Adverse experience in early life can affect the formation of neuronal circuits during postnatal development and exert long-lasting influences on neural functions that can lead to the development of a variety of psychiatric disorders... more
Adverse experience in early life can affect the formation of neuronal circuits during postnatal development and exert long-lasting influences on neural functions that can lead to the development of a variety of psychiatric disorders including depression, anxiety disorders, and post-traumatic stress disorder. Many studies have demonstrated that daily repeated maternal separation, an animal model of early-life stress, can induce impairments in emotional behaviours and cognitive function during adolescence and adulthood. However, the behavioural phenotypes of maternally separated mice under long-term group-housing conditions are largely unknown. In this study, we applied our newly developed assay system to investigate the effects of maternal separation on behaviours under group-housing conditions during four days of continuous observations. Using our system, we found that repeated maternal separation resulted in inappropriate social distance from cagemates, altered approach preferences...
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OBJECTIVE Transplantation of bone marrow stromal cells (BMSCs) is a theoretical potential as a therapeutic strategy in the treatment of spinal cord injury (SCI). Although a scaffold is sometimes used for retaining transplanted cells in... more
OBJECTIVE Transplantation of bone marrow stromal cells (BMSCs) is a theoretical potential as a therapeutic strategy in the treatment of spinal cord injury (SCI). Although a scaffold is sometimes used for retaining transplanted cells in damaged tissue, it is also known to induce redundant immunoreactions during the degradation processes. In this study, the authors prepared cell sheets made of BMSCs, which are transplantable without a scaffold, and investigated their effects on axonal regeneration, glial scar formation, and functional recovery in a completely transected SCI model in rats. METHODS BMSC sheets were prepared from the bone marrow of female Fischer 344 rats using ascorbic acid and were cryopreserved until the day of transplantation. A gelatin sponge (GS), as a control, or BMSC sheet was transplanted into a 2-mm-sized defect of the spinal cord at the T-8 level. Axonal regeneration and glial scar formation were assessed 2 and 8 weeks after transplantation by immunohistochemi...
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Early-life stress has long-lasting effects on the stress response, emotions, and behavior throughout an individual's life. Clinical reports have demonstrated that child abuse victims exhibit impairments in reward-associated behavior;... more
Early-life stress has long-lasting effects on the stress response, emotions, and behavior throughout an individual's life. Clinical reports have demonstrated that child abuse victims exhibit impairments in reward-associated behavior; yet, the mechanism for this effect remains unclear. Maternal separation (MS) or MS coupled with social isolation (SI) (MS+SI) is widely used as a model for early-life stress in rodent studies. We employed mice subjected to MS+SI to clarify the long-term effect of early-life stress on reward-seeking involving palatable foods by a conditioned place-preference (CPP) paradigm. Prior MS+SI experience decreased exploration time in a chocolate-paired compartment in adult female mice, but not in male mice. We then focused on the mesolimbic dopamine pathway associated with reward-seeking behavior and measured both mRNA and protein levels of tyrosine hydroxylase (TH) in the ventral tegmental area (VTA) and dopamine D1 and D2 receptors in the nucleus accumbens...
Research Interests: Psychology, Cognitive Science, Epigenetics, Medicine, Nucleus Accumbens, and 15 moreReward, Dopamine, Female, Animals, Male, DNA methylation, Social Isolation, Early life stress, Tyrosine Hydroxylase, Sex Factors, Receptor, Ventral Tegmental Area, Neurosciences, Maternal Deprivation, and Conditioned place preference
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Early-life stress during the prenatal and postnatal periods affects the formation of neural networks that influence brain function throughout life. Previous studies have indicated that maternal separation (MS), a typical rodent model... more
Early-life stress during the prenatal and postnatal periods affects the formation of neural networks that influence brain function throughout life. Previous studies have indicated that maternal separation (MS), a typical rodent model equivalent to early-life stress and, more specifically, to child abuse and/or neglect in humans, can modulate the hypothalamic–pituitary–adrenal (HPA) axis, affecting subsequent neuronal function and emotional behavior. However, the neural basis of the long-lasting effects of early-life stress on brain function has not been clarified. In the present review, we describe the alterations in the HPA-axis activity—focusing on serum corticosterone (CORT)—and in the end products of the HPA axis as well as on the CORT receptor in rodents. We then introduce the brain regions activated during various patterns of MS, including repeated MS and single exposure to MS at various stages before weaning, via an investigation of c-Fos expression, which is a biological mar...
