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This study aimed to investigate the expression of type VI collagen α3 chain (COL6a3) in neoplastic cells of canine mammary gland carcinomas (CMGCs) using immunohistochemistry (IHC) and to evaluate the association between COL6a3 expression and tumor histological features, histological grades, and the differentiation status of neoplastic epithelial cells. COL6a3 expression in carcinoma cells was significantly associated with histologically low malignancy and low mitotic indices. In addition, COL6a3+ carcinoma cells were more frequently detected in simple carcinomas (tubular and tubulopapillary types) than in solid carcinomas. These findings indicate that reduced expression of COL6a3 in carcinoma cells contributes to the malignant phenotype in CMGCs. We also showed that COL6a3 expression in the carcinoma cells was more frequently detected in CK19+/CD49f + and/or CK19+/CK5+ tumors. In addition, COL6a3+/CK19+/CD49f + and COL6a3+/CK19+/CK5+ tumors consisted of CK19+/CD49f + and CK19+/CD49f- cells, and CK19+/CK5+ and CK19+/CK5- cells, respectively. Most of these tumors more frequently expressed GATA3, but not Notch1. These results indicate that COL6a3 is expressed in CMGCs containing both luminal progenitor-like and mature luminal-like cells and showing differentiation ability into mature luminal cells. It is possible that COL6 may be involved in the differentiation of luminal progenitor-like carcinoma cells into mature luminal-like carcinoma cells in CMGCs, which may suppresses the development of malignant phenotypes in CMGCs., Competing Interests: Declaration of Competing Interest None of the authors of this paper have a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper., (Copyright © 2023. Published by Elsevier Ltd.)

This study aimed to investigate the expression of receptor-type tumour endothelial marker 8 (TEM8RT) in canine mammary gland carcinomas (CMGCs) using immunohistochemistry and to evaluate the association between carcinoma cell TEM8RT expression and tumour histological features, histological grades and the differentiation status of neoplastic epithelial cells. TEM8RT expression was more frequently detected in simple carcinomas (tubular and tubulopapillary) than in solid carcinomas, and it was significantly correlated with histological grade Ⅰ tumours and a low mitotic index. Additionally, TEM8RT + carcinoma cells were more frequently found in CMGCs showing luminal progenitor-like phenotypes, such as Notch1 + , CK19 + /CK5 + /CD49f + and CK19 + /CK5 - /CD49f + . Double-labelling immunofluorescence detection techniques confirmed that most TEM8RT + carcinoma cells expressed CD49f, Notch1 and CK19. However, TEM8RT immunoreactivity was not found in carcinoma cells expressing GATA3, which upregulates mature luminal cell differentiation. Furthermore, TEM8RT + carcinoma cells were detected in a few CMGCs showing basal/stem cell-like phenotypes such as CK19 - /CK5 + /CD49f + and CK19 - /CK5 + /CD49f - . These findings indicate that TEM8RT is expressed in luminal progenitor-like carcinoma cells in CMGCs. Since TEM8 enhances self-renewal in human mammary stem/progenitor cells, it also may be involved in maintenance of luminal progenitor-like carcinoma cells, resulting in prevention of their transition to basal/stem cell-like carcinoma cells and development of less malignant CMGCs. Therefore, TEM8RT may be useful for indicating prognostic outcomes and identifying the possible ontogeny of carcinoma cells in mammary gland tumours., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Amyloid-β and tau pathologies are important factors leading to neurodegeneration in Alzheimer's disease (AD); however, the molecular mechanisms that link these pathologies remain unclear. Assuming that important though as yet unidentified factors inhibit/accelerate tau pathology and neuronal cell death under amyloid pathology, we sought to isolate and identify tau-interacting proteins from mouse brains with or without amyloid pathology. Among the proteins that were identified, we focused on protein arginine methyltransferase 8 (PRMT8), which interacts with tau specifically in the absence of amyloid pathology. To investigate the role of PRMT8 in the pathogenesis of AD, we conducted Prmt8 gene deletion and overexpression experiments in AppNL-G-F/MAPT double knock-in mice and analysed the resulting pathological alterations. PRMT8-knockout did not alter the AD pathology in double knock-in mice, whereas PRMT8-overexpression promoted tau phosphorylation, neuroinflammation and vacuole degeneration. To evaluate if such a PRMT8-induced vacuole degeneration depends on tau pathology, PRMT8 was overexpressed in tau-KO mice, which were consequently found to exhibit vacuole degeneration. In addition, proteomic analyses showed that PRMT8 overexpression facilitated the arginine methylation of vimentin. Abnormal protein methylation could be involved in PRMT8-induced brain pathologies. Taken together, PRMT8 may play an important role in the formation of tau pathology and vacuole degeneration., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Purpose To investigate the accuracy of implant-supported connecting crowns fabricated with digital definitive casts and conventional definitive casts.Methods Using a master model with two implant bodies inserted into the right mandibular molar area, 10 digital definitive casts were fabricated. Additionally, 10 conventional definitive casts were fabricated. The distance and angle between the two abutments of each definitive cast were compared. To compare the amount of lift of the incisal pin, each of the 10 superstructures was fabricated via computer-aided design/computer-aided manufacturing and then returned to the master model.Results No significant difference was observed for either the angle or the distance between the two abutments. The amount of lift of the incisal pin when the superstructure obtained via the optical method was returned to the master model was significantly larger than that when the superstructure obtained via the conventional method was returned to the definitive cast and the master model. No significant difference was observed after occlusal adjustment of the superstructures obtained using the conventional method.Conclusions The precision of definitive casts obtained via the optical method was virtually equal to that of definitive casts obtained via the conventional method. The accuracy of implant-supported connecting crowns fabricated using the optical method was significantly lower than that of implant-supported connecting crowns fabricated using the conventional method. This is attributed to the alignment of the maxillary and mandibular digital definitive casts.

