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Investigates the use of Broyden method and direct inversion in the iterative subspace in solving N-dimensional nonlinear equations. Improvement of rate of convergence in iterative calculations; Evaluation of efficiency using the reference interaction site model and Ornstein-Zernike integral equation; Determination of the converged solution.

The lifetime of II-VI-based blue-green laser diodes on GaAs substrates is limited by rapid degradation in the active layers. This degradation has been observed as dark defects in the active layer during the laser operation, where defects occurred due to stacking faults that originated from the Ga[sub 2]Se[sub 3] compounds at the ZnSe/GaAs interface. The reported value of the density of stacking faults of the II-VI lasers was in the order of 10[sup 5] cm[sup -2]. To extend the lifetime, surface treatment of the GaAs substrate and control of the interface reaction are necessary. We investigated a new treatment technique using hydrogen-radical and Zn/As fluxes. We fabricated ZnSe-based double-hetero (DH) structures on a treated GaAs substrate and measured the density of dark defects in the light emitter area by electroluminescence microscopy. Chemical bonds at the interface were evaluated by x-ray photoelectron spectroscopy. A dark defect density of less than 10[sup 5] cm[sup -2] was obtained when the As-terminated GaAs surface was Zn treated. The Zn treatment prevented the formation of the Ga[sub 2]Se[sub 3] layers. When we alternated the exposure between Zn and As fluxes, excess ZnAs[sub x] interfacial layers were formed and the quality of the DH structure was unacceptable. However, hydrogen-radical exposure before and during the Zn/As treatment effectively removed the excess ZnAs[sub x] compounds, and the density of dark defects fell to 2 × 10[sup 4] cm[sup -2]. [ABSTRACT FROM AUTHOR]

Background: Decellularized porcine small intestinal submucosa (SIS) is a biological scaffold used surgically for tissue repair. Here, we demonstrate a model of SIS as a scaffold for human adipose-derived stem cells (ASCs) in vitro and apply it in vivo in a rat ventral hernia repair model.Study Design: ASCs adherence was examined by confocal microscopy and proliferation rate was measured by growth curves. Multipotency of ASCs seeded onto SIS was tested using adipogenic, chondrogenic, and osteogenic induction media. For in vivo testing, midline abdominal musculofascial and peritoneal defects were created in Sprague-Dawley rats. Samples were evaluated for tensile strength, histopathology and immunohistochemistry.Results: All test groups showed cell adherence and proliferation on SIS. Fibronectin-treated scaffolds retained more cells than those treated with vehicle alone (p < 0.05). Fresh stromal vascular fraction (SVF) pellets containing ASCs were injected onto the SIS scaffold and showed similar results to cultured ASCs. Maintenance of multipotency on SIS was confirmed by lineage-specific markers and dyes. Histopathology revealed neovascularization and cell influx to ASC-seeded SIS samples following animal implantation. ASC-seeded SIS appeared to offer a stronger repair than plain SIS, but these results were not statistically significant. Immunohistochemistry showed continued presence of cells of human origin in ASC-seeded repairs at 1 month postoperation.Conclusion: Pretreatment of the scaffold with fibronectin offers a method to increase cell adhesion and delivery. ASCs maintain their immunophenotype and ability to differentiate while on SIS. Seeding freshly isolated SVF onto the scaffold demonstrated that minimally manipulated cells may be useful for perioperative surgical applications within the OR suite. We have shown that this model for a "living mesh" can be successfully used in abdominal wall reconstruction. [ABSTRACT FROM AUTHOR]

The lumbar spinal cord contains local neural circuits that are important in regulating male sexual behaviours, but the molecular mechanisms underlying these systems remain elusive. Gastrin-releasing peptide (GRP) is a member of the bombesin-like peptide family first isolated from the porcine stomach. Despite extensive pharmacological studies on the activity of bombesin-like peptides administered to mammals, little is known about the physiological functions of GRP in the spinal cord. We review recent findings on a system of neurones in the upper lumbar spinal cord, within the recently reported ejaculation generator, projecting axons containing GRP to the lower lumbar spinal cord and innervating regions known to control erection and ejaculation. [ABSTRACT FROM AUTHOR]

Studies with green fluorescent protein (GFP) have revealed the subcellular distribution of many steroid hormone receptors to be much more dynamic than previously thought. Fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) are powerful techniques with which to examine protein–protein interaction and the mobility of tagged proteins, respectively. FRET analysis revealed that steroid treatment (with corticosterone or testosterone) induces direct interaction of the glucocorticoid receptor (GR) and importin α in the cytoplasm and that, shortly after nuclear entry, the GR detaches from importin α. The mineralocorticoid receptor (MR) and androgen receptor (AR) show the same trafficking. Upon oestradiol treatment, ERα and ERβ in the same cell are relocalised to form a discrete pattern and are localised in the same discrete cluster (subnuclear foci). FRAP analysis showed that nuclear ERα and ERβ are most dynamic and mobile in the absence of the ligand, and that mobility decreases slightly after ligand treatment. Genomic as well as non-genomic actions of steroid hormones influence the cellular function of target tissues spacio-temporally. [ABSTRACT FROM AUTHOR]