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This study was undertaken to evaluate the short-term effects of glucocorticoid on the morphological characteristics of cultured rat dentate gyrus (DG) cells using immunocytochemistry for a cytoskeletal protein, micro-tubule-associated... more
This study was undertaken to evaluate the short-term effects of glucocorticoid on the morphological characteristics of cultured rat dentate gyrus (DG) cells using immunocytochemistry for a cytoskeletal protein, micro-tubule-associated protein 2 (MAP2). A selective cell culture system of DG neurons was successfully established. Synthetic glucocorticoid, dexamethasone (DEX) increased the total length of immunoreactive (IR) processes of the DG neurones in a dose-dependent manner. DEX also increased the ratio of multipolar neurones with more than three processes. The area of cell body of immunoreactive processes increased with 5 microM DEX treatment. Our results suggest that cytological features of DG cells are strongly influenced by the action of glucocorticoid.
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Androgen and estrogen act not only in a sex-specific manner but also interactively and synergistically. In the present study, to examine the possible interaction between androgen receptor (AR) and estrogen receptor-alpha (ERalpha), we... more
Androgen and estrogen act not only in a sex-specific manner but also interactively and synergistically. In the present study, to examine the possible interaction between androgen receptor (AR) and estrogen receptor-alpha (ERalpha), we investigated the subcellular dynamics of AR and ERalpha fused with green fluorescent protein color variants in single living cells using time-lapse microscopy and the technique of fluorescence recovery after photobleaching. AR and ERalpha showed punctate colocalization in the nucleus with estrogen, but not androgen. N-terminal AR deletion mutant did not form a nuclear punctate pattern with either androgen or estrogen. In the presence of AR, but not ERalpha, N-terminal AR deletion mutant formed a punctate nuclear pattern with androgen. AR had different mobility depending on the ligand and the presence of ERalpha. On the other hand, AR had little effect on the stability of ERalpha. ERalpha mutant that does not bind coactivators did not alter the mobility of AR. Taken together, using an imaging technique, we clarified that possible homo/hetero dimerization between AR and ERalpha could be attributed to androgen-estrogen interaction in living cells.
Research Interests: Fluorescence Microscopy, Image Analysis, Confocal Microscopy, Molecular endocrinology, Estrogen Receptor, and 14 moreBiological Sciences, Molecular, Cercopithecus aethiops, Cell line, Animals, Green Fluorescent Protein, Molecular cloning, Cell nucleus, Androgen Receptor, Site-directed Mutagenesis, Estrogen receptor alpha, Immunoblotting, Restriction Mapping, and Medical and Health Sciences
Adrenal corticosteroids (cortisol in humans/corticosterone in rodents) readily enter the brain and exert markedly diverse effects, such as the stress response of target neural cells. These effects are regulated via two receptor systems,... more
Adrenal corticosteroids (cortisol in humans/corticosterone in rodents) readily enter the brain and exert markedly diverse effects, such as the stress response of target neural cells. These effects are regulated via two receptor systems, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), both of which are ligand-inducible transcription factors. It is generally accepted that GR and MR predominantly reside in the cytoplasm in the absence of corticosterone (CORT), and are quickly translocated into the nucleus upon binding CORT. Then these receptors form dimers to bind hormone-responsive elements and regulate the expression of target genes. Given the different actions of MR and GR in the central nervous system, it is important to elucidate how the trafficking of these receptors between the cytoplasm and nucleus and their interaction are regulated by ligands or other molecules to exert transcriptional activity. However, the precise mechanisms of these processes are still not completely clarified. To address these issues, we have tried to observe more dynamic subcellular trafficking processes in living cells by employing a green fluorescent protein. In this review, we describe our recent studies of corticosteroid receptor dynamics in living cells focusing on three points: (1) the effects of a ligand, corticosteroid; (2) the carrier molecules involved in active nuclear transport, importins, and (3) the possibility of heterodimer formation. These studies demonstrate that GR and MR were quickly translocated from the cytoplasm to the nucleus after CORT treatment by associating with importin molecules. GR and MR differed in their response to the concentration of CORT in neural cells and non-neural cells. In the nuclear region, we detected GR-MR heterodimers, which were affected by changes in CORT concentrations in response to various hormonal milieus such as circadian rhythm and stress.