Vascular endothelial growth factor-A (VEGF-A) is a principal regulator of hematopoiesis as well as angiogenesis. However, the functions of VEGF-A and its receptors (VEGFRs) in the differentiation of mast cells (MCs) in the skin remain unclear. The aim of this study was to determine the expression patterns of two VEGFRs (Flk1 and Flt1) in the skin MCs during development and maturation in rats. From the 17th days of embryonic development (E17) to 1 day after birth (Day 1), most of skin MCs were immature cells containing predominant alcian blue (AB) + rather than safranin O (SO) + granules (AB>SO MCs). AB>SO MC proportions gradually decreased, while mature AB+ MC proportions increased from E20 and reached to approximately 90% from Day 1 to 21, thereafter decreased to about 10% at Day 60 and 90. Flk1 + MC proportions changed almost in parallel with the numbers of MCs and Ki67 + MC proportions from E17 to Day 90. The proportions of MCs with both nuclear and cytoplasmic Flt1-immunoreactivity were markedly increased at Day 28, when the proportions of nuclear Flk1 + , Ki67 + , and AB>SO MCs had significantly decreased, and AB

The aim of this study was to investigate the expression of tumour endothelial marker 8 (TEM8) in canine mammary gland tumours (MGTs) by immunohistochemistry and to evaluate the association between tumour cell TEM8 expression and tumour histological features, histological grades and expression of luminal and basal/myoepithelial cell markers. TEM8 expression was detected in >60 % of neoplastic epithelial cells in all simple adenomas (n = 25), simple carcinomas (n = 43) and invasive micropapillary carcinomas (n = 5) studied. Six of the 18 solid carcinomas studied showed TEM8 expression in >60% of carcinoma cells present in solid structures and in 12 of the 18 solid carcinomas, <30% of the luminal structure-forming carcinoma cells showed TEM8 expression. TEM8 expression in the neoplastic cells was not associated with histological malignancy in canine MGTs. TEM8 + tumour cells frequently showed the luminal-like phenotype cytokeratin (CK)19 + /p63 - /α-smooth muscle actin (SMA) - , while most TEM8 - tumour cells exhibited the basal-like phenotype CK19 - /p63 + /αSMA - . These findings indicate that TEM8 may be involved in maintaining the characteristics of luminal cells in canine MGTs and that TEM8 would be useful in identifying the type of neoplastic epithelial cell in MGTs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Weather changes accompanied by decreases in barometric pressure are suggested to trigger meteoropathy, i.e., weather-related pain. We previously reported that neuropathic pain-related behavior in rats is aggravated by lowering barometric pressure, and that this effect is abolished by inner ear lesions. These results suggest that mechanisms that increase vestibular neuronal activity may parallel those that contribute to meteoropathy generation. However, it remains unknown whether changes in barometric pressure activate vestibular neuronal activity. To address this issue, we used expression of c-Fos protein as a marker for neural activation. Male and female mice were placed in a climatic chamber, and the barometric pressure was lowered by 40 hPa, from 1013 hPa, for 50 min (LP stimulation). The total number of c-Fos-positive cells in the vestibular nuclei was counted bilaterally after LP stimulation. We also video-recorded mouse behaviors and calculated the total activity score during the LP stimulation. LP stimulation resulted in significant c-Fos expression in the superior vestibular nucleus (SuVe) of male and female mice. There was no effect of LP stimulation on the total activity score. These data show that distinct neurons in the SuVe respond to LP stimulation. Similar mechanisms may contribute to the generation of meteoropathy in humans., Competing Interests: The authors have declared that no competing interests exist.

