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Neonicotinoid pesticides (NNs) have been reported to have neurobehavioral effects on offspring after fetal and lactational exposure. In this study, clothianidin (CLO), an NN, was administered orally as a single dose (6.5 mg/kg: 1/10 of the no-observed-adverse-effect level in the current Pesticide Evaluation Report) to 10-day post-partum ICR mice, and CLO and its metabolites desmethyl-CLO (dm-CLO) were quantified using liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI/MS/MS) after collecting maternal breast milk and blood samples over time (1, 3, 6, 9, 12, and 24 h after administration). CLO and dm-CLO were detected in the breast milk at 1 h after the administration, and their concentrations were significantly higher than those in blood at all time points. The concentrations of CLO and dm-CLO in the breast milk were at their highest levels at 1 and 3 h, respectively, and then decreased over time to become almost undetectable at 24 h after the administration. These results show that CLO is metabolized in the mother's body and is rapidly transferred to and concentrated in the breast milk. Since CLO concentrations in breast milk are higher than those in the blood, there is concern about the effects of CLO during lactation.

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

Recently, developmental exposure to clothianidin (CLO) has been shown to cause reproductive toxicity in male mice, but the effects in female mice remain to be clarified. Pregnant C57BL/6N mice were given a no-observed-adverse-effect-level (NOAEL) dose of CLO until weaning. We then examined ovaries of 3- or 10-week-old female offspring. In the CLO-administered group, morphological changes, a decrease in the immunoreactivity of the antioxidant enzyme glutathione peroxidase 4 (GPx4), and activation of genes in the steroid hormone biosynthesis pathway were observed in 3-week-old mice, and decreases of GPx4 immunoreactivity, 17OH-progesterone and corticosterone levels were observed in 10-week-old mice, along with high rates of infanticide and severe neglect, providing new evidence that developmental exposure to CLO affects juvenile and adult mice differently.

Recently, it has been reported that neonicotinoid pesticides (NNs) are transferred from mother to child and are assumed to affect the next generation, but the behavioral effects of NN exposure at different developmental stages have not been investigated. We exposed mice to no-observed-adverse-effect level (NOAEL) doses of clothianidin (CLO) during the fetal and lactational period, and then evaluated the neurobehavioral effects in juvenile and adult mice. Significant increases in anxiety-like behavior and locomotor activity were observed in juveniles and adults, respectively, and neuronal activity and neurogenesis in the hippocampal dentate gyrus were affected in both stages. These results suggest that fetal and lactational exposure to CLO may inhibit neurogenesis and cause different behavioral abnormalities at different developmental stages.

Fipronil (FPN) is a systemic insecticide that antagonizes the gamma-aminobutyric acid type A (GABAA) receptors in insects. Recently, adverse effects of FPN on mammals have been reported, but most of those were caused by high doses of FPN and additives in the products. We investigated the effects of low-dose pure FPN on the emotional behavior of mice. Nine-week-old male mice conducted behavioral tests 24 hr after FPN administration by gavage at doses of 0.05 or 5 mg/kg based on the no-observed-effect level (NOEL), showed a significant increase in locomotor activity and dose-dependent responses on the time they spent in the central zone in the open field test. Pure FPN below the NOEL dose may affect the emotional behavior of mice.

Neonicotinoid pesticides (NNs) cause behavioral abnormalities in mammals, raising concerns about their effects on neural circuit activity. We herein examined the neurological effects of the NN clothianidin (CLO) by in vivo Ca2+ imaging using two-photon microscopy. Mice were fed the no-observed-adverse-effect-level (NOAEL) dose of CLO for 2 weeks and their neuronal activity in the primary somatosensory cortex (S1) was observed weekly for 2 weeks. CLO exposure caused a sustained influx of Ca2+ in neurons in the Si 2/3 layers, indicating hyperactivation of neurons. In addition, microarray gene expression analysis suggested the induction of neuroinflammation and changes in synaptic activity. These results demonstrate that exposure to the NOAEL dose of CLO can overactivate neurons and disrupt neuronal homeostasis.

The comprehensive targets of innervation in the intestinal mucosa are unknown, partly because of the diversity of cell types and the complexity of the neural circuits. Herein, we investigated the comprehensive targets of neural connectivity and analyzed the precise characteristics of their contact structures in the mucosa of rat ileum. We examined target cells of neural connections and the characteristics of their contact structures by serial block-face scanning electron microscopy at four portions of the rat ileal mucosa: the apical and basal portions in the villi, and the lateral and basal portions around/in the crypts. Nerve fibers were in contact with several types of fibroblast-like cells (FBLCs), macrophage-like cells, eosinophils, lymphocyte-like cells, and other types of cells. The nerve fibers almost always ran more inside of lamina propria than subepithelial FBLC, and thus contacts with epithelial cells were very scarce. The contact structures of the nerve fibers were usually contained synaptic vesicle-like structures, and we classified them into patterns based on the number of nerve fiber contacting the target cells at one site, the maximum diameter of the contact structures, and the relationship between nerve fibers and nerve bundles. The contact structures for each type of cells occasionally dug into the cellular bodies of the target cells. We revealed the comprehensive targets of neural connectivity based on the characteristics of contact structures, and identified FBLCs, immunocompetent cells, and eosinophils as the candidate targets for innervation in the rat ileal mucosa.