Studies with green fluorescent protein (GFP) have revealed the subcellular distribution of many steroid hormone receptors to be much more dynamic than previously thought. Fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) are powerful techniques with which to examine protein–protein interaction and the mobility of tagged proteins, respectively. FRET analysis revealed that steroid treatment (with corticosterone or testosterone) induces direct interaction of the glucocorticoid receptor (GR) and importin α in the cytoplasm and that, shortly after nuclear entry, the GR detaches from importin α. The mineralocorticoid receptor (MR) and androgen receptor (AR) show the same trafficking. Upon oestradiol treatment, ERα and ERβ in the same cell are relocalised to form a discrete pattern and are localised in the same discrete cluster (subnuclear foci). FRAP analysis showed that nuclear ERα and ERβ are most dynamic and mobile in the absence of the ligand, and that mobility decreases slightly after ligand treatment. Genomic as well as non-genomic actions of steroid hormones influence the cellular function of target tissues spacio-temporally. [ABSTRACT FROM AUTHOR]

Antibodies reactive with a murine teratocarcinoma cell line (F9) were detected in immunofluorescence staining in sera from patients bearing ovarian germ cell tumour, Immunochemical studies revealed that antibodies binding to the cell surface of F9 cells react with large glycopeptides which are known to be components of F9 antigens defined by marine anti-F9 antibodies. In addition, treatment of F9 cells with retinoic acid, which induces differentiation of embryonal carcinoma cells, distinctly reduced both the ability of these antibodies to stain F9 cells and the biosynthesis of large glycopeptides by the cells. These findings indicate that the large glycopeptides precipitable by the patients' antibodies are differentiation associated antigens on characteristic embryonic cells. [ABSTRACT FROM AUTHOR]

Understanding the relative importance of selection and stochastic factors in population divergence of adaptive traits is a classical topic in evolutionary biology. However, it is difficult to separate these factors and detect the effects of selection when two or more contrasting selective factors are simultaneously acting on a single locus. In the damselfly Ischnura senegalensis, females exhibit color dimorphism and morph frequencies change geographically. We here evaluated the role of selection and stochastic factors in population divergence of morph frequencies by comparing the divergences in color locus and neutral loci. Comparisons between population pairwise FST for neutral loci and for the color locus did not detect any stochastic factors affecting color locus. Although comparison between population divergence in color and neutral loci using all populations detected only divergent selection, we detected two antagonistic selective factors acting on the color locus, that is, balancing and divergent selection, when considering geographical distance between populations. Our results suggest that a combination of two antagonistic selective factors, rather than stochastic factors, establishes the geographic cline in morph frequency in this system. [ABSTRACT FROM AUTHOR]

Androgens are known to affect cognitive and mnemonic aspects of spatial processing. The cholinergic system is thought to play an important role in cognition and memory, but little is known about the interaction between androgen and cholinergic neurons. The present study focused on the effects of testosterone on the cholinergic neurons in the anterior cingulate cortex, the posterior parietal cortex, the hippocampus, and the basal forebrain including the medial septum, i.e., regions related to spatial processing. We examined choline acetyltransferase (ChAT) immunoreactivity in three groups of adult male rats: sham-operated (Sham), 28-day gonadectomized (GDX), and 28-day gonadectomized with immediate implantation of testosterone propionate (GDX+TP). Comparison of the Sham and GDX+TP groups demonstrated that the GDX group had significantly decreased cell counts of ChAT-immunoreactive neurons in anterior cingulate cortex layer II/III, posterior parietal cortex layer II/III, and the medial septum, but not in the other basal forebrain subregions examined (the horizontal part of the diagonal band of Broca and the substantia innominata). The GDX group also had significantly reduced hippocampal ChAT-immunoreactive fiber pixel density. The GDX+TP group maintained ChAT-immunoreactive cell counts in the anterior cingulate cortex, posterior parietal cortex, and medial septum equivalent to those in the Sham group. Less than 1% of identified cells showed colocalization of immunoreactivity for ChAT and androgen receptor in the cell bodies of the cortex and basal forebrain.Our observations demonstrate that the presence or absence of testosterone for 4 weeks influenced the cholinergic population region-specifically in the adult rat brain. [Copyright &y& Elsevier]

Background: Implementation of appropriate oral anticoagulant treatment for the prevention of stroke in very elderly patients with atrial fibrillation is challenging because of concerns regarding bleeding.Methods: We conducted a phase 3, multicenter, randomized, double-blind, placebo-controlled, event-driven trial to compare a once-daily 15-mg dose of edoxaban with placebo in elderly Japanese patients (≥80 years of age) with nonvalvular atrial fibrillation who were not considered to be appropriate candidates for oral anticoagulant therapy at doses approved for stroke prevention. The primary efficacy end point was the composite of stroke or systemic embolism, and the primary safety end point was major bleeding according to the definition of the International Society on Thrombosis and Haemostasis.Results: A total of 984 patients were randomly assigned in a 1:1 ratio to receive a daily dose of 15 mg of edoxaban (492 patients) or placebo (492 patients). A total of 681 patients completed the trial, and 303 discontinued (158 withdrew, 135 died, and 10 had other reasons); the numbers of patients who discontinued the trial were similar in the two groups. The annualized rate of stroke or systemic embolism was 2.3% in the edoxaban group and 6.7% in the placebo group (hazard ratio, 0.34; 95% confidence interval [CI], 0.19 to 0.61; P<0.001), and the annualized rate of major bleeding was 3.3% in the edoxaban group and 1.8% in the placebo group (hazard ratio, 1.87; 95% CI, 0.90 to 3.89; P = 0.09). There were substantially more events of gastrointestinal bleeding in the edoxaban group than in the placebo group. There was no substantial between-group difference in death from any cause (9.9% in the edoxaban group and 10.2% in the placebo group; hazard ratio, 0.97; 95% CI, 0.69 to 1.36).Conclusions: In very elderly Japanese patients with nonvalvular atrial fibrillation who were not appropriate candidates for standard doses of oral anticoagulants, a once-daily 15-mg dose of edoxaban was superior to placebo in preventing stroke or systemic embolism and did not result in a significantly higher incidence of major bleeding than placebo. (Funded by Daiichi Sankyo; ELDERCARE-AF ClinicalTrials.gov number, NCT02801669.). [ABSTRACT FROM AUTHOR]