Although the involvement of two types of vasopressin (AVP) receptors, v1a and v1b, in neural regulation of social behavior is well documented in rodents, there is no report on combined actions of them in regulation of social behavior. In this study, we investigated behavioral differences between wild-type (WT) and v1a and v1b double knockout (dKO) mice. For this, we measured olfactory preference, sexual behavior with receptive females (four weekly tests) in an enriched large observation cage, and anxiety-like behaviors. No difference between WT and dKO mice was found in olfactory preferences for estrous female odor to male odor. Over all four mating tests, the number of mounts and pursuits after receptive females was significantly greater in dKO mice than in WT mice. In the elevated plus maze and the open field test, dKO mice showed lower anxiety-like behavior than WT mice. Finally, we measured approach behavior to several types of objects, figurines, and caged anestrous or estrous females placed in the open field apparatus. The only difference observed was that dKO mice spent longer in the vicinity of estrous females than did WT mice. These findings suggest that vasopressin receptors are involved in the regulation of sociosexual behavior, presumably partly mediated by emotional responses, in male mice., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The nasal cavity of turtles is composed of the upper and lower chambers, lined by the upper and lower chamber epithelia, respectively. In many turtles including the Reeve's turtle Mauremys reevesii, the upper chamber epithelium contains ciliated olfactory receptor neurons (ORNs) and the lower chamber epithelium contains microvillous ORNs. However, in the olfactory organ of the Chinese soft-shelled turtle Pelodiscus sinensis, both the upper and lower chamber epithelia contain ciliated ORNs. In the present study, we immunohistochemically examined the developmental process of olfactory organs in soft-shelled turtle and the Reeve's turtle to clarify the developmental origins of the lower chamber epithelium in these turtles. Obtained data indicate that olfactory organs of these turtles have identical origin and follow similar process of development, suggesting that, in the lower chamber epithelium of the nasal cavity, ciliated ORNs differentiate in soft-shelled turtle whereas microvillous ORNs differentiate in the Reeve's turtle., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Endoplasmic reticulum (ER) stress is believed to play an important role in the etiology of Alzheimer's disease (AD). The accumulation of misfolded proteins and perturbation of intracellular calcium homeostasis are thought to underlie the induction of ER stress, resulting in neuronal dysfunction and cell death. Several reports have shown an increased ER stress response in amyloid precursor protein (APP) and presenilin1 (PS1) double-transgenic (Tg) AD mouse models. However, whether the ER stress observed in these mouse models is actually caused by AD pathology remains unclear. APP and PS1 contain one and nine transmembrane domains, respectively, for which it has been postulated that overexpressed membrane proteins can become wedged in a misfolded configuration in ER membranes, thereby inducing nonspecific ER stress. Here, we used an App -knockin (KI) AD mouse model that accumulates amyloid-β (Aβ) peptide without overexpressing APP to investigate whether the ER stress response is heightened because of Aβ pathology. Thorough examinations indicated that no ER stress responses arose in App -KI or single APP-Tg mice. These results suggest that PS1 overexpression or mutation induced a nonspecific ER stress response that was independent of Aβ pathology in the double-Tg mice. Moreover, we observed no ER stress in a mouse model of tauopathy (P301S-Tau-Tg mice) at various ages, suggesting that ER stress is also not essential in tau pathology-induced neurodegeneration. We conclude that the role of ER stress in AD pathogenesis needs to be carefully addressed in future studies., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

To measure regional saturation of oxygen ( rSO 2 ) of hemoglobin and total hemoglobin index (HbI) in the brain (through the molera of the head) and skeletal muscle (musculus gracilis) of conscious Chihuahua dogs using an examiner’s finger-mounted near-infrared spectroscopy (NIRS) device, Toccare, we investigated brain and skeletal muscle NIRS in 48 Chihuahuas without severe disease. To measure rSO 2 and total HbI, a Toccare probe was placed on the molera of the head and musculus gracilis of each dog for real-time recording. Stable NIRS values were obtained within 10 s. We also examined the effect of anesthesia on rSO 2 and total HbI of a Chihuahua. Cerebral rSO 2 values ( 59 % ± 7 % ) were significantly lower than those obtained at femoral regions ( 67 % ± 6 % ), whereas total HbI values in the brain ( 0.38 ± 0.09 ) were significantly higher than those of the musculus gracilis ( 0.20 ± 0.05 ). Sedation with a combination of medetomidine and ketamine decreased cerebral rSO 2 along with a corresponding reduction in heart rate. Sevoflurane anesthesia with 100% O 2 maintained rSO 2 in the brain with a

The male red-bellied newt (Cynops pyrrhogaster) approaches the female's cloaca prior to performing any courtship behaviour, as if he is using some released substance to gauge whether she is sexually receptive. Therefore, we investigated whether such a female sexual attractiveness pheromone exists. We found that a tripeptide with amino acid sequence Ala-Glu-Phe is secreted by the ciliary cells in the epithelium of the proximal portion of the oviduct of sexually developed newts and confirmed that this is the major active substance in water in which sexually developed female newts have been kept. This substance only attracted sexually developed male newts and acted by stimulating the vomeronasal epithelial cells. This is the first female sexual attractiveness peptide pheromone to be identified in a vertebrate.

Several drug-metabolizing cytochrome P450 (CYP) enzymes exhibit sexual dimorphism depending on the pituitary growth hormone (GH) secretory patterns. However, the mechanism underlying CYP sexual dimorphism remains unclear. We previously established a transgenic (Alb-DsRed2 Tg) rat that expressed red fluorescent DsRed2 protein, particularly in hepatocytes, to visualize cell differentiation and multiplication and found that hepatic DsRed2 expression exhibited sexual dimorphism that was limited to adult males. In this study, we compared the expression patterns between sexual dimorphic Cyps and DsRed2 in Tg rats after experimentally reversing the GH secretory patterns in males and females. Postnatal day 1 male and female Tg rats were gonadectomized and then testosterone propionate (0.25 mg/rat) was subcutaneously administered to ovariectomized females immediately after surgery. Cyp mRNA and DsRed2 expression levels were quantified using RT-PCR and an in vivo imaging system, respectively. GH-dependent Cyps and hepatic DsRed2 expression patterns were reversed in males and females at 9 weeks after birth and were significantly correlated (P<0.05). This suggested that DsRed2 expression in these Tg rats depended on GH secretory patterns. Based on DsRed2 fluorescence, this Tg rat model could become a tool to readily and effectively evaluate changes in GH-dependent Cyp expression.