Research Interests: Imaging, Neuroendocrinology, Fluorescence, Stress, Fluorescence Resonance Energy Transfer, and 15 moreHippocampus, Dynamics, Humans, Circadian Rhythm, Stress response, Animals, Green Fluorescent Protein, Diagnostic Imaging, Live cell Imaging, Clinical Sciences, Cell nucleus, Fluorescent Antibody Technique, Mineralocorticoid Receptor, Neurosciences, and Nuclear Transport
Adrenal corticosteroids readily enter the brain and exert markedly diverse effects, such as stress response of the target neural cells. These effects are regulated via two receptor systems, the mineralocorticoid receptor (MR; type I... more
Adrenal corticosteroids readily enter the brain and exert markedly diverse effects, such as stress response of the target neural cells. These effects are regulated via two receptor systems, the mineralocorticoid receptor (MR; type I corticosteroid receptor) and the glucocorticoid receptor (GR; type II corticosteroid receptor), both of which are ligand-dependent transcription factors. MR is localized mainly in the hippocampus, and GR is distributed throughout the brain. MR and GR, however, are highly colocalized in the hippocampus, which is a target of the stress hormone and an especially plastic and vulnerable region of the brain. MR has a high affinity for corticosterone/cortisol, a common endogenous ligand for MR and GR, and is extensively bound at low levels of circulating corticosterone/cortisol. GR has a lower affinity and is extensively bound at high levels of circulating corticosterone/cortisol. These findings suggest that MR plays major roles under physiological conditions, whereas GR is mainly effective at high concentrations of corticosterone/cortisol, such as stressful conditions. The interactions of MR and GR in the hippocampal regions could be important for regulating stress response in the brain.
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Adrenal corticosteroids (cortisol in humans/corticosterone in rodents) readily enter the brain and exert markedly diverse effects, such as the stress response of target neural cells. These effects are regulated via two receptor systems,... more
Adrenal corticosteroids (cortisol in humans/corticosterone in rodents) readily enter the brain and exert markedly diverse effects, such as the stress response of target neural cells. These effects are regulated via two receptor systems, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), both are ligand-inducible transcription factors. GR and MR predominantly reside in the cytoplasm in the absence of corticosterone (CORT), but are quickly translocated into the nucleus upon binding CORT. Then these receptors form dimers to bind hormone responsive elements and regulate the expression of target genes. Given the different actions of MR and GR in the central nervous system, it is important to elucidate how the trafficking of these receptors between the cytoplasm and nucleus and their interaction are regulated by ligands or other molecules to exert transcriptional activity. However, these processes have still not been completely clarified. To address these issues, we...
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Adrenal corticosteroids (cortisol in humans or corticosterone (CORT) in rodents) exert numerous effects on the central nervous system that regulates the stress response, mood, learning and memory, and various neuroendocrine functions.... more
Adrenal corticosteroids (cortisol in humans or corticosterone (CORT) in rodents) exert numerous effects on the central nervous system that regulates the stress response, mood, learning and memory, and various neuroendocrine functions. CORT actions in the brain are mediated via two receptor systems: the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). It has been shown that GR and MR are highly colocalized in the hippocampus. These receptors are mainly distributed in the cytoplasm without hormones and translocated into the nucleus after treatment with hormones to act as transcriptional factors. Thus, the subcellular dynamics of both receptors are one of the most important issues. Given the differential action of MR and GR in the central nervous system, it is of great consequence to clarify how these receptors are trafficked between cytoplasm and nucleus, and their interactions are regulated by hormones and/or other molecules to exert their transcriptional activity. In this chapter, we describe our recent studies of corticosteroid receptor dynamics in living cells focusing on three points: (1) time-lapse imaging of GFP-labeled corticosteroid receptors; (2) intranuclear dynamics of GFP-labeled corticosteroid receptors using the fluorescence recovery after photobleaching technique; and (3) the possibility of heterodimers formation using the fluorescence resonance energy transfer technique. We discuss various factors affecting the dynamics of these receptors. Further, we would like to present the future directions of in vivo molecular imaging of corticosteroid receptors at the whole brain level.