The effects of annealing on a ZnCdSe single quantum well (SQW) structure with ZnCdSSe/ZnSSe superlattice optical guiding layers are investigated. X-ray diffraction and photoluminescence (PL) measurements showed disordering of a ZnCdSSe/ZnSSe superlattice after annealing at about 500 °C. The PL peak energy of the SQW shifted to the higher energy side, and the linewidth narrowed in the sample annealed at 300 °C. Cadmium diffusion was confirmed by secondary ion mass spectrometry. We found that the disordering of the ZnCdSSe/ZnSSe superlattice and the changes in the emissions from the SQW were due to the Cd diffusion. © 1996 American Institute of Physics. [ABSTRACT FROM AUTHOR]

Focuses on a study which examined the nonalloyed ohmic contact to p-type gallium arsenide from the 2DEG-heterojunction bipolar transistor emitter electrode application point of view. Epitaxial layer structures of gallium arsenide; Description of the transmission line method device; Thermal stability of gallium arsenide.

The article discusses various reports published within the issue, including one on the classical mechanisms of sex steroid hormone action in the brain, and another on the developmental effects of steroid hormones.

The population structure of the Pacific cod Gadus macrocephalus was examined using 15 microsatellite loci and mitochondrial DNA ( ND2 region). In total, 274 individuals were sampled from 16 locations around Japan to estimate the level of genetic differentiation and effective population size ( Ne). Pairwise FST, analysis of molecular variance and Bayesian clustering analysis suggested the presence of two genetically distinct groups in waters around Japan, with a higher Ne value in the eastern group than in the western group. A possible factor that restricts gene flow between groups may be related to the water temperature differences in the south-western part of the Sea of Japan, where the Tsushima Warm Current flows around the area inhabited by the western group, which may limit migration between the west and east. [ABSTRACT FROM AUTHOR]

The guppy is known to exhibit remarkable interindividual variations in spectral sensitivity of middle to long wavelength-sensitive (M/LWS) cone photoreceptor cells. The guppy has four M/LWS-type opsin genes (LWS-1, LWS-2, LWS-3 and LWS-4) that are considered to be responsible for this sensory variation. However, the allelic variation of the opsin genes, particularly in terms of their absorption spectrum, has not been explored in wild populations. Thus, we examined nucleotide variations in the four M/LWS opsin genes as well as blue-sensitive SWS2-B and ultraviolet-sensitive SWS1 opsin genes for comparison and seven non-opsin nuclear loci as reference genes in 10 guppy populations from various light environments in Trinidad and Tobago. For the first time, we discovered a potential spectral variation (180 Ser/Ala) in LWS-1 that differed at an amino acid site known to affect the absorption spectra of opsins. Based on a coalescent simulation of the nucleotide variation of the reference genes, we showed that the interpopulation genetic differentiation of two opsin genes was significantly larger than the neutral expectation. Furthermore, this genetic differentiation was significantly related to differences in dissolved oxygen (DO) level, and it was not explained by the spatial distance between populations. The DO levels are correlated with eutrophication that possibly affects the color of aquatic environments. These results suggest that the population diversity of opsin genes is significantly driven by natural selection and that the guppy could adapt to various light environments through color vision changes. [ABSTRACT FROM AUTHOR]

Natural hybrids between the boreal species Hexagrammos octogrammus and two temperate species Hexagrammos agrammus and Hexagrammos otakii were observed frequently in southern Hokkaido, Japan. Previous studies revealed that H. octogrammus is a maternal ancestor of both hybrids; the hybrids are all fertile females and they frequently breed with paternal species. Although such rampant hybridization occurs, species boundaries have been maintained in the hybrid zone. Possible explanations for the absence of introgressions, despite the frequent backcrossing, might include clonal reproduction: parthenogenesis, gynogenesis and hybridogenesis. The natural hybrids produced haploid eggs that contained only the H. octogrammus genome (maternal ancestor) with discarded paternal genome and generated F1‐hybrid type offspring by fertilization with the haploid sperm of H. agrammus or H. otakii (paternal ancestor). This reproductive mode was found in an artificial backcross hybrid between the natural hybrid and a male of the paternal ancestor. These findings indicate that the natural hybrids adopt hybridogenesis with high possibility and produce successive generations through hybridogenesis by backcrossing with the paternal ancestor. These hybrids of Hexagrammos represent the first hybridogenetic system found from marine fishes that widely inhabit the North Pacific Ocean. In contrast with other hybridogenetic systems, these Hexagrammos hybrids coexist with all three ancestral species in the hybrid zone. The coexistence mechanism is also discussed. [ABSTRACT FROM AUTHOR]