Selective estrogen receptor modulators (SERMs) are a class of therapeutic chemicals which present tissue-specific estrogen receptor modulating activity. Neonatal exposure to SERMs has been reported to adversely affect central nervous system development, however, mechanism and involvement of hypothalamic kisspeptin neurone in this impairment remains undetermined. To clarify this uncertainty, neonates from female Donryu rats were subcutaneously injected with raloxifene (RLX) at 0.1, 1, and 10mg/kg or tamoxifen (TMX) at 10mg/kg on postnatal day 0, and then hypothalamic KiSS1 mRNA expression and gonadotropin levels were investigated during young adulthood and estrous cycling was monitored until middle age. Treatment with RLX or TMX at 10mg/kg significantly depressed luteinizing hormone surge levels and KiSS1 mRNA expression in the anteroventral periventricular nucleus (AVPV), the control center of estrous cyclicity. The 10mg/kg TMX group also showed decreased levels of follicle-stimulating hormone and KiSS1 mRNA expression in the arcuate nucleus (ARC). Early cessation of normal estrous cycling was observed in the 10mg/kg RLX group, while the estrous cycle in the 10mg/kg TMX group had ceased by the start of the analysis. The same dose of tamoxifen or raloxifene had either weak-estrogenic or anti-estrogenic activity on the uterus, respectively; however, treatment in adulthood with both SERMs did not affect KiSS1 mRNA expression in either the AVPV or ARC in the present study. These results indicate that neonatal exposure to SERMs could disrupt neuroendocrine development and postnatal reproductive function through the alteration of kisspeptin neurons., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In general, the nasal cavity of turtles is divided into two chambers: the upper chamber, lined with the olfactory epithelium containing ciliated olfactory receptor cells, and the lower chamber, lined with the vomeronasal epithelium containing microvillous receptor cells. In the nasal cavity of soft-shelled turtles, however, differences between the upper and lower chamber epithelia are unclear due to the presence of ciliated receptor cells in both epithelia. In the olfactory organ of vertebrates, the surface of sensory epithelium is covered with secretory products of associated glands and supporting cells, playing important roles in the olfaction by dissolving odorants and transporting them to the olfactory receptors. Here, the associated glands and supporting cells in the olfactory organ of soft-shelled turtles were analyzed histochemically and ultrastructurally. The upper chamber epithelium possessed associated glands, constituted by cells containing serous secretory granules; whereas, the lower chamber epithelium did not. In the upper chamber epithelium, secretory granules filled the supranuclear region of supporting cells, while most of the granules were distributed near the free border of supporting cells in the lower chamber epithelium. The secretory granules in the supporting cells of both epithelia were seromucous, but alcian blue stained them differently from each other. In addition, distinct expression of carbohydrates was suggested by the differences in lectin binding. These data indicate the quantitative and qualitative differences in the secretory properties between the upper and lower chamber epithelia, suggesting their distinct roles in the olfaction.

In turtles, the epithelia lining the upper and lower chambers of the nasal cavity project axons to the ventral and dorsal parts of the olfactory bulbs, respectively. In a semi-aquatic soft-shelled turtle, Pelodiscus sinensis, more than 1,000 odorant receptor genes have been found, but it is not known where they are expressed. In this study, we aimed to clarify the distribution of cells expressing these genes in the olfactory organs of soft-shelled turtles. Immunoreactions for the Gαolf, the α subunit of G protein coupled to the odorant receptors, were detected on the surface of epithelia lining both the upper and lower chambers of the nasal cavity. The receptor cells in the epithelium of both chambers possessed cilia on the tip of their dendrites, whereas microvillous, non-ciliated, receptor cells were not found. These data suggest that the odorant receptor genes are expressed by the ciliated receptor cells in the upper and lower chamber epithelia. Precise location of the vomeronasal epithelium is not known at present.

Neonatal exposure to estrogens is known to cause delayed effects, a late-occurring adverse effect on adult female reproductive functions, such as early onset of age-matched abnormal estrous cycling. However, the critical period in which neonates are sensitive to delayed effects inducible by exogenous estrogen exposure has not been clearly identified. To clarify this window, we examined the intensity and timing of delayed effects using rats exposed to ethynylestradiol (EE) at various postnatal ages. After subcutaneous administration of a single dose of EE (20 μg/kg, which induces delayed effects) on Postnatal Day (PND) 0, 5, 10, or 14 in Wistar rats, hypothalamic and hormonal alterations in young adults and long-term estrous cycling status were investigated as indicators of delayed effects. In young adults, peak luteinizing hormone concentrations at the time of the luteinizing hormone surge showed a decreasing trend, and KiSS1 mRNA expression of the anterior hypothalamus and number of KiSS1-positive cells in the anteroventral periventricular nucleus were significantly decreased in the PND 0, 5, and 10 groups. The reduction in KiSS1 mRNA and KiSS1-postive cells was inversely correlated with age at time of exposure. These groups also exhibited early onset of abnormal estrous cycling, starting from 17 wk of age in the PND0 group and 19 wk of age in the PND5 and 10 groups. These indicators were not apparent in the PND14 group. Our results suggest that PND0-PND10 is the critical window of susceptibility for delayed effects, and PND14 is presumed to be the provisional endpoint of the window., (© 2015 by the Society for the Study of Reproduction, Inc.)