Few histological studies have been performed to evaluate infectious diseases in honeybees, as their water-resistant exoskeletons make the preparation of whole-body specimens difficult by blocking the permeation of fixative and substitution solutions. Additionally, it remains difficult to fix samples when specimens are collected far from the laboratory, as delays in fixation lead to the autolysis of abdominal organs. In this study, we propose an intraperitoneal fixative injection method to prevent fixation delay and a method for preparing honeybee tissue specimens for histopathological examinations.

Organ culture systems are useful for elucidating the process of testicular differentiation from mammalian undifferentiated genetically male gonads, as they permit various experiments, including experiments involving the control of gene expression. However, without addition of testicular differentiation-related factors, it is difficult to induce the formation of testis cord from immature gonads by a time point earlier 12 tail somites (ts) that corresponding to 11.0 days post coitum (dpc). In this study, we attempted to establish an organ culture system that induces testis formation from immature gonads (around 8 ts: 10.5 dpc) just before Sry (sex-determining region of the Y chromosome) expression. A paired gonad-mesonephros complex of around 8 ts was placed in the groove of an agarose gel block and put the semi-cylindrical piece of agarose gel to maintain the gonad morphology. The gonads were cultured in the gas phase for 96 hr. As a result, testis cord-like structures appeared in many genetically male gonads. Cells expressing the Sertoli cell markers Sox9 (SRY-box 9) and Amh (anti-Mullerian hormone) were observed, while granulosa cell marker Foxl2 (forkhead box L2) was not detected. In addition, Sox9- and Amh-expressing cells were observed throughout the entire gonad in many individuals. Amh mRNA expression was also upregulated. Surprisingly, formation of a partial testicular structure was observed from more immature gonads (6 ts). These results show that our gonadal organ culture system is useful for elucidating the regulation mechanism of Sry expression in undifferentiated bipotential gonads.

Dinotefuran (DIN) belongs to the neonicotinoids (NNs), a class of globally applied pesticides originally developed to exhibit selective toxicity in insects. However, several reports have suggested that NNs also exert neurotoxic effects in mammals. We previously demonstrated neurobehavioral effects of DIN on mice under non-stressful conditions. For further toxicity assessments in the present study, we investigated the effects of DIN on mice exposed to stressful conditions. After subacutely administering a no-observed-effect-level (NOEL) dose of DIN and/or chronic unpredictable mild stress (CUMS) to mice, we conducted three behavioral tests (i.e., open field test [OFT], tail suspension test [TST] and forced swimming test [FST]). In addition, serotonin (5-HT) and tryptophan hydroxylase 2 (TPH2) of the dorsal raphe nuclei (DRN) and median raphe nuclei (MRN) and tyrosine hydroxylase (TH) of the ventral tegmental area and substantia nigra (SN) were evaluated immunohistochemically. A NOEL dose of DIN or CUMS alone increased of the total distance in OFT, decreased or increased the immobility time in TST or FST, respectively, and increased the positive intensity of 5-HT and TPH2 in the DRN/MRN, and TH in the SN. These changes were suppressed under the conditions of combined exposure to DIN and CUMS, though the blood corticosterone level was increased depending on the blood DIN values and the presence of CUMS. The present study suggests the multifaceted toxicity of the neurotoxin DIN.

Neonicotinoid pesticides (NNs) act as agonists on nicotinic acetylcholine receptors (nAChRs) of insects, and there have been concerns about the effects of NNs on the health of mammals. Since nAChRs are expressed in immune cells, it is possible that NNs disturb the immune system. However, few reports have examined the immunotoxicity of clothianidin (CLO), a widely-used NN. Here, we report the effects of CLO on immune organs and type IV allergic reactions in ear auricles. We orally administered CLO at 0, 30 and 300 mg/kg/day (CLO-0, 30 and 300) to Sprague-Dawley rats for 28 days. The effects were evaluated by organ and body weights, histopathology, and immunohistochemistry (TCRαβ, CD4, CD8, CD11b, CD68, CD103). In addition, some cecal contents were subjected to preliminary gut microbiota analysis, because microbiota contribute to host homeostasis, including the immunity. Our results showed loose stool, suppression of body weight gain, significant changes in organ weights (thymus: decreased; liver: increased) and changes of the gut microbiota in the CLO-300 group. There were no obvious histopathological changes in immune organs. Granulomas of the ear auricles were found in one rat of each of the CLO-30 and 300 groups, but CLO had no apparent effect on the thickness or immunohistochemistry in the ear auricles. We present new evidence that CLO affects the thymus and intestine, and might enhance the local inflammatory response. These findings should contribute to the appropriate evaluation of the safety of NNs in the future.