Abstract: Dramatic changes in the cytoskeleton and the morphology of oligodendrocytes (OLs) occur during various stages of the myelination process. OLs in culture produce large membrane sheets containing cytoskeletal veins of microtubules and actin filaments. We recently showed that estrogen receptors (ER) related to ERα/β were expressed in the membrane sheets of mature OLs in culture. Ligation of these or other membrane ERs in OLs with both 17β- and 17α-estradiol mediated rapid non-genomic signaling. Here, we show that estrogens also mediate rapid non-genomic remodeling of the cytoskeleton in mature OLs in culture. 17β-Estradiol caused a rapid loss of microtubules and the actin cytoskeleton in the OL membrane sheets. It also increased phosphorylation of the actin filament-severing protein cofilin, thus inactivating it. Staining for actin barbed ends with rhodamine-actin showed that it decreased the amount of actin barbed ends. 17α-Estradiol, on the other hand, increased the percentage of cells with abundant staining of actin filaments and actin barbed ends, suggesting that it stabilized and/or increased the dynamics of the actin cytoskeleton. The specific ERα and ERβ agonists, 4,4′,4″-(4-propyl-(1H)-pyrazole-1,3,5-triyl) trisphenol (PPT) and diarylpropionitrile 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN), respectively, also caused the rapid phosphorylation of cofilin. Estrogen-induced phosphorylation of cofilin was inhibited by Y-27632, a specific inhibitor of the Rho-associated protein serine/threonine kinase (ROCK). The Rho/ROCK/cofilin pathway is therefore implicated in actin rearrangement via estrogen ligation of membrane ERs, which may include forms of ERα and ERβ. These results indicate a role for estrogens in modulation of the cytoskeleton in mature OLs, and thus in various processes required for myelinogenesis. [Copyright &y& Elsevier]

Follicular development and ovulation are strongly suppressed during lactation in mammals via a profound suppression of gonadotrophin secretion. The present study aimed to examine the role of oestrogen feedback action in suppressing luteinising hormone (LH) secretion and hypothalamic kisspeptin expression during the latter half of lactation. Plasma LH concentrations kept at low levels throughout the lactating period in intact and oestrogen-replaced ovariectomised (OVX) lactating rats, whereas plasma LH concentrations gradually elevated from day 10 postpartum in lactating OVX rats. OVX lactating rats showed frequent LH pulses at late lactation, although the LH pulses were significantly inhibited by an oestrogen replacement, which is much less effective on LH release in nonlactating rats. Oestrogen replacement in lactating OVX rats significantly reduced the number of Kiss1 mRNA-expressing cells in the arcuate nucleus (ARC) at late lactation, although the same oestrogen treatment did not affect the number of Kiss1-expressing cells in nonlactating controls. Exogenous kisspeptin challenge (0.2 nmol) into the third cerebroventricle significantly increased LH secretion in lactating OVX, lactating OVX + subcutaneous 17β-oestradiol and intact lactating rats at day 16 postpartum. These results suggest that LH pulse suppression during late lactation could be a result of the enhanced oestrogen-dependent suppression of ARC kisspeptin expression. [ABSTRACT FROM AUTHOR]

The sagittalis nucleus (SGN) of the hypothalamus is a newly-identified nucleus that is located in the interstitial area between the arcuate and ventromedial nuclei of the rat hypothalamus and for which the long axis of the nucleus is oriented sagittally. Interestingly, the SGN exhibits structural and physiological sex differences, as defined by Nissl staining and oestrogen receptor (ER)α immunoreactivity (-ir), being larger in males than females. The structural sex difference is established by sex steroid action in neonates because the treatment of female pups with testosterone propionate masculinised the SGN. The phenotypical sex difference in ERα-ir is mediated hormonally in adulthood. Ovariectomy of female rats caused a significant increase in ERα-ir in the SGN, and eliminated the physiological sex difference, but with recovery to the level of gonad-intact females when given oestradiol replacement. Adult females have oestrous cycle-related variations in ERα-ir in the SGN, with levels at a nadir during the evening of pro-oestrous. The discovery of the SGN, a target of sex steroid action, provides a new opportunity for explaining hormonal regulation of sexually-differentiated behavioural and endocrine functions. [ABSTRACT FROM AUTHOR]

Abstract: Importin-α and β1 mediate the translocation of macromolecules bearing nuclear localization signals across the nuclear pore complex. Five importin-α isoforms have been identified in mice and six in human. Some of these importins play an important role in neural activity such as long term potentiation, but the functional differences of each isoform in the CNS are still unclear. We performed in situ hybridization (ISH) using non-isotopic probes to clarify the expression patterns of importin-α subtypes (α5, α7, α1, α4, α3) and importin-β1 in the mouse CNS of adult and early postnatal stages. The mRNAs of the importin-α subtypes and importin β1 were expressed throughout the CNS with specific patterns; importin-α5, α7, α3, and β1 showed moderate to high expression levels throughout the brain and spinal cord; importin-α4 showed a lack of expression in limited regions; and importin-α1 showed a low expression level throughout the brain and spinal cord but with a moderate expression level in the olfactory bulb and reticular system. We also demonstrated that importin-αs and β1 mRNAs were predominantly expressed in neurons in the adult mouse brain by using double-labeling fluorescence ISH and immunohistochemistry. Moreover, importin-αs and β1 mRNAs were detected throughout the CNS of postnatal mice and were highly expressed in the external granule layer of the cerebellar cortex on postnatal days 0, 4, and 10. This is the first report of importin-αs and β1 expression throughout the CNS of adult mice, as well as in the developing brain, including cell type specific localization. [Copyright &y& Elsevier]