Research Interests: Confocal Microscopy, Optical Imaging, Fluorescence Resonance Energy Transfer, Hippocampus, Cercopithecus aethiops, and 14 moreHumans, Mice, Animals, Neurons, Cell nucleus, Rats, Transfection, fluorescence recovery after photobleaching (FRAP), Cell Survival, Cytoplasm, Glucocorticoid Receptors, Protein Transport, Biochemistry and cell biology, and Cell Tracking
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We examined the response and regulation of 5-HT1A receptor on hippocampal cultured fetal neurons grown in the absence of serotonin and steroids using three experimental designs: 1) functional response using an antibody against... more
We examined the response and regulation of 5-HT1A receptor on hippocampal cultured fetal neurons grown in the absence of serotonin and steroids using three experimental designs: 1) functional response using an antibody against phosphorylated cyclic adenosine monophosphate response element binding protein (pCREB); 2) transcriptional regulation using in situ hybridization; and 3) translational expression using antipeptide 5-HT1A receptor antibody. Pretreatment of cultured hippocampal cells with the agonist 8-hydroxy-2-(di-N-propylamino)-tetralin (8-OH-DPAT) (10(-8) M) or ipsapirone (IPS) (10(-9) M) for 10 min blocked the forskolin-stimulated increase in pCREB immunoreactivity. In situ hybridization radioautography revealed that IPS (10(-9) M) decreased the 5-HT1A receptor mRNA expression (-33%) after a 24-h treatment. The decrease in 5-HT1A receptor mRNAwas accompanied by a change in protein immunoreactivity using a 5-HT1A receptor antipeptide antibody. Computer-assisted morphometric analyses showed a reduction in the 5-HT1A receptor immunoreactive (IR) intensity as compared to control 24 h after treatment with 8-OH-DPAT (10(-7)-10(-12) M) and IPS (10(-9) M). Thus, fetal hippocampal neurons have a functional 5-HT1A receptor that is downregulated at both the transcription and translation levels. In addition, we found increased 5-HT1A receptor-IR intensity (+17% approximately +39%) 24 h after treatment with the antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide (WAY 100635) (10(-7)-10(-12) M). Our results indicate that the 5-HT1A receptor is sensitive to both agonists (downregulation) and antagonists (upregulation) in hippocampal fetal neurons grown in the absence of serotonin and steroids.
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Adrenal corticosteroids readily enter the brain and exert markedly diverse effects, such as stress responses in the target neural cells. These effects are regulated by two receptor systems via the mineralocorticoid receptor (MR) and the... more
Adrenal corticosteroids readily enter the brain and exert markedly diverse effects, such as stress responses in the target neural cells. These effects are regulated by two receptor systems via the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), which are both ligand-dependent transcription factors. Several steroid hormone receptors including GR, estrogen receptor, and androgen receptor, have been shown to move rapidly in the nucleus even after ligand treatment, supposedly corresponding to transcriptional "fine-tuning". We applied fluorescence recovery after photobleaching (FRAP) to assess the mobility of green fluorescent protein (GFP)-tagged GR and -MR in the nucleus of transiently transfected cultured hippocampal neurons. FRAP results showed high mobility of GR and MR in the nucleus. Half-recovery time of GR was longer than that of MR in the presence of 10(-6)M corticosterone (CORT), but shorter in the presence of 10(-9)M CORT. Proteasome inhibition reduced the subnuclear mobility of GR and MR, and increased the transcriptional activity at both concentrations of CORT. We also investigated the differential effects of CORT concentration and proteasome inhibition on the nuclear retention level of these receptors. Our findings may provide intriguing new insights into the dynamics of corticosteroid receptors in neural cells and the molecular basis of stress regulation by these receptors in the hippocampus.