Although neonicotinoids are among the major classes of pesticides that affect mammalian nervous systems, little is known about sex differences in their effects. This study aimed to examine whether the neurobehavioral effects of a neonicotinoid, clothianidin (CLO), differed between sexes. Male and female C57BL/6N mice were orally administered CLO (5 or 50 mg/kg) at or below the chronic no-observed-adverse-effect-level (NOAEL) and sub- jected to behavioral tests of emotional and learning functions. Changes in neuroactivity in several brain regions and the concentrations of CLO and its metabolites in blood and urine were measured. Acute CLO exposure caused sex-related behavioral effects; decreases in locomotor activities and elevation of anxiety-like behaviors were more apparent in males than in females. In addition, male-specific impairment of short- and long-term learning memory by CLO exposure was observed in both the novel recognition test and the Barnes maze test. Male- dominant increases in the number of c-fos positive cells were observed in the paraventricular thalamic nu- cleus in the thalamus and in the dentate gyrus in the hippocampus, which are related to the stress response and learning function, respectively. The concentrations of CLO and most metabolites in blood and urine were higher in males. These results support the notion that male mice are more vulnerable than females to the neuro- behavioral effects of CLO and provide novel insights into the risk assessment of neonicotinoids in mammalian neuronal function.

Our previous studies using immunohistochemistry and serial block-face scanning electron microscopy (SBF-SEM) clarified that fibroblast-like cells (FBLCs) in the rat ileal mucosa are classifiable into several subtypes, but their characteristics throughout the large intestine remain unknown. In this study, we investigated the region-specific characteristics of FBLCs in the rat large intestine using histological analysis including SBF-SEM. Immunohistochemistry revealed that CD34

The composition of fecal bacteria is reported to change throughout the day, whereas the circadian rhythmicity of indigenous bacteria that settle on the epithelium is mostly unknown. The present study aimed to clarify the diurnal changes in the settlement of indigenous bacteria in the rat alimentary tract using histological analysis. The settlement of indigenous bacteria on the mucosal epithelium throughout the day and the diurnal changes in settlement levels were observed in the esophagus, the nonglandular area of the stomach, and the ileum. The peak of zeitgeber time (ZT) in the settlement level differed by segment: ZT 12 in the esophagus, ZT 6 in the nonglandular area of the stomach, and ZT 0 in the ileum. Moreover, 16S rRNA amplicon sequencing using tissue sections revealed that the compositions of the indigenous bacteria in the ileum differed among ZT. In the intervillous spaces of the ileum, the formation level of the mucus layer, one of the most fundamental host defenses against bacteria, was lowest at ZT 0. Bacteria were preferentially adjacent to the villous epithelium in the area without coverage by the mucus layer at ZT 0. These findings collectively suggest that the settlement level and possibly the composition of the indigenous bacteria changed diurnally in various segments of the alimentary tract, and the formation of the mucus layer might be the most likely to lead to such diurnal changes in indigenous bacteria, at least in the ileum.

C57BL/6J-XYPOS (B6J-XYPOS) mice, which have the Y chromosome derived from Mus musculus poschiavinus on a B6J genetic background, form ovotestes or ovaries. Previously, we replaced the genetic background of B6J-XYPOS mice with B6N and found that individuals with testes also appeared in addition to those with ovaries or ovotestes. To investigate the effect of the B6J genetic sequence on the testis differentiation, the genetic background of B6N-XYPOS mice was replaced with B6J again. The recovery of the B6J genetic background significantly decreased the incidence of testes; only ovaries developed. These results indicate that the testicular differentiation process tends to be perturbed especially in the B6J substrain. This shows the importance of substrain differences in mice usually treated as B6 collectively.

The effect of bacterial colonies expanded into the intervillous spaces on the localization of several lymphocyte lineages was immunohistochemically investigated in two types of mucosa: ordinary mucosa of rat ileum, which consists of mucosa without any mucosal lymphatic tissue; and follicle-associated mucosa (FAM), which accompanies the parafollicular area under the muscularis mucosae in the rat ileal Peyer's patch. The results showed that bacterial colonies in the intervillous spaces induced increased populations of CD8(+) cells in the epithelium of the intestinal villus in ordinary mucosa (IV) and intestinal villus in FAM (IV-FAM). Bacterial colonies in the intervillous spaces were also associated with increased numbers of IgA(+) cells, which were mainly localized in the lamina propria of basal portions of IV and IV-FAM, and with expanded localization of IgA(+) cells into the villous apex in both IV and IV-FAM. Moreover, IgA(+) cells around the intestinal crypts adjacent to IV or IV-FAM were also increased in response to bacterial colonies. In the IV-FAM, but not IV, L-selectin(+) cells, which were found to be immunopositive for TCRαβ or CD19, were drastically increased in the lamina propria from the crypt to middle portion of IV-FAM and in the lumen of central lymph vessel of IV-FAM in response to the bacterial colonies in the intervillous spaces. These findings revealed that the expansion of bacterial colonies into the intervillous spaces accompanies the change of histological localization of the lymphocyte lineage in both the ordinary mucosa and FAM.