Abstract: Vasopressin (AVP) plays an important role in anxiety-related and social behaviors. Single-prolonged stress (SPS) has been established as an animal acute severe stress model and has been shown to induce a lower adrenocorticotropic hormone (ACTH) response upon cortisol challenge. Here, we show results from immunoassays for AVP, ACTH, and corticosterone (CORT), and in situ hybridizations for AVP mRNA performed 7 days after SPS exposure. Immunofluorescence for AVP was also performed during the 7-day period following SPS exposure and after an additional forced swimming stress paradigm. We observed that the plasma concentrations of AVP, ACTH, and CORT were not altered by SPS; ACTH content in the pituitary and AVP mRNA expression in the supraoptic nucleus (SON) were significantly reduced by SPS. During the 7-day period following SPS, the intensity of immunoreactivity, the size of the soma, and the immunoreactive optical density of the dendrites of AVP neurons in the SON all increased. An apparent reduction in the intensity of AVP immunoreactivity was observed in the SON at 4 h after additional stress. Additional forced swimming led to a rapid increase in the dendritic AVP content only in the controls and not in the SPS-treated rats. These findings suggest that AVP is a potential biomarker for past exposure to severe stress and that alterations in AVP may affect the development of pathogenesis in stress-related disorders. [Copyright &y& Elsevier]

The anti-diuretic hormone arginine vasopressin (AVP) is synthesised in the magnocellular neurosecretory cells (MNCs) in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the hypothalamus. AVP-containing MNCs that project their axon terminals to the posterior pituitary can be identified using immunohistochemical techniques with specific antibodies recognising AVP and neurophysin II, and by virtue of their electrophysiological properties. Recently, we generated transgenic rats expressing an AVP-enhanced green fluorescent protein (eGFP) fusion gene in AVP-containing MNCs. In this transgenic rat, eGFP mRNA was observed in the PVN and the SON, and eGFP fluorescence was seen in the PVN and the SON, and also in the posterior pituitary, indicating transport of transgene protein down MNC axons to storage in nerve terminals. The expression of the AVP-eGFP transgene and eGFP fluorescence in the PVN and the SON was markedly increased after dehydration and chronic salt-loading. On the other hand, AVP-containing parvocellular neurosecretory cells in the PVN that are involved in the activation of the hypothalamic-pituitary adrenal axis exhibit robust AVP-eGFP fluorescence after bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide. In the median eminence, the internal and external layer showed strong fluorescence for eGFP after osmotic stimuli and stressful conditions, respectively, again indicating appropriate transport of transgene traslation products. Brain slices and acutely-dissociated MNCs and axon terminals also exhibited strong fluorescence, as observed under fluorescence microscopy. The AVP-eGFP transgenic animals are thus unique and provide a useful tool to study AVP-secreting cells in vivo for electrophysiology, imaging analysis such as intracellular Ca2+ imaging, organ culture and in vivo monitoring of dynamic change in AVP secretion. [ABSTRACT FROM AUTHOR]

The anti-diuretic hormone arginine vasopressin (AVP) is synthesised in the magnocellular neurosecretory cells (MNCs) in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the hypothalamus. AVP-containing MNCs that project their axon terminals to the posterior pituitary can be identified using immunohistochemical techniques with specific antibodies recognising AVP and neurophysin II, and by virtue of their electrophysiological properties. Recently, we generated transgenic rats expressing an AVP-enhanced green fluorescent protein (eGFP) fusion gene in AVP-containing MNCs. In this transgenic rat, eGFP mRNA was observed in the PVN and the SON, and eGFP fluorescence was seen in the PVN and the SON, and also in the posterior pituitary, indicating transport of transgene protein down MNC axons to storage in nerve terminals. The expression of the AVP-eGFP transgene and eGFP fluorescence in the PVN and the SON was markedly increased after dehydration and chronic salt-loading. On the other hand, AVP-containing parvocellular neurosecretory cells in the PVN that are involved in the activation of the hypothalamic-pituitary adrenal axis exhibit robust AVP-eGFP fluorescence after bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide. In the median eminence, the internal and external layer showed strong fluorescence for eGFP after osmotic stimuli and stressful conditions, respectively, again indicating appropriate transport of transgene traslation products. Brain slices and acutely-dissociated MNCs and axon terminals also exhibited strong fluorescence, as observed under fluorescence microscopy. The AVP-eGFP transgenic animals are thus unique and provide a useful tool to study AVP-secreting cells in vivo for electrophysiology, imaging analysis such as intracellular Ca2+ imaging, organ culture and in vivo monitoring of dynamic change in AVP secretion. [ABSTRACT FROM AUTHOR]

Abstract: The amygdala modulates memory consolidation with the storage of emotionally relevant information and plays a critical role in fear and anxiety. We examined changes in neuronal morphology and neurotransmitter content in the amygdala of rats exposed to a single prolonged stress (SPS) as a putative animal model for human post-traumatic stress disorder (PTSD). Rats were perfused 7 days after SPS, and intracellular injections of Lucifer Yellow were administered to neurons of the basolateral (BLA) and central amygdala (CeA) to analyze morphological changes at the cellular level. A significant increase of dendritic arborization in BLA pyramidal neurons was observed, but there was no effect on CeA neurons. Neuropeptide Y (NPY) was abundant in BLA under normal conditions. The local concentration and number of immunoreactive fibers of NPY in the BLA of SPS-exposed rats were increased compared with the control. No differences were observed in this regard in the CeA. Double immunostaining by fluorescence and electron microscopy revealed that NPY immunoreactive terminals were closely associated with calcium/calmodulin II-dependent protein kinase (CaMKII: a marker for pyramidal neurons)–positive neurons in the BLA, which were immunopositive to glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). SPS had no significant effect on the expression of CaMKII and MR/GR expression in the BLA. Based on these findings, we suggest that changes in the morphology of pyramidal neurons in the BLA by SPS could be mediated through the enhancement of NPY functions, and this structural plasticity in the amygdala provides a cellular and molecular basis to understand for affective disorders. [Copyright &y& Elsevier]