Research Interests: Psychology, Cognitive Science, Estrogen Receptor, Hippocampus, Proteasome, and 15 moreMice, Stress response, Animals, Corticosterone, Neurons, Green Fluorescent Protein, Transcription Factor, Cell nucleus, Analysis of Variance, Androgen Receptor, Gr, GFP, Mineralocorticoid Receptor, Neurosciences, and Frap
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The expression of glucocorticoid receptor (GR) in rat adrenocorticotropin (ACTH)-containing neurons in rat brain was immunohistochemically investigated. ACTH-containing cell bodies were found mainly in the arcuate nucleus. Most of these... more
The expression of glucocorticoid receptor (GR) in rat adrenocorticotropin (ACTH)-containing neurons in rat brain was immunohistochemically investigated. ACTH-containing cell bodies were found mainly in the arcuate nucleus. Most of these neurons exhibited GR immunoreactivities in their nuclei. ACTH-containing nerve fibers were distributed in the bed nucleus of the stria terminalis, periventricular nucleus, retrochiasmatic nucleus, parvocellular part of paraventricular nucleus and dorsomedial hypothalamic nucleus. After adrenalectomy there was a marked decrease of ACTH immunoreactivity, as well as GR immunoreactivity, in neurons of the arcuate nucleus, but ACTH immunoreactivity in the fibers was not affected. These results indicate that glucocorticoids up-regulate ACTH and GR production in hypothalamic arcuate neurons, but that glucocorticoid-induced changes could be delayed in the fibers derived from these neurons.
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... Page 2. 238 Nishi et a1 J Anesth 1993 ... These results suggest that volatile anesthetics have considerable neuromuscular blocking effect in MG patients. Various factors may affect the sen-sitivities of MG patients to volatile... more
... Page 2. 238 Nishi et a1 J Anesth 1993 ... These results suggest that volatile anesthetics have considerable neuromuscular blocking effect in MG patients. Various factors may affect the sen-sitivities of MG patients to volatile anesthetics. ...
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In an attempt to investigate the subcellular trafficking of beta(2)-adrenergic receptor (beta(2)AR) in living cells, we performed real-time imaging of beta(2)AR tagged with green fluorescent protein (GFP). We transiently transfected a... more
In an attempt to investigate the subcellular trafficking of beta(2)-adrenergic receptor (beta(2)AR) in living cells, we performed real-time imaging of beta(2)AR tagged with green fluorescent protein (GFP). We transiently transfected a chimera construct of beta(2)AR and GFP (beta(2)AR-GFP) into HEK 293 cells, primary cultured rat hippocampal neurons and cortical neuronal cells, and then compared the dynamic changes in subcellular localization of beta(2)AR-GFP in these live cells. In the absence of ligands, beta(2)AR-GFP fluorescence was detected predominantly on the plasma membrane in HEK 293 cells as well as on the surface of cell somata and dendrites in cortical neuronal cells. In contrast, in hippocampal neurons, beta(2)AR-GFP was diffusely distributed not only on the surface of cells but in the whole cell somata and dendrites. In HEK 293 cells, cortical neuronal cells and cortical glial cells, time-lapse images showed the rapid appearance of a punctate distribution pattern that became more numerous over the 15-min course of agonist exposure. Semiquantitative analysis revealed the time-course internalization of beta(2)AR-GFP in a single living cell. In hippocampal neurons, beta(2)AR-GFP distribution became scattered both in cell somata and dendrites following agonist exposure. Three-dimensional analysis of time-lapse images revealed a significant portion of beta(2)AR-GFP was distributed in endosomal compartments, along with Alexa 546-labeled transferrin, in all types of cells. Our results demonstrate spatial and temporal redistribution pattern of beta(2)AR in living non-neuronal cells and neuronal cells.
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Serotonergic innervation of the central nervous system has a sexual dimorphism. The serotonin level in the hypothalamus was modulated by estrogen, and the formation of sexual dimorphism of serotonergic fiber innervation in the... more
Serotonergic innervation of the central nervous system has a sexual dimorphism. The serotonin level in the hypothalamus was modulated by estrogen, and the formation of sexual dimorphism of serotonergic fiber innervation in the hypothalamus has been shown by the effect of sexual hormone during the critical perinatal period. In this study, we examined the direct effect of estrogen on the neurite growth of serotonergic neurons in primary culture from embryonic day 14 (E14) of rat mesencephalon. The total neurite length of serotonin-immunoreactive (IR) cells was significantly decreased by estradiol benzoate (E2, 10(-8)M) treatment for 7 days, compared with the case of no treatment. Moreover, the presence of estrogen receptor (ER) alpha and ERbeta mRNA in the E14 mesencephalon with reverse transcription-polymerase chain reaction (RT-PCR), and the ERalpha or ERbeta protein in the cultured serotonin-IR cells with double fluorescence immunohistochemistry were also demonstrated. Our results suggest that the inhibitory effects of E2 on the neurite growth of serotonergic cells expressing ERalpha or ERbeta might be involved in the formation of the sexual dimorphic distribution of serotonergic innervation.