Serial block-face scanning electron microscopy (SBF-SEM) is useful for three-dimensional observation of tissues or cells at high-resolution. In this study, SBF-SEM was used to three-dimensionally analyze the characteristics of fibroblast-like cells (FBLCs) in the rat ileal lamina propria (LP). The results revealed that FBLCs in LP could be divided into four types, tentatively named FBLC type I-IV, based on the external cellular appearance, abundance or shape of each organelle, detailed distribution in the LP and relationship with surrounding cells. FBLC-I and -II localized around the intestinal crypt (InC), FBLC-III localized from the lateral portion of InC to the apical portion of the intestinal villus (InV), and FBLC-IV localized in the InV. FBLC-I, -II and -III, but not FBLC-IV, localized beneath the epithelium. FBLC-II possessed thin lamellar-shaped endoplasmic reticula, whereas FBLC-I possessed expanded endoplasmic reticula that occasionally showed a spherical shape. FBLC-III and -IV possessed a cytoplasmic region with high-electron density and no organelles immediately beneath the cellular membrane; this region was found at the epithelial sides in FBLC-III and scattered in FBLC-IV. FBLC-IV were in constant close proximity to villous myocytes throughout the InV and also in contact with FBLC-III especially in the apical portion of the InV. FBLC-I, -II and -IV, but not -III, were constantly found to be in contact with various immunocompetent cells in the LP. Three-dimensional analysis using SBF-SEM indicates that four types of FBLC localized in the rat ileal LP.

Neonicotinoids, which act as agonists of the nicotinic acetylcholine receptors of insects, are widely used pesticides worldwide. Although epidemiological studies revealed that the detection amounts of neonicotinoids in urine are higher in the elderly population than other age-groups, there is no available information regarding the risks of neonicotinoids to older mammals. This study was aimed to investigate aging-related differences in the behavioral effects of the neonicotinoid pesticide clothianidin (CLO). We acutely administered a sub-NOAEL level (5 mg/kg) of CLO to adult (12-week-old) and aging (90-week-old) mice and conducted four behavioral tests focusing on the emotional behavior. In addition, we measured the concentrations of CLO and its metabolites in blood, brain and urine. There were age-related changes in most parameters in all behavioral tests, and CLO significantly decreased the locomotor activity in the open field test and elevated plus-maze test in the aging group, but not in the adult group. The concentrations of most CLO and its metabolites were significantly higher in the blood and brain and were slightly lower in the urine in the aging group compared to the adult group. These findings should contribute to our understanding of age-related differences in the adverse effects of neonicotinoids in mammals.

Several types of macrophages have been reported in the intestinal mucosa, but their histological localization remains ambiguous. Here, we obtained detailed information about ultrastructural and phenotypical diversity of macrophage-like cells (MLCs) in the rat ileal mucosa using immunofluorescent analysis and serial block-face scanning electron microscopy (SBF-SEM). The results revealed that the cells immunopositive for CD68, the pan-macrophage marker, included CD163-CD4+, CD163+CD4+, and CD163-CD4- cells in the lamina propria (LP) of the intestinal villus and around the crypt. CD68+CD4+CD163- cells seemed to be preferentially localized in the intestinal villus, whereas CD68+CD163+CD4+ cells were frequently localized around the crypt. SBF-SEM analysis identified three types of MLCs in the ileal mucosa, which were tentatively named types I-III MLC based on aspects of the 3D-ultrastructure, such as the localization, quantity of lysosomes, endoplasmic reticulum, and exoplasm. Type I and II MLCs were localized in the villous LP, while type III MLCs were localized around the crypt, although type II MLCs were a minor population. All three MLC types extended their cellular processes into the epithelium, with type I MLCs showing the greatest abundance of extended processes. Type I MLCs in the upper portion of the intestinal villus showed a higher level of attachment to intraepithelial lymphocytes (IELs) compared to type III MLCs around the crypt. These findings suggest that macrophages of the rat ileal mucosa differed by region along the longitudinal axis of the villous tip-crypt from the perspective of ultrastructure, cellular composition, localization, and interactions with IELs.

We developed a new 2.5 &mu, m global shutter (GS) pixel using a 65 nm process with an advanced light pipe (LP) structure. This is the world&rsquo, s smallest charge domain GS pixel reported so far. This new developed pixel platform is a key enabler for ultra-high resolution sensors, industrial cameras with wide aperture lenses, and low form factors optical modules for mobile applications. The 2.5 &mu, m GS pixel showed excellent optical performances: 68% quantum efficiency (QE) at 530 nm, &plusmn, 12.5 degrees angular response (AR), and quite low parasitic light sensitivity (PLS)&mdash, 10,400 1/PLS with the F#2.8 lens. In addition, we achieved an extremely low memory node (MN) dark current 13 e&minus, /s at 60 &deg, C by fully pinned MN. Furthermore, we studied how the LP technology contributes to the improvement of the modulation transfer function (MTF) in near infrared (NIR) enhanced GS pixel. The 2.8 &mu, m GS pixel using a p-substrate showed 109 lp/mm MTF@50% at 940 nm, which is 1.6 times better than that without an LP. The MTF can be more enhanced by the combination of the LP and the deep photodiode (PD) electrically isolated from the substrate. We demonstrated the advantage of using LP technology and our advanced stacked deep photodiode (SDP) technology together. This unique combination showed an improvement of more than 100% in NIR QE while maintaining an MTF that is close to the theoretical Nyquist limit (MTF @50% = 156 lp/mm).