Male wild house mice selected for a long (LAL) or a short (SAL) latency to attack a male intruder generally show opposing behavioural coping responses to environmental challenges. LAL mice, unlike SAL mice, adapt to novel challenges with a highly reactive hypothalamic-pituitary-adrenal axis and show an enhanced expression of markers for hippocampal plasticity. The present study aimed to test the hypothesis that these features of the more reactive LAL mice are reflected in parameters of hippocampal cell proliferation. The data show that basal cell proliferation in the subgranular zone (SGZ) of the dentate gyrus, assessed by the endogenous proliferation marker Ki-67, is lower in LAL than in SAL mice. Furthermore, application of bromodeoxyuridine (BrdU) over 3 days showed an almost two-fold lower cell proliferation rate in the SGZ in LAL versus SAL mice. Exposure to forced swimming resulted, 24 h later, in a significant reduction in BrdU + cell numbers in LAL mice, whereas cell proliferation was unaffected by this stressor in SAL mice. Plasma corticosterone and dentate gyrus glucocorticoid receptor levels were higher in LAL than in SAL mice. However, no differences between the SAL and LAL lines were found for hippocampal NMDA receptor binding. In conclusion, the data suggest a relationship between coping responses and hippocampal cell proliferation, in which corticosterone may be one of the determinants of line differences in cell proliferation responses to environmental challenges. [ABSTRACT FROM AUTHOR]

Abstract: The hippocampal formation is a plastic brain structure important for certain types of learning and memory, and also vulnerable to the effects of stress and trauma. Since hippocampal neurons express high levels of corticosteroid receptor, the morphological changes, including alterations in the size of soma, and the length and number of neurites and spines, in response to glucocorticoids released as a result of stress are intriguing. In order to highlight the morphology of neurons that express glucocorticoid receptor (GR), we have generated a transgenic mouse line expressing green fluorescent protein (GFP) under the control of the GR promoter. We found strong green fluorescence in the pyramidal cell layer of the CA1 and CA2 regions and the granule cell layer of the dentate gyrus of the hippocampus in brain sections of the transgenic mice. GFP fluorescence was observed not only in somas, but also in neurites including both dendrites and axons. In dissociated culture, we also observed GFP fluorescence in the soma, neurites including both dendrites and axons, and dendritic spines. Microtubule-associated protein 2 immunopositive pyramidal-shaped neurons clearly showed two different populations, GFP positive and GFP negative neurons. These results indicate that this transgenic mouse line should be useful for live imaging of neuronal structure in animals as well as GR-positive cultured cells using GFP as a specific indicator. [Copyright &y& Elsevier]

The paraventricular nucleus (PVN) in the hypothalamus receives inputs from the hippocampus. The present study explored the influence of the hippocampus on genes mediating glucocorticoid feedback in the PVN. Accordingly, the expression of mRNAs for corticotrophin-releasing hormone (CRH), the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) in the PVN was examined by in situ hybridisation in rats subjected to transection of the fornix. Significant increases in CRH, MR and GR mRNAs were observed in the parvocellular PVN after fornix transection (FT). FT-animals subjected to adrenalectomy also showed an increase in the number of cells positive for CRH and GR mRNAs. CRH, MR and GR mRNA expression was also increased by bilateral adrenalectomy, and GR mRNA expression was further enhanced in the parvocellular PVN of the FT transected animals. However, no such changes were evident in the magnocellular PVN. These results suggest that the input from the hippocampus to the PVN, particularly to its parvocellular region, has distinct and differential inhibitory effects on the expression of MR, GR and CRH mRNAs that may operate independently from the feedback actions of corticosterone. [ABSTRACT FROM AUTHOR]

Arginine vasopressin (AVP) plays an important role in stress-induced activation of the hypothalamic-pituitary adrenal axis. In the present study, AVP-enhanced green fluorescent protein (eGFP) transgenic rats were used to investigate changes in AVP-eGFP expression in the hypothalamic paraventricular nucleus (PVN) and the median eminence (ME) upon exposure to stress conditions. The eGFP fluorescence in the parvocellular division of the PVN (pPVN) was markedly increased 5 days after bilateral adrenalectomy (ADX) and it was colocalised with corticotrophin-releasing hormone-like immunoreactivity in the pPVN. Peripheral administration of dexamethasone completely suppressed the increase of eGFP fluorescence in the pPVN and the external layer of the ME (eME) after bilateral ADX. Significant increases of eGFP fluorescence were observed in the pPVN 6, 12, 24 and 48 h after intraperitoneal (i.p.) administration of lipopolysaccharide (LPS). In the eME, eGFP fluorescence was significantly increased 48 h after i.p. administration of LPS. By contrast, eGFP fluorescence changed neither in the magnocellular division of the PVN, nor the internal layer of the ME after i.p. administration of LPS. Our results indicate that AVP-eGFP transgenic rats are useful animal model to study dynamic changes of AVP expression in the hypothalamus under stressful conditions. [ABSTRACT FROM AUTHOR]