Research Interests: Neuroscience, Cognitive Science, Immunohistochemistry, Estrogen Receptor, Sexual dimorphism, and 12 moreSerotonin, Cell Differentiation, Hypothalamus, Animals, Fluorescent Antibody Technique, Estrogens, Rats, Neural pathways, Neurosciences, Estrogen receptor-beta, Down-Regulation, and Estrogen receptor alpha
Steroid hormones easily cross the blood-brain barrier because of their physicochemical lipid solubility. The hormones act through nuclear receptor-mediated mechanisms and modulate gene transcription. In contrast to their genomic actions,... more
Steroid hormones easily cross the blood-brain barrier because of their physicochemical lipid solubility. The hormones act through nuclear receptor-mediated mechanisms and modulate gene transcription. In contrast to their genomic actions, the non-genomic rapid action of steroid hormones, acting via various types of membrane-associated receptors, reveals pharmacological properties that are distinct from the actions of the intracellular nuclear receptors. As a result, non-genomic rapid actions have gained increased scientific interest. However, insight into the phylogenic and/or comparative actions of steroids in the brain is still poorly understood. In this review, we summarize recent findings concerning the rapid, non-genomic signaling of steroid hormones in the vertebrate central nervous system, and we discuss (using a comparative view from fish to mammals) recently published data regarding the mechanism underlying physiology and behavior.
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... Corticosterone treatment induced GFP-GR translocation from the cell soma and neurites to the nucleus in a time-dependent manner. Bar. ... The authors are grateful to Ms. Makiko Yamamoto and Ryoko Nishida for preparing this manuscript.... more
... Corticosterone treatment induced GFP-GR translocation from the cell soma and neurites to the nucleus in a time-dependent manner. Bar. ... The authors are grateful to Ms. Makiko Yamamoto and Ryoko Nishida for preparing this manuscript. ...
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Steroid hormones regulate several important functions of the brain by altering the expression of particular genes through their receptors. First in this paper the localization of glucocorticoid receptor immunoreactivity and mRNA in the... more
Steroid hormones regulate several important functions of the brain by altering the expression of particular genes through their receptors. First in this paper the localization of glucocorticoid receptor immunoreactivity and mRNA in the brain was examined. Second biphasic effects of glucocorticoid on the hippocampus was described and particular emphasis was given on the apoptosis. Third the significance of estrogen receptor in the sexually dimorphic areas was discussed. These results suggest that steroids modulate the gene expression along with the alteration of cell structures in a different manner in a tissue-specific pattern.
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Adrenal corticosteroids readily enter the brain and exert markedly diverse effects, including stress responses in the target neural cells via two receptor systems, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR).... more
Adrenal corticosteroids readily enter the brain and exert markedly diverse effects, including stress responses in the target neural cells via two receptor systems, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). It has been shown that the GR and MR are highly colocalized in the hippocampus. Given the differential action of the MR and GR in the hippocampal region, it is important to elucidate how these receptors interact with each other in response to corticosteroids. We investigated the heterodimerization of the MR and GR with green fluorescent protein-based fluorescence resonance energy transfer (FRET) microscopy in living cells with spatiotemporal manner. FRET was evaluated in three ways: (1) ratio imaging; (2) emission spectra; and (3) acceptor photobleaching. FRET analysis demonstrated that cyan fluorescent protein-GR and yellow fluorescent protein-MR form heterodimers after corticosterone (CORT) treatment both in the nucleus of cultured hippocampal neurons and COS-1 cells, whereas they do not form heterodimers in the cytoplasm. The content of the GR-MR heterodimer was higher at 10(-6) m CORT than at 10(-9) m CORT and reached a maximum level after 60 min of CORT treatment in both cultured hippocampal neurons and COS-1 cells. The distribution pattern of heterodimers in the nucleus of cultured hippocampal neurons was more restricted than that in COS-1 cells. The present study using mutant fusion proteins in nuclear localization signal showed that these corticosteroid receptors are not translocated into the nucleus in the form of heterodimers even after treatment with ligand and thus allow no heterodimerization to take place in the cytoplasm. These results obtained with FRET analyses give new insights into the sites, time course, and effects of ligand concentration on heterodimersization of the GR and MR.