Neonicotinoids (NNs), a widely used class of systemic pesticides, are regarded as exhibiting selective toxicity in insects. However, NNs are suspected of exerting adverse effects on mammals as well, including humans. To date, only adult male animal models have been subjected to general toxicity studies of NNs; fetuses have yet to be considered in this context. Here, we focused on the NN clothianidin (CLO) for the first quantitative LC–MS/MS analysis of maternal-to-fetal transfer and residual property of once-daily (single or multiple days), orally administered CLO and its metabolites in mice. The results revealed the presence of CLO and its five metabolites at approximately the same respective blood levels in both dams and fetuses. In the dams, CLO showed a peak value 1 h after administration, after which levels rapidly decreased at 3 and 6 h. In the fetuses of each group, levels of CLO were almost the same as those observed in the corresponding dams. The present results clearly demonstrated rapid passage of CLO through the placental barrier. However, metabolite-dependent differences observed in blood pharmacokinetics and residual levels. This is the first quantitative demonstration of the presence of CLO and its metabolites in fetal mouse blood.

Stable reference genes are important for gene expression analyses such as quantitative PCR. The stability of 15 candidate reference genes that can be used to developing mouse gonads was thoroughly verified using combinations of multiple algorithms. The expression of these genes fluctuated greatly depending on the analysis period and/or gender. Peptidylprolyl isomerase A (Ppia) and polymerase (RNA) II (DNA directed) polypeptide A (Polr2a) were the reference genes that were used stably for a wide analysis period in developing mouse gonads. Furthermore, the stable reference genes corresponding to the analysis period and/or gender were ranked. These results are useful for the selection of the optimal reference gene required for high-precision measurements.

Although neonicotinoid pesticides are expected to have harmful influence on mammals, there is little animal experimental data to support the effect and mechanisms. Since acetylcholine causes the release of dopamine, neonicotinoids may confer a risk of developmental disorders via a disturbance in the monoamine systems. Male mice were peripubertally administered dinotefuran (DIN) referring to no observed effect level (NOEL) and performed behavioral and immunohistological analyses. In an open field test, the total locomotor activity was increased in a dose-dependent manner. The immunoreactivity of tyrosine hydroxylase in the substantia nigra was increased in DIN-exposed mice. These results suggest that exposure to DIN in peripubertal male mice causes hyperactivity and a disturbance of dopaminergic signaling.

It has been suggested that an increase in the use of pesticides affects neurodevelopment, but there has been no animal experiment showing a causal relation between neonicotinoid pesticides (NNs) and depression. We examined whether dinotefuran (DIN), the most widely used NN in Japan, induces depression. Male mice were administered DIN between 3 and 8 weeks of age, referring to the no-observed-effect level (NOEL). The mice were then subjected to a tail suspension test (TST) and a forced swimming test (FST). After these tests, their brains were dissected for immunohistochemical analyses of serotonin (5-HT). Antidepressant activity in TST and no decrease in 5-HT-positive cells were observed. The subchronic exposure to DIN alone in juvenile male mice may not cause depression-like indication.

The distributions of β-defensin 1 and 2 in secretory host defense system throughout respiratory tract of healthy rats were immunohistochemically investigated. In the nasal epithelium, a large number of non-ciliated and non-microvillous cells (NCs) were immunopositive for both β-defensin 1 and 2, whereas a small number of goblet cells (GCs) were immunopositive only for β-defensin 1. Beta-defensin 2-immunopositive GCs were few. In the nasal glands, a small number of acinar cells and a large number of ductal epithelial cells were immunopositive for both β-defensins. In the laryngeal and tracheal epithelia, a very few NCs and GCs were immunopositive for both β-defensins. In laryngeal and tracheal glands, a very few acinar cells and a large number of ductal epithelial cells were immunopositive for both β-defensins. In the extra-pulmonary bronchus, a small number of NCs were immunopositive for both β-defensins. A small number of GCs were immunopositive for β-defensin 1, whereas few GCs were immunopositive for β-defensin 2. From the intra-pulmonary bronchus to alveoli, a very few or no epithelial cells were immunopositive for both β-defensins. In the mucus and periciliary layers, β-defensin 1 was detected from the nose to the extra-pulmonary bronchus, whereas β-defensin 2 was weakly detected only in the nose and the larynx. These findings suggest that the secretory sources of β-defensin 1 and 2 are mainly distributed in the nasal mucosa and gradually decrease toward the caudal airway in healthy rats.

Paneth cells secrete bactericidal substances in response to bacterial proliferation on the mucosal surface without directly contacting bacteria. However, the induction mechanism of this transient secretion has not been clarified, although nervous system and/or immunocompetent cells in the lamina propria (LP) might be involved. In this study, we ultrastructurally and immunohistochemically investigated which LP cells are localized beneath Paneth cells and examined the relationship between the Paneth cell-derived cellular processes which extended into the LP and the LP cells. The results showed that various cells-including blood capillary, subepithelial stromal cell, and nerve fiber-were present in the LP beneath Paneth cells. Endothelial cells of blood capillary were the cells most frequently found in this location; they were situated within 1 μm of the Paneth cells and possessed fenestration on the surfaces adjacent to Paneth cells. The Paneth cells rarely extended the cellular processes toward the LP across the basal lamina. Most of the cellular processes of Paneth cells contacted the subepithelial stromal cells. Immunohistochemistry revealed that the CD34+ CD31- αSMA- stromal cells preferentially localized in the LP beneath the intestinal crypt base, while PDGFRαhi αSMA+ stromal cells mainly localized around the lateral portions of the intestinal crypt and PDGFRαhi αSMA- stromal cells localized in the intestinal villus. From these findings, the existence of blood capillaries beneath Paneth cells might reflect the active exocrine function of Paneth cells. Furthermore, subepithelial stromal cells, probably with a CD34+ CD31- αSMA- PDGFRα-/lo phenotype, beneath the crypt base might affect Paneth cell activity by interacting with their cellular processes. Anat Rec, 301:1074-1085, 2018. © 2018 Wiley Periodicals, Inc.