The objective of this study was to quantify end-organ damage caused by bacteremic sepsis. Twelve adult swine were divided into two groups. The anesthesia control group (n = 6) received general anesthesia for 4 hours. The septic shock group (n = 6) received an infusion of Aeromonas hydrophila under general anesthesia for 4 hours. Swine were sacrificed at the end of the 4-hour procedure. Tissues from lungs, kidneys, livers, and hearts were stained with hematoxylin and eosin. Images of tissues were studied with digital image analysis. In lungs, cytoplasmic area (CA), nuclear area (NA), intra-alveolar hemorrhage (IAH), total airspace (TAS), and alveolar septum thickness (ST) were measured. Nuclear and cytoplasmic intensities (NI and CI) were measured in integrated optical density units (IOD). In kidneys, livers, and hearts, CA, CI, NA, and NI were measured similarly. Sinusoidal blood in the liver and vacuolization (VAC) in the kidney were also measured. In septic lungs, CI, NA, NI, ST, IAH, TAS, and ratios of NA/CA, NI/CI, and IAH/TAS were significantly increased compared with the control (P < 0.02). In septic kidneys, CI, NA, VAC, NA/CA, and NI/CI were significantly increased (P < 0.0005). In livers, CA, CI, and NI/CI were significantly increased (P < 0.005). In hearts, the ratios of NA/CA and NI/CI were statistically significant. End organs from septic swine, with exception of the heart, showed significantly higher levels of cellular damage. Digital image analysis provides an objective, precise, and accurate method of quantifying image characteristics. Automating these tasks is a high priority in the research and clinical community in providing a reproducible method for longitudinal analysis of various biological studies. [ABSTRACT FROM AUTHOR]

We examined the effects of chronic salt loading on the hypothalamic expressions of the enhanced green fluorescent protein (eGFP), arginine vasopressin (AVP) and oxytocin (OXT) genes in AVP-eGFP transgenic rats that expressed eGFP in the hypothalamic AVP-containing neurones. In these rats, salt loading for 5 days caused a marked increase of the eGFP fluorescence in the magnocellular divisions of the paraventricular nucleus (PVN), the supraoptic nucleus (SON) and the internal layer of the median eminence. Expression of the eGFP gene was increased seven- to eight-fold in the PVN and SON of salt-loaded rats in comparison with euhydrated rats. By contrast, none of these changes were observed in the suprachiasmatic nucleus. The expression of the AVP and OXT genes was increased 1.5- to two-fold in the PVN and SON of salt-loaded nontransgenic (control) and transgenic rats. There were no differences in the expression levels of the AVP and OXT genes in the PVN and SON between nontransgenic (control) and transgenic animals under normal conditions and after salt loading. In the posterior pituitary gland, the intensity of the eGFP fluorescence did not change after salt loading for 5 days, but increased after 10 days of salt loading. Upon salt loading, significant increases in the plasma AVP concentrations, plasma osmolality and plasma Na+ were observed. Furthermore, there were no significant differences in changes of water intake, food intake, urine volume, urine osmolality, urine Na+ concentrations, and the body weights in both models under normal or salt-loaded conditions. Our results show that the response of the AVP-eGFP fusion gene to chronic salt loading is exaggerated, and humoral responses such as AVP and OXT and the body fluid homeostasis are maintained in AVP-eGFP transgenic rats. The AVP-eGFP transgenic rat gives us a new opportunity to study the dynamics of the AVP system in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Glucocorticoids exert various neuroendocrinological effects, including stress response, in the central nervous system via glucocorticoid receptor (GR). GRs are transported from the cytoplasm to the nucleus upon ligand binding, and then exert the transcriptional activity. Although it is important for unraveling the actual property of the GR in vivo, subcellular dynamics of the GR are still unclear within the brain tissue in which the neuronal circuitry is maintained. To address this issue, we generated green fluorescent protein (GFP)–GR knockin mice, whose GR has been replaced by a GFP–GR fusion protein that is functionally indistinguishable from endogenous GR. In fixed brain sections of the GFP–GR knockin mice, the distribution of the green fluorescence was similar to that of GR immunoreactivity. By subtracting autofluorescence using fluorescent emission fingerprinting method with confocal laser scanning microscope, nuclear localization of GFP–GR was identifiable in the hippocampal CA3 subregion, where subcellular localization of the GR has been unsolved compared with other areas. To examine the subcellular trafficking of GFP–GR in vivo, we performed adrenalectomy on the GFP–GR knockin mice. GFP–GR was translocated from the nucleus to the cytoplasm and neurites two days after adrenalectomy. Furthermore, laser scanning cytometry by which fluorescence intensity in situ can be quantitatively measured revealed the entire GFP–GR expression level was increased. We then examined the dynamic changes in the subcellular localization of GFP–GR in living hippocampal neurons both in dissociated culture and in tissue slices. GFP–GR was localized in not only the perikarya but also neurites in the absence of ligand, and nuclear translocation following ligand treatment was observed. This is the first report visualizing subcellular trafficking of the GR in the mouse brain in more physiological condition. The present results propose new avenues for the research of the GR dynamics both in vitro and in vivo. [Copyright &y& Elsevier]