Neonatal exposure to 17alpha-ethynylestradiol (EE) causes delayed effect, a late-occurring irreversible damage to reproductive functions characterized by the early onset of age-matched abnormal estrous cycling. To clarify the involvement of a hypothalamic key cycling regulator KiSS1/GPR54 in the delayed effect, we investigated artificially induced LH surges and KiSS1 mRNA expression in the anteroventral periventricular nucleus (AVPV) of cycling young adult rats neonatally exposed to EE, and compared these parameters to those in about 5 months old middle-aged rats. KiSS1 mRNA expression, the number of KiSS1-positive cells and KiSS1/ERα co-expressing cells in the AVPV decreased in both EE-exposed and middle-aged rats. The peak area and levels of LH surge dose-dependently decreased in EE-exposed rats, and reduction was more evident in middle-aged rats. These results indicate that the prior attenuation of KiSS1 and consequent depression of LH surges plays a key role in the onset of abnormal estrous cycling in the delayed effect., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We analyzed the expression of G protein α subunits and the axonal projection into the brain in the olfactory system of the semiaquatic newt Cynops pyrrhogaster by immunostaining with antibodies against Gαolf and Gαo , by in situ hybridization using probes for Gαolf , Gαo , and Gαi2 , and by neuronal tracing with DiI and DiA. The main olfactory epithelium (OE) consists of two parts, the ventral OE and dorsal OE. In the ventral OE, the Gαolf - and Gαo -expressing neurons are located in the apical and basal zone of the OE, respectively. This zonal expression was similar to that of the OE in the middle cavity of the fully aquatic toad Xenopus laevis. However, the Gαolf - and Gαo -expressing neurons in the newt ventral OE project their axons toward the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB), respectively, whereas in Xenopus, the axons of both neurons project solely toward the MOB. In the dorsal OE of the newt, as in the principal cavity of Xenopus, the majority of the neurons express Gαolf and extend their axons into the MOB. In the vomeronasal organ (VNO), the neurons mostly express Gαo . These neurons and quite a few Gαolf -expressing neurons project their axons toward the AOB. This feature is similar to that in the terrestrial toad Bufo japonicus and is different from that in Xenopus, in which VNO neurons express solely Gαo , although their axons invariably project toward the AOB. We discuss the findings in the light of diversification and evolution of the vertebrate olfactory system., (© 2014 Wiley Periodicals, Inc.)

Puberty onset in mammals is tightly coupled to the animal's nutritional and metabolic state. In the present study, we evaluated the effects of a high-fat diet on leptin and adiponectin levels, leptin mRNA expression and puberty onset in female rats. On day 21, female rats were divided into 2 groups, normal food (NF) and high-fat food (HF). The HF group showed a significantly earlier (P<0.001) date of vaginal opening and lower body weight (P<0.001) than the NF group. The rats fed the HF food had a significantly heavier uterus (P<0.05) than those fed the NF food, whereas the serum leptin and adiponectin levels and leptin mRNA expression were not significantly different between the NF and HF groups. We speculate that the fat-induced nutritional imbalance in young females may lead to neuroendocrine dysfunction during adolescence.

The aim of this study was to investigate food intake, serum leptin levels, and leptin mRNA expression during the sexual cycle in rats. Female Wistar-Imamichi rats aged 8-10 weeks were used in this experiment. Food intake was measured during the light and dark phases (light on at 07:00 and off at 19:00) of the 4-day estrous cycle in female rats. Serum leptin levels were measured by ELISA, and leptin mRNA expression levels were analyzed using real-time PCR on diestrous- and proestrous-stage rats. Our results revealed that during the sexual cycle, food intake was significantly higher in the dark phase compared with the light phase. Food intake in proestrous females was significantly lower in the light and dark phases compared with the other groups. Serum leptin concentrations were significantly higher in both phases in proestrous rats compared with diestrous rats. There was a significant increase in leptin mRNA expression in adipose tissue during the proestrous period compared with the diestrous period. These findings suggest that increased leptin mRNA expression and serum leptin levels, which are induced by estrogen during the proestrous stage, may play a role in regulating appetitive behavior.

Neurons in the medial nucleus of the amygdala (MeA) play a key role in the innate maternal, reproductive, defensive, and social behaviors. However, it is unclear how activation of the vomeronasal system leads to the behavioral outputs that are associated with pheromones. Here, we characterized the electrophysiological and morphological properties of MeA neurons using whole-cell recordings in mice slice preparations. Biocytin labeling revealed that MeA neurons possessed bipolar to multipolar cell bodies and dendritic fields covering projection areas from the accessory olfactory bulb. In 70% of recorded MeA neurons, monosynaptic excitatory postsynaptic currents (EPSCs) were evoked from the accessory olfactory bulb afferent in which the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate component was dominant and was rarely followed by the N-methyl-d-aspartic acid component. Norepinephrine increased the frequency of spontaneous inhibitory postsynaptic currents in some neurons, whereas α-methyl-5-hydroxytryptamine increased spontaneous EPSCs in other neurons. Morphologically and physiologically, heterogeneous MeA neurons appear likely to produce multiplex outputs of instinctive behaviors., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In mammals, the vomeronasal system (VS) originating from the vomeronasal organ (VNO; also called "Jacobson's organ") is considered to be a chemosensory system that recognizes "pheromone" signals. In the accessory olfactory bulb (AOB), the primary center of the VS, the glomerular cell layer (GL) of the AOB is regarded as an important functional area in the transmission of pheromone signals from vomeronasal sensory neurons (VSNs) of the VNO. In mice, the most frequently used animal model for the study of the VS, the GL of the AOB has several unique histological properties when compared with the main olfactory bulb (MOB): (i) each glomerular size is far smaller than in the MOB; (ii) many juxtaglomerular cells (JGCs) are GABA immunopositive, but subpopulations of cells distributed in the AOB are tyrosine hydroxylase- or calcium-binding protein immunopositive; and (iii) the dendritic branching pattern of the JGC in the AOB is heteromeric. The biological significance of the mammalian VS is still debated. The unique histological properties of the mouse AOB summerized in the present review may give some useful information that may help in understanding the function of the mammalian VS.