Cerebral palsy (CP) is a major neuronal disease and the most common movement disorder in children. Although environmental factors leading to CP have been greatly investigated, the genetic mechanism underlying CP is not well understood. Here we focused on two clinical reports that characterized a deletion involving the KANK1 gene locus in the 9p24.3 region. One report shows spastic CP and the other shows no spastic CP phenotype. Based on the epigenetic status and evolutionary conservation, we first found a functional genomic element at the noncoding region that was deleted only in patients with spastic CP. This element contains the retinoic acid receptor/retinoid X receptor (RAR/RXR) complex-binding motif that is widely conserved among placental mammals. RAR/RXR ChIP-seq data from mouse F9 embryonal carcinoma cells that were treated with trans-retinoic acids showed that the element has a binding ability. In addition, data regarding chromosome conformation capture from mouse neural progenitor and ES cells suggested that the element spatially interacts with the Doublesex and mab-3 related transcription factor 3 (Dmrt3) gene promoter that is located approximately 120 kb downstream of the RAR/RXR-binding site. Dmrt3 is detected in the developing mouse forebrain and in some interneurons in the spinal cord, and it works as a locomotion coordinator in horses and mice. Thus, the deletion of the cis-regulatory element for DMRT3 in humans may cause impaired development of the forebrain and gait abnormalities, resulting in spastic CP. In conclusion, this study provides new mechanistic insights into the genetic basis of CP.

Neonicotinoids are pesticides used worldwide. They bind to insect nicotinic acetylcholine receptors (nAChRs) with high affinity. We previously reported that clothianidin (CTD), one of the latest neonicotinoids, reduced antioxidant expression and induced germ cell death in the adult testis of vertebrates. Here, we investigated the male reproductive toxicity of prenatal and early postnatal exposure to CTD, because it is likely that developmental exposure more severely affects the testis compared to adults due to the absence of the blood-testis barrier. Pregnant C57BL/6 mice were given water gel blended with CTD (0, 10 or 50 mg/kg/day; noobserved- adverse-effect-level [NOAEL for mice]: 47.2 mg/kg/day) between gestational day 1 and 14 days post-partum. We then examined the testes of male offspring at postnatal day 14. The testis weights and the numbers of germ cells per seminiferous tubule were decreased in the CTD-50 group, and abnormal tubules containing no germ cells appeared. Nevertheless, the apoptotic cell number and proliferative activity were not significantly different between the control and CTD-exposed groups. There were no significant differences in the androgen-related parameters, such as the Leydig cell volume per testis, the Sertoli cell number and the tubule diameter. The present study is the first demonstration that in utero and lactational exposures to CTD at around the NOAEL for mice reduce the germ cell number, but our findings suggest that these exposures do not affect steroidogenesis in Leydig cells during prenatal or early postnatal life.

BACKGROUND The left male gonad in the chicken embryo has a thickened cortical layer, but it eventually becomes flattened after the onset of testicular development. Because the destination of the cortical cells migrating from the left gonad remains unclear, we examined this issue herein. RESULTS The testis-inducing gene doublesex- and mab-3-related transcription factor 1 (DMRT1) was detected in a proportion of the columnar and cubic epithelial cells in the cortex of the left testis as well as Sertoli cells in both testes. Interestingly, some of the DMRT1-expressing cortical cells were contiguous with Sertoli cells in the testis cord. Some cortical cells exhibited a vimentin-positive cytoplasm that was elongated all the way to the medulla. In addition, a desmosome-like structure was observed between the elongated cytoplasm in these cells and the adjacent Sertoli cell. After the organ culture, a few cells labeled with a fluorescent dye that stained only the cortical cells at the beginning of the culture were located in the testis cord of the left testis. CONCLUSIONS Some cortical cells expressing DMRT1 were suggested to contribute to the Sertoli cells in the testis cord only after the onset of testicular development and only in the left testis. Developmental Dynamics 246:148-156, 2017. © 2016 Wiley Periodicals, Inc.

The mechanism by which indigenous bacteria on the follicle-associated epithelium (FAE) of lymphatic follicles (LFs) accelerate the differentiation of microvillous columnar epithelial cells (MV) into M-cells was immunohistochemically investigated in rat Peyer's patches. The results showed that the number of Toll-like receptor (TLR) -4(+) M-cells was greater in the FAE with expansion of bacterial colonies (LFs with bacterial colonies on the FAE: b-LF) than the FAE without expansion of bacterial colonies (nb-LF). TLR-4 was also expressed in the striated borders of MV upstream next to M-cells in the FAE of the b-LF. TLR-4(+) vesicles were frequently detected in the cytoplasms of MV with TLR-4(+) striated borders upstream next to TLR-4(+) M-cells in the FAE of b-LF. These findings suggest that TLR-4(+) MV take up TLR-4 ligands and differentiate into M-cells in the b-LF. Neither the distribution of RANK nor that of RANKL was coincident with that of M-cells in the b-LF. Moreover, RANK, but not RANKL, was expressed in intestinal villi, whereas cleaved caspase-3 was immunonegative in the MV and M-cells of the FAE, unlike in villous epithelial cells. Therefore, RANK/RANKL signaling in the LF might contribute to the down-regulation of epithelial apoptosis to facilitate the differentiation of MV into M-cells in rat Peyer's patches.