Neurosteroids are synthesized de novo in the brain and the cerebellar Purkinje cell is a major site for neurosteroid formation. We have demonstrated that the rat Purkinje cell actively produces progesterone de novo from cholesterol only during neonatal life and progesterone promotes dendritic growth, spinogenesis and synaptogenesis via its nuclear receptor in this neuron. On the other hand, 25-Dx, a putative membrane progesterone receptor, has been identified in the rat liver. In this study, we therefore investigated the expression and localization of 25-Dx in the Purkinje cell to understand the mode of progesterone actions in this neuron. Reverse transcription-PCR and Western immunoblot analyses revealed the expressions of 25-Dx mRNA and 25-Dx-like protein in the rat cerebellum, which increased during neonatal life. By immunocytochemistry, the expression of 25-Dx-like protein was localized in the Purkinje cell and external granule cell layer. At the ultrastructural level, we further found that 25-Dx-like immunoreactivity was associated with membrane structures of the endoplasmic reticulum and Golgi apparatus in the Purkinje cell. These results indicate that the Purkinje cell expresses the putative membrane progesterone receptor, 25-Dx during neonatal life. Progesterone may promote dendritic growth, spinogenesis and synaptogenesis via 25-Dx as well as its nuclear receptor in the Purkinje cell in the neonate. [Copyright &y& Elsevier]

Abstract In the pituitary gland, annexin-1 (lipocortin-1) located in folliculo-stellate (FS) cells has been advocated as one of the candidates for paracrine agents produced by FS cells that modulate the release of pituitary hormones. However, the expression and distribution pattern of annexin-1 in FS cells under different circulating corticosteroid conditions has not been examined. Thus, by means of pre-embedding immunoelectron microscopy, we investigated the expression of annexin-1 in FS cells under different corticosteroid conditions. Annexin-1-immunoreactivity was observed in the cytoplasm; especially intense immunoreactivity was detected in the follicle surface of FS cells under control conditions. After adrenalectomy, annexin-1-immunoreactivity almost disappeared, but the immunoreactivity recovered with corticosterone replacement. The expression of glucocorticoid receptor immunoreactivity in the nucleus of FS cells also showed a similar pattern to annexin-1 associated with the changes in the corticosteroid conditions. However, S-100 immunoreactivity, a marker for FS cells, was not changed whatever the corticosteroid conditions. These results confirm that glucocorticoids regulate the annexin-1 expression and demonstrate the translocation of annexin-1 from intracellular to pericellular sites in the FS cells of the rat anterior pituitary gland. [ABSTRACT FROM AUTHOR]

Prolactin secretion is controlled by the hypothalamus, and by circulatingsteroids; oestrogens stimulate, but glucocorticoids inhibit prolactin release.Lactotrophs express intracellular receptors for oestrogens, but apparentlynot glucocorticoids. Therefore, a genomic effect of oestrogens could be direct,but that of glucocorticoids appears to be indirect. Lactotrophs are not ahomogeneous cell population: some have large irregular dense-cored vesicles,others have small round vesicles, but the functional significance of thisinhomogeneity is far from clear. Oestradiol and testosterone can stimulate rapid release of prolactin selectivelyfrom type II lactotrophs characterised by small round vesicles. Progesteroneand other steroids do not exert this effect, which results from a non-genomicaction of oestradiol and testosterone. Glucocorticoid inhibition of secretagogue-inducedprolactin secretion is mimicked by annexin 1 (lipocortin 1), a protein inducedby glucocorticoids in the pituitary and many other tissues, and can be blockedby annexin 1 immunoneutralisation and antisense. Glucocorticoid inhibitionof ACTH and growth hormone secretion also involves annexin 1. Pituitary annexin1 is located in folliculo-stellate cells; these express glucocorticoid receptors,and glucocorticoids induce annexin-1 synthesis. Annexin 1 is externalisedfrom folliculo-stellate cells in response to glucocorticoids, despite thefact that it lacks a secretory signal sequence and is not packaged in vesicles.Inhibition of annexin 1 externalisation by glyburide suggests involvementof an ABC (ATP-binding cassette) transporter in externalisation. Both oestradioland glucocorticoids therefore influence the secretion of prolactin by noveldirect and indirect mechanisms, in addition to their much better understoodeffects on transcription via classical intracellular steroid receptors. [ABSTRACT FROM AUTHOR]

AbstractSerotonin is involved in female sexual behaviour in which the medial preoptic area (MPA) has a pivotal role. The present study used immunohistochemistry, in situ hybridization and retrograde transport analysis to investigate whether serotonin neurones in the dorsal raphe nucleus (DRN) of females projecting into the MPA contained oestrogen receptor α or β. The projection of serotonin neurones from the DRN to the MPA was confirmed using the microinjection of Fluoro-Gold (FG), a fluorescent retrograde tracer, into the MPA of ovariectomized (OVX-group) and OVX-rats treated with oestradiol benzoate (E2-group). A number of serotonin neurones in the DRN were labelled with FG, indicating that these serotonin neurones in DRN project their terminals into the MPA. FG-labelled serotonin neurones expressed ERβ mRNA in the DRN, and the number of the serotonin neurones containing ERβ mRNA between the OVX-group and the E2-treated group was not significantly different. Serotonin neurones in the DRN did not express ERα-immunoreactivity. Since previous findings showed that the density of serotonin-immunoreactive fibres and the concentration of serotonin within the MPA was significantly lower in the E2-group than the OVX-group, our present observations suggested that the regulatory effects of E2 on the serotonergic neurone system in the MPA may be via ERβ within the serotonin-containing cells in the DRN of female rats. [ABSTRACT FROM AUTHOR]

Abstract: Mn K-edge spin-polarized X-ray absorption near edge structure (SPXANES) spectra of MnO have been examined by use of a spin-dependent full multiple-scattering theory. Theoretical calculations for MnO show good agreement with the observed spectra on the basis of the paramagnetic model, whereas antiferromagnetic model should be taken into account to obtain better results even above Néel temperature . [Copyright &y& Elsevier]