Purpose: The dopamine-derived endogenous compound, R-salsolinol (SAL), was recently identified as a putative endogenous prolactin (PRL)-releasing factor. However, how SAL influences copulatory behavior is unknown. In this study, we examined the relationship between SAL and copulatory behavior in male rats., Methods: Male Sprague-Dawley rats administered SAL were exposed to female rats in estrus, the plasma PRL concentration was measured, and the behavioral frequency and time during copulatory behavior were noted., Results: In the control and SAL groups, plasma PRL concentrations at 15 min before exposure to the female were 7.3 ± 2.0 and 8.0 ± 1.5 ng/ml, respectively. Moreover, plasma PRL concentrations in males immediately after exposure to the female were 7.4 ± 1.2 and 68.0 ± 5.9 ng/ml, respectively ( P < 0.05). All (8/8) of the control animals ejaculated in the presence of the female, whereas only 33% (2/6) of the SAL group ejaculated. An increasing tendency for mount latency and intromission latency and a decreasing tendency for intromission frequency were observed in the SAL group., Conclusions: Copulatory behavior was inhibited in male rats after SAL injection, suggesting that SAL is a copulatory behavior inhibiting factor.

Estradiol plays an essential role in sexual differentiation of the rodent hypothalamus. Endocrine disruptors with estrogenic activity such as bisphenol A (BPA) are reported to disturb sexual differentiation of the hypothalamus. The purpose of the present study was to examine in vitro effects of BPA on developing hypothalamic neurons by focusing on a presynaptic protein synapsin I and microtubule-associated protein 2 (MAP2). In cultured hypothalamic cells from fetal rats, treatment with BPA enhanced both dendritic and synaptic development, as evidenced by increases in the area of dot-like staining of synapsin I and MAP2-positive area. An estrogen receptor (ER) antagonist, ICI 182,780, only partially blocked BPA-induced increase in the synapsin I-area, while it suppressed the MAP2-area increased by BPA. A specific ERK inhibitor, U0126, reduced the synapsin I-area without affecting the MAP2-area. BPA significantly decreased protein levels of synapsin I phosphorylated at Ser-9 and Ser-603. These findings indicate that BPA-inducing effects on dendritic and synaptic development are mediated by different molecular pathways., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Estradiol (17beta-estradiol, E(2)) plays an essential role in sexual differentiation of the rodent brain. The purpose of the present study was to investigate the effects of E(2) on developing hypothalamic neurons by focusing on a presynaptic protein, synapsin I. We applied E(2) to cultured hypothalamic cells removed from fetal rats and investigated resultant effects upon synapsin I. Our immunocytochemical study revealed that administration of E(2) increased the dendritic area ('MAP2-area') and synaptic area detected as dot-like staining of synapsin I ('synapsin I-area'). However, immunoblotting and real-time PCR showed that E(2) did not increase both protein and mRNA expression levels of synapsin I. Studies with cyclohexamide (CHX), membrane impermeable E(2) (E(2)-BSA), and an estrogen receptor (ER) antagonist ICI 182,780 indicated that E(2) affected the synapsin I-area mainly via a non-genomic pathway mediated by membrane ER. Immunoblotting showed that E(2) suppressed phosphorylation of synapsin I at residues Ser-9, Ser-553, and Ser-603. On the other hand, E(2) did not affect phosphorylation of synapsin I at Ser-62, Ser-67 and Ser-549. The present study suggests that E(2) affects localization of synapsin I in hypothalamic neurons by altering site-specific phosphorylation of synapsin I, which is likely mediated by membrane ER.

We investigated the effects of a bioartificial endocrine pancreas (Bio-AEP) produced by mouse beta cells on sexual dysfunction of streptozotocin (STZ)-induced diabetic female rats. Female rats were administered STZ (60 mg/kg BW, i.v.) at the age of 10 weeks and transplanted with a Bio-AEP including mouse beta cells at the age of 14 weeks (STZ+Bio-AEP group). Lordosis and proceptive sexual behavior of female rats were observed. The results showed that after the Bio-AEP transplant blood glucose recovered from 380-450 mg/dl induced by streptozotocin to 140-230 mg/dl and suppressed lordosis and proceptive behavior also recovered. These results suggest that it is possible to reverse sexual dysfunction by continuous administration of mouse insulin.