Eosinophils are abundantly present in intestinal mucosa. However, the morphological characteristics of their cellular population are still largely unknown. In this study, we examine their characteristics in the rat ileal mucosa using histological and ultrastructural methods. The results indicated that ileal eosinophils could be distinguished into two main groups based on their nuclear shapes and distribution: eosinophils with spheric or reniform nuclei mainly localized in the villous region and eosinophils with annular or bacilliform nuclei as the major population around crypts. Immunohistochemical analysis revealed that all eosinophils in the lamina propria (LP) were immunopositive for CD11b, whereas eosinophils in LP of the intestinal villus but not those in LP around the crypt, were immunopositive for CD11c. Three-dimensional ultrastructural analysis using serial block-face scanning electron microscopy showed that the eosinophils with spheric or reniform nuclei were abundant in the upper portions of the intestinal villus, whereas those with annular nuclei were abundant in the lower portions of the intestinal villus and around crypts. The eosinophils with spheric or reniform nuclei possessed broader cellular bodies with greater abundance of surface projections compared with those with annular nuclei. Eosinophils in the upper portions of intestinal villus frequently extended their cellular bodies into the intraepithelial space. The number of total and eosinophil-specific granules was positively correlated with the minor axis of the nuclear holes in the annular nuclei. These data suggest that ileal eosinophils exhibit not homogenous but rather diverse characteristics, possible due to the mixture of eosinophils at different maturation and/or activation stages.

C57BL/6J-XYPOS (B6J-XYPOS) mice, which have the Y chromosome derived from Mus musculus poschiavinus on a B6J genetic background, form ovotestes or ovaries. Previously, we replaced the genetic background of B6J-XYPOS mice with B6N and found that individuals with testes also appeared in addition to those with ovaries or ovotestes. To investigate the effect of the B6J genetic sequence on the testis differentiation, the genetic background of B6N-XYPOS mice was replaced with B6J again. The recovery of the B6J genetic background significantly decreased the incidence of testes; only ovaries developed. These results indicate that the testicular differentiation process tends to be perturbed especially in the B6J substrain. This shows the importance of substrain differences in mice usually treated as B6 collectively.

We developed a 2.5um global shutter (GS) CMOS image sensor pixel using an advanced Light-Pipe (LP) structure designed with novel guidelines. To the best of our knowledge, it is the smallest reported GS pixel in the world. The developed pixel shows an excellent Quantum Efficiency (QE), Angular Responses (AR) and very low Parasitic Light Sensitivity (PLS). Also, even in oblique light condition of 10 degrees, the 1/PLS is maintained to about half value. These key characteristics allow development of ultra-high resolution sensors, industrial cameras with wide aperture lenses and low form factors optical modules for GS mobile applications.

The expressions of Toll-like receptor (TLR) -2, -4 and -9 were immunohistochemically investigated in the follicle-associated epithelium (FAE), and epithelia of the follicle-associated intestinal villus (FAIV) and ordinary intestinal villus (IV) in rat Peyer's patch regions with no bacterial colonies on the mucous membranes. TLR-2 was expressed in the striated borders of microvillous columnar epithelial cells (MV) in both FAIV and IV except in the apices. However, TLR-2 expression in the striated borders was weaker in the epithelium of the follicular side of FAIV (f-FAIV) than in epithelia of IV and the anti-follicular side of FAIV. TLR-4 and -9 were not expressed in the FAIV and IV. In the FAE, TLR-2, -4 and -9 were not expressed in the striated borders of MV, but the roofs of some typical M-cells were immunopositive for all TLRs. Especially, no TLR-positive MV were found at the FAE sites where M-cells appeared most frequently. In the follicle-associated intestinal crypt (FAIC), immunopositivity for all TLRs was observed in the striated borders of MV and the luminal substances. In conclusion, the lower levels of TLR-2 in both FAE and the epithelium of f-FAIV probably reduce recognition of indigenous bacteria. TLR-2, -4 and -9 appear not to participate directly in differentiation of MV into M-cells, because TLRs were not expressed in any MV in the upstream region of M-cells in FAE with no settlement of indigenous bacteria in the rat Peyer's patches.

We developed a low parasitic light sensitivity (PLS) and low dark current 2.8 μm global shutter pixel. We propose a new inner lens design concept to realize both low PLS and high quantum efficiency (QE). 1/PLS is 7700 and QE is 62% at a wavelength of 530 nm. We also propose a new storage-gate based memory node for low dark current. P-type implants and negative gate biasing are introduced to suppress dark current at the surface of the memory node. This memory node structure shows the world smallest dark current of 9.5 e−/s at 60 °C.

