nerve were distinct from pOPCs and had a greater number of processes and more complicated Golgi apparatus morphology. All pOPCs and astrocytes contained a pair of centrioles (basal bodies). Among them, 45% of pOPCs extended a short cilium, and 20% of pOPCs had centrioles accompanied by vesicles, whereas all astrocytes with basal bodies had cilia with invaginated ciliary pockets. These results suggest that the fine structures of pOPCs during the developing and immature stages may account for their distinct behavior. Additionally, the vesicular transport of the centrioles, along with a short cilium length, suggests active ciliogenesis in pOPCs.

CTCF loss induces giant lamellar bodies in Purkinje cell dendrites.

CCCTC-binding factor (CTCF) has a key role in higher-order chromatin architecture that is important for establishing and maintaining cell identity by controlling gene expression. In the mature cerebellum, CTCF is highly expressed in Purkinje cells (PCs) as compared with other cerebellar neurons. The cerebellum plays an important role in motor function by regulating PCs, which are the sole output neurons, and defects in PCs cause motor dysfunction. However, the role of CTCF in PCs has not yet been explored. Here we found that the absence of CTCF in mouse PCs led to progressive motor dysfunction and abnormal dendritic morphology in those cells, which included dendritic self-avoidance defects and a proximal shift in the climbing fibre innervation territory on PC dendrites. Furthermore, we found the peculiar lamellar structures known as "giant lamellar bodies" (GLBs), which have been reported in PCs of patients with Werdnig-Hoffman disease, 13q deletion syndrome, and Krabbe disease. GLBs are localized to PC dendrites and are assumed to be associated with neurodegeneration. They have been noted, however, only in case reports following autopsy, and reports of their existence have been very limited. Here we show that GLBs were reproducibly formed in PC dendrites of a mouse model in which CTCF was deleted. GLBs were not noted in PC dendrites at infancy but instead developed over time. In conjunction with GLB development in PC dendrites, the endoplasmic reticulum was almost absent around the nuclei, the mitochondria were markedly swollen and their cristae had decreased drastically, and almost all PCs eventually disappeared as severe motor deficits manifested. Our results revealed the important role of CTCF during normal development and in maintaining PCs and provide new insights into the molecular mechanism of GLB formation during neurodegenerative disease.