The brown-eared bulbul (Hysipetes amaurotis) is commonly found in Japan where it is regarded as a harmful bird that causes damage to agricultural products. Few studies have investigated the sensory apparatus of this bird, and consequently little is known of the sensory modalities it uses. Here we analyzed the anatomical and histological properties of the nasal cavity and olfactory bulb (OB) of the bulbul in order to investigate the functional level of olfaction in this species. Although both anterior and maxillary conchae were observed in the bulbul nasal cavity, there was no structure equivalent to the posterior concha. The OB located on the ventral side of the anterior extremity of the cerebrum and the ratio of olfactory bulb size to that of the cerebral hemisphere were very small. Interestingly, the left and right OBs were completely fused at the midline of the cerebrum. Furthermore, certain types of lectins that bind to the olfactory nerve of vertebrates with a well-developed sense of smell also bound positively to the olfactory nerve and glomerular layers of the bulbul OB. These findings suggest that the brown-eared bulbul has an anatomically and functionally less well developed sense of smell compared to other avian species. Although the molecular and developmental mechanisms underlying the fusion of the OB remain unknown, we suggest that the fused OB may offer a unique model for studying the evolution and development of the central olfactory nervous system in vertebrates.

The nasal cavity and olfactory bulb (OB) of the Japanese jungle crow (Corvus macrorhynchos) were studied using computed tomography (CT) and histochemical staining. The nasal septum divided the nasal cavity in half. The anterior and maxillary conchae were present on both sides of the nasal cavity, but the posterior concha was indistinct. A small OB was present on the ventral surface of the periphery of the cerebrum. The OB-brain ratio--the ratio of the size of the OB to that of the cerebral hemisphere--was 6.13. The olfactory nerve bundles projected independently to the OB, which appeared fused on gross examination. Histochemical analysis confirmed the fusion of all OB layers. Using a neural tracer, we found that the olfactory nerve bundles independently projected to the olfactory nerve layer (ONL) and glomerular layer (GL) of the left and right halves of the fused OB. Only 4 of 21 lectins bound to the ONL and GL. Thus, compared with mammals and other birds, the jungle crow may have a poorly developed olfactory system and an inferior sense of olfaction. However, it has been contended recently that the olfactory abilities of birds cannot be judged from anatomical findings alone. Our results indicate that the olfactory system of the jungle crow is an interesting research model to evaluate the development and functions of vertebrate olfactory systems.

As members of Western societies age, sexual function of older (elderly) individuals becomes an important issue, particularly for men. Specifically, copulatory behavior increases circulatory load, which may be related to reports of cardiac sudden death following ejaculation. To further examine this relationship, we compared heart rate (HR) before and after ejaculation in 48-week-old (aged) and 10-week-old (young) male rats. As compared with resting HR, HR after ejaculation was increased by 54.2 +/- 3.5 and 41.7 +/- 2.7%, respectively, among aged and young male rats. These values were significantly higher than baseline levels (P<0.01). The increases in HR at each time point during copulation were significantly higher in aged male rats than in young male rats (P<0.05 or P<0.01). We also studied decreases in HR following ejaculation and found that aged male rats had a significantly higher HR at 1 and 2 min after ejaculation than young rats (P<0.01). These results suggest that the circulatory load on the aged rat heart is greater than that on a young rat heart during copulatory behavior, especially at ejaculation. In addition, the decrease in HR in aged rats after ejaculation was more gradual than in young male rats. These results suggest that there is a higher risk of sudden cardiac death during sexual behavior in older males.

Aim: Elucidation of the neural mechanism of maternal behaviors is a medically and biologically important research task. The rat is the laboratory animal most extensively analyzed for maternal behaviors. However, the neural mechanism that maintains the motivation of postpartum rats for maternal behaviors has not yet been elucidated. In this study, we aimed to identify brain regions involved in the maintenance of motivation for maternal behaviors by detecting brain regions that exhibit changes in nerve activity when the mother rat is separated from her pups., Methods: Lactating mother rats were separated from their pups on postpartum day 3 and kept away from the pups for a certain period of time, and brain regions that exhibited changes in nerve activity when the rats were separated from their pups and those that exhibited changes in nerve activity when the pups are returned were detected by immunohistochemistry using anti-c-Fos antibody, a marker for increased nerve activity., Results: Rats that were separated from their pups and with the pups returned later showed increases in the number of c-Fos immunoreactive (c-Fos-IR) cells in the medial preoptic area (MPA), the bed nucleus of the stria terminalis (BST), the caudal portion of posterior hypothalamic area (PH) and the supramamillary nucleus (SUM). In mother rats permanently separated from their pups, only the PH and SUM exhibited an increase in the number of c-Fos-IR cells., Conclusion: In rats, the SUM is involved in aversive memory and changes in the postpartum anxiety level. The observed increase in the number of c-Fos-IR cells in the SUM of mother rats separated from their pups suggests that the nerve activity change in the SUM, which is involved in aversive memory and anxiety, is involved in the maintenance of maternal behaviors.

Purpose: Sudden cardiac death after ejaculation has been reported in humans and highlights the important relationship between sexual behavior and the heart. The rat is an extremely useful animal model for investigating reproductive function in male mammals. In this study, we examined the relationship between autonomic nervous system activity and the circulatory system during sexual behavior in male rats., Methods: Male Wistar-Imamichi rats were exposed to female rats in estrous and heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), and plasma noradrenaline (NA) and adrenaline (Ad) concentrations were measured by telemetry during copulation., Results: The resting HR was 365.5 ± 18.4 beats/min (mean ± SE), which increased to 531.2 ± 21.1 beats/min at ejaculation and decreased to 404.6 ± 30.7 beats/min 1 min after ejaculation. At rest, SBP and DBP were 123.8 ± 6.6 and 81.5 ± 4.1 mmHg, respectively, which increased to 154.5 ± 5.9 and 112.1 ± 7.3 mmHg at ejaculation. Baseline plasma Ad and NA concentrations were 151.6 ± 32.0 and 248.5 ± 22.5 pg/ml, respectively, and these increased to 393.8 ± 89.9 and 792.7 ± 154.0 pg/ml at ejaculation, after which they decreased to resting levels. The rate of increase in NA at ejaculation differed significantly from that of Ad., Conclusions: The load on the circulatory and autonomic nervous systems is controlled by a rapid decrease in HR and NA concentration immediately after ejaculation, such that the male rat is prepared for the next copulation.