The host defense system with lysozyme and secretory phospholipase A2 (sPLA2) was immunohistochemically investigated in rat respiratory tract under healthy conditions. In the nasal epithelium, a large number of non-ciliated and non-microvillous cells (NC) and a small number of goblet cells (GC) were immunopositive for lysozyme and sPLA2. A few acinar cells and almost all epithelial cells of intercalated ducts were immunopositive for both bactericidal substances in the nasal glands. In the laryngeal and tracheal epithelia, few NC and GC were immunopositive for both bactericidal substances. In the laryngeal and tracheal glands, a few acinar cells and most ductal epithelial cells were immunopositive for both bactericidal substances. In extra-pulmonary bronchus, small numbers of NC and GC were immunopositive for lysozyme and sPLA2, whereas few NC and no GC were immunopositive in the intra-pulmonary bronchus. No secretory source of either bactericidal substance was located in the bronchioles. In the alveolus, many glandular epithelial cells and alveolar macrophages were immunopositive for lysozyme but immunonegative for sPLA2. Moreover, lysozyme and sPLA2 were detected in the mucus layer and in the periciliary layer from the nose to the extra-pulmonary bronchus. These findings suggest that secretory sources of lysozyme and sPLA2 are distributed in almost all the respiratory tract. Their secretory products are probably transported to the pharynx and contribute to form the first line of defense against inhaled bacteria throughout the respiratory tract.

Mammalian sexual fate is determined by the presence or absence of sex determining region of the Y chromosome (Sry) in the “bipotential” gonads. Recent studies have demonstrated that both male and female sexual development are induced by distinct and active genetic pathways. Breeding the Y chromosome from Mus m. domesticus poschiavinus (POS) strains into C57BL/6J (B6J) mice (B6J-XYPOS) has been shown to induce sex reversal (75%: bilateral ovary, 25%: true hermaphrodites). However, our B6N-XYPOS mice, which were generated by backcrossing of B6J-XYPOS on an inbred B6N-XX, develop as males (36%: bilateral testis with fertility as well as bilateral ovary (34%), and the remainder develop as true hermaphrodites. Here, we investigated in detail the expressions of essential sex-related genes and histological features in B6N-XYPOS mice from the fetal period to adulthood. The onsets of both Sry and SRY-box 9 (Sox9) expressions as determined spatiotemporally by whole-mount immunohistochemistry in the B6N-XYPOS gonads occurred 2–3 tail somites later than those in B6N-XYB6 gonads, but earlier than those in B6J-XYPOS, respectively. It is possible that such a small difference in timing of the Sry expression underlies testicular development in our B6N-XYPOS. Our study is the first to histologically show the expression and ectopic localization of a female-related gene in the XYPOS testes and a male-related gene in the XYPOS ovaries. The results from these and previous experiments indicate that the interplay between genome variants, epigenetics and developmental gene regulation is crucial for testis development.

Indigenous bacteria in the alimentary tract are exposed to various bactericidal peptides and digestive enzymes, but the viability status and morphological changes of indigenous bacteria are unclear. Therefore, the present study aimed to ultrastructurally clarify the degeneration and viability status of indigenous bacteria in the rat intestine. The majority of indigenous bacteria in the ileal mucous layer possessed intact cytoplasm, but the cytoplasm of a few bacteria contained vacuoles. The vacuoles were more frequently found in bacteria of ileal chyme than in those of ileal mucous layer and were found in a large majority of bacteria in both the mucous layer and chyme throughout the large intestine. In the dividing bacteria of the mucous layer and chyme throughout the intestine, the ratio of area occupied by vacuoles was almost always less than 10%. Lysis or detachment of the cell wall in the indigenous bacteria was more frequently found in the large intestine than in the ileum, whereas bacterial remnants, such as cell walls, were distributed almost evenly throughout the intestine. In an experimental control of long-time-cultured Staphylococcus epidermidis on agar, similar vacuoles were also found, but cell-wall degeneration was never observed. From these findings, indigenous bacteria in the mucous layer were ultrastructurally confirmed to be the source of indigenous bacteria in the chyme. Furthermore, the results suggested that indigenous bacteria were more severely degenerated toward the large intestine and were probably degraded in the intestine.

A portion of the minute chylomicrons less than 75 nm in diameter are transcytosed from the extravascular tissue into the subepithelial blood capillaries (sBC) in the villous apices of the rat jejunum. However, the details of the transportation mechanism have not been clarified. In this study, the endothelial receptor involved in the transportation of minute chylomicrons into the sBC’s lumina was immunohistochemically and histoplanimetrically examined in intestinal villi of the rat jejunum. Immunopositivity for very low density lipoprotein (VLDL) receptor was detected on the luminal and basal surfaces of the endothelial cells of sBC in approximately 68% of those apices of jejunal villi that possessed numerous chylomicrons in the lamina propria, while VLDL receptor was detected on the endothelial cells of sBC in only approximately 8% of intestinal villi that possessed few or no chylomicrons in the lamina propria. No immunopositivity for LDL receptor was detected in the sBC of all intestinal villi. These findings suggest that VLDL receptor is expressed by the endothelial cells of the sBC in conjunction with the filling of the lamina propria of jejunal villi with many chylomicrons produced by the villous columnar epithelial cells and that the VLDL receptor mediates the transportation of minute chylomicrons, maybe VLDL, into the subepithelial portal blood from the extravascular tissue of the rat jejunal villi.