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To examine the distribution of mineralocorticoid receptor (MR) and the interactions with glucocorticoid receptor (GR) in the brain, we raised a polyclonal antibody against the transcriptional modulation domain of rat MR using the... more
To examine the distribution of mineralocorticoid receptor (MR) and the interactions with glucocorticoid receptor (GR) in the brain, we raised a polyclonal antibody against the transcriptional modulation domain of rat MR using the GST-fusion system. Immunoblotting analysis revealed that this antibody recognized a band with the molecular mass of MR in MR-transfected COS-1 cells and in a homogenate of rat hippocampus, and showed no cross-reactivity with GR. In vitro immunocytochemistry of both primary cultured hippocampal neurons and MR-transfected cells revealed immunoreactivity detected by this antibody in both the cytoplasm and nucleus in the absence of aldosterone (ALD), a specific agonist of MR. After 1 h of treatment with 10(-7) M ALD, the MR-immunoreactivity was accumulated in the nuclear region. In the case of GR-transfected cells, our anti-MR antibody either detected no immunopositive cells in the presence or absence of GR agonist. In our in vivo study, MR-immunoreactivity was observed in the rat hippocampus, where cell nuclei showed immunopositive reactions. These results suggest that our antibody against rat MR shows high specificity for the receptor both in liganded and unliganded forms, with no cross-reactivity to GR, and will be useful for cell biological and neuroanatomical investigations of MR.
Research Interests: Neuroscience, Cognitive Science, Immunohistochemistry, Antibodies, Hippocampus, and 15 moreAldosterone, Female, Animals, Subcellular Localization, Rats, Wistar Rats, Molecular Mass, Primary Culture, Rabbits, Amino Acid Sequence, Subcellular Fractions, Fusion Protein, Mineralocorticoid Receptor, Neurosciences, and Immunoblotting
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We investigated the distribution and colocalization pattern of the two corticosteroid receptors, mineralocorticoid receptor (MR) and glucocorticoid receptor (GR), in the hippocampus and hypothalamus, the main target regions of... more
We investigated the distribution and colocalization pattern of the two corticosteroid receptors, mineralocorticoid receptor (MR) and glucocorticoid receptor (GR), in the hippocampus and hypothalamus, the main target regions of corticosterone in the rat brain, using double immunofluorescence histochemistry in conjunction with specific polyclonal antibodies against MR and GR. In the hippocampus, MR- and GR-immunoreactivity (ir) were colocalized in CA1 and CA2 pyramidal neurons and granule cells of the dentate gyrus, while only MR-ir was seen in the CA3 pyramidal neurons. Colocalization of MR- and GR-ir was also observed in the parvocellular region, but not in the magnocellular region of the paraventricular nucleus (PVN). Subcellular distribution of MR-ir was more cytoplasmic in comparison with that of GR-ir, while the ratio of cytoplasmic to nuclear distribution of these receptors was different among the regions. After adrenalectomy (ADX), the distribution pattern of both receptors was changed to cytoplasmic, although the degree of the change of distribution was different among each region. Replacement of corticosterone after ADX recovered the distribution pattern to that of the sham-operated animals. These results suggest that the balance of MR and GR in the cell underlies the potential regulation of corticosteroid through the hippocampus and hypothalamus.
Research Interests: Neuroscience, Cognitive Science, Immunohistochemistry, Hippocampus, Hypothalamus, and 15 moreAnimals, Corticosterone, Male, Rat Brain, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Paraventricular Nucleus, Anti-inflammatory agents, Dentate Gyrus, Fluorescent Antibody Technique, Rats, Wistar Rats, Mineralocorticoid Receptor, Neurosciences, Adrenalectomy, and Immunoblotting
Steroid hormones substantially influence brain development, reproduction sexual differentiation and emotion. These effects are mediated by steroid hormone receptors and cofactors, which directly regulate gene expression. Deciphering how... more
Steroid hormones substantially influence brain development, reproduction sexual differentiation and emotion. These effects are mediated by steroid hormone receptors and cofactors, which directly regulate gene expression. Deciphering how and where these transcriptional activators occur in a cell provides the groundwork for elucidating the influence of these small hydrophobic signal molecules on various brain functions. This paper describes some of the recent investigations into the subcellular localization of steroid hormone receptors and cofactors using GFPs and other immunocytochemical methods.