The purpose of the present study was to examine the in vitro effects of low-dose cadmium (Cd) on developing cortical cells. The cortical cells removed from fetuses (embryonic day 15) were treated with 10nM of Cd for 24h. The effects of Cd on dendritic and synaptic development were immunocytochemically observed with anti-microtubule associated protein-2 (MAP2) and anti-synapsin I antibodies, respectively. Administration of Cd suppressed dendritic as well as synaptic development at 10nM. By two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometric (LC/MS/MS) analysis, we identified three proteins with different expression after Cd-treatment; dihydropyrimidinase-related protein 2 (DRP-2/CRMP-2), 14-3-3-epsillon and calmodulin (CaM). Though the number of identified proteins was small, these proteins are known to be involved in neuronal development. The present study demonstrated the morphological effects as well as affected proteins in Cd-treated cortical cells, providing tools and insights in elucidating mechanisms how low-dose Cd distorts brain development.

In this study, in order to clarify the kinetics of leptin, we focused on the ratio of leptin concentrations in cerebrospinal fluid and serum in aged male rats, and examined the weight of epididymal fat, and the passage rate of leptin through the blood-brain barrier. In the lighter animals, the epididymal fat weight was low, while leptin concentrations in the serum and cerebrospinal fluid were also low. Conversely, in the heavier animals, the weight of epididymal fat and leptin concentrations in the serum and cerebrospinal fluid were higher. With regard to the ratio of leptin in the cerebrospinal fluid and serum, the passage rate of leptin through the blood-brain barrier was lower in the heavier animals than in the lighter animals.

Efficient reproduction using natural mating and reproduction technology [in vitro fertilization (IVF) and embryo transfer (ET)] was investigated in IRS2 deficient mice with C57BL/6JJcl genetic background (Irs2(-/-) mice) as a typical type 2 diabetes model. From the results using various combinations of Irs2(-/-) and Irs2(-/+) mice, the combination of female Irs2(-/+) x male Irs2(-/-) was found to be more efficient than other combinations. In applications of reproduction technology using IVF and ET, the combination of female Irs2(-/+) x male Irs2(-/-) involves the possibility of Irs2(-/-) production by repeats using female Irs2(-/+) mice. However, reproductive continuity using this combination is difficult because of dependence on human technique and the cost of ET. Therefore, we concluded that Irs2(-/-) mice should be produced by embryo transfer using Irs2(-/-) mice from a colony consisting of female Irs2(-/+) x male Irs2(-/-).

Aim:  In the rat, intraperitoneal injection of p -chloroamphetamine (PCA), which releases central 5-hydroxytryptamine (5-HT) from serotonergic nerve terminals, induces ejaculation, even in the absence of an estrus female or female-related smell information. It is well known that the medial preoptic nucleus (MPN) and the medial nucleus amygdala (MEA) play a major role in the control of male sexual behavior in mammals. We examined whether or not neuronal activity of the MPN and/or the MEA was associated with PCA-induced ejaculation. Methods:  Using c-Fos immunohistochemistry, we demonstrated a difference in the neural activities of the MPN and the MEA for ejaculation during copulation with an estrus female and ejaculation by PCA injection. Results:  Increased numbers of c-Fos-immunoreactive (c-Fos-IR) cells were found in the MPN and the MEA in the brains of the mating animals, whereas in the brains of the animals undergoing PCA-induced ejaculation there was no increase in the number of c-Fos-IR cells in the MPN and a small increase in the MEA. Conclusion:  Based on these results, ejaculation induced by PCA is not associated with the MPN. Moreover, the MEA is not the main act for this ejaculation. (Reprod Med Biol 2008; 7 : 37-43).

We investigated effects of 17beta-estradiol (E(2)) and endocrine disrupters, nonylphenol (NP) and bisphenol-A (BPA), focusing on the neuronal development in cultures of fetal rat hypothalamic cells. We applied different concentrations of E(2), NP or BPA to the cultured hypothalamic cells and observed their effects on dendritic and synaptic development by immunocytochemistry using anti-microtubule associated protein-2 (MAP2) and anti-synapsin I antibodies, respectively. Administration of E(2) for 7 days affected MAP2-positive area as well as synapsin I-positive area. NP and BPA also influenced neuronal developments. The significant increase both in MAP2- and synapsin I-positive areas was observed at 10 and/or 100 nM of them, while 1 microM of them reduced the positive areas. Synaptic densities calculated from synapsin I-positive area/MAP2-positive area were not constant among different doses of three chemicals, but increased at 10 and/or 100 nM and decreased at 1 microM. Furthermore, immunostaining of NP-treated cells with the antibody against glial fibrillary acidic protein (GFAP) revealed that glial development was similarly influenced by NP. Therefore, the present results demonstrated that not only E(2) but also the environmental estrogenic chemicals, NP and BPA, affect development of fetal rat hypothalamic cells in vitro.