Kazunari Yuri

Local secretion of corticotropin-releasing hormone by enterochromaffin cells in human colon

BACKGROUND/AIMS Corticotropin-releasing hormone (CRH) is a regulator of the hypothalamic-pituitary-adrenal axis and a coordinator of the gastrointestinal response to stress. In addition to its central effects, CRH has peripheral effects on the immune system. CRH is present in several human tissues, such as the brain, spinal cord, adrenal medulla, lung, liver, peripheral blood leukocytes, as well as the gastrointestinal tract. The current study examined the local production of CRH in the normal human colon. METHODS Normal human colonic tissues obtained by endoscopic biopsy were immunostained with anti-CRH and anti-5-hydroxytryptamine antibody and analyzed for CRH messenger (m)RNA by a reverse-transcribed polymerase chain reaction method and by in situ hybridization. RESULTS Immunoreactive CRH and CRH mRNA were detected in the colonic mucosal cells in the neighborhood of the base of the crypts. The mucosal cells that expressed CRH mRNA also immunostained with anti-5-hydroxytryptamine antibody. CONCLUSIONS Normal human colonic mucosal enterochromaffin cells produce CRH. CRH in the colonic mucosa may play a role in the modulation of the intestinal immune system and/or other gastrointestinal functions basally during stressful conditions.

The effect of estrogen on the estrogen receptor-immunoreactive cells in the rat medial preoptic nucleus

The localization of estrogen receptor-immunoreactive (ER-IR) cells in the medial preoptic nucleus (MPN) and the effect of estrogen upon these cells were quantitatively analyzed by immunohistochemistry using a monoclonal antibody to estrogen receptor (ER) protein in rat. ER was distributed in the nucleus of MPN neurons and was not detected in either cytoplasm or glial cells. There were more ER-IR cells with higher immunoreactivities in ovariectomized (OVX) rats than in estradiol (E2)-treated rats. The number of ER-IR cells in E2-treated compared with OVX rats was reduced by 43%. In particular, the number of ER-IR cells in the central part of the MPN was largely decreased. Our data indicate that the central part of the MPN is most sensitive to estrogen.

Immunocytochemical analysis of sex differences in calcitonin gene-related peptide in the rat dorsal root ganglion, with special reference to estrogen and its receptor

Previous studies have shown that the calcitonin gene-related peptide (CGRP) immunoreactivity in the central nervous system (CNS) of adult rats is sexually dimorphic and regulated by sex steroid. In the present study, we used immunocytochemistry to investigate the sex difference in CGRP-immunoreactive (IR) neurons in rat dorsal root ganglia (DRG). The numbers of CGRP-IR neurons at the cervical, lumbar and sacral levels in the female rats were significantly lower than those of the male rats. We also found that the number of CGRP-IR neurons at the lumbar level was increased in ovariectomized (OVX) rats, but was decreased in estradiol (E2)-treated rats (OVX+E2). A large number of estrogen receptor (ER)-IR neurons at the lumbar level were found in the female rats, and its number was greater than that in the male rats. We also investigated the change in the number of ER-IR neurons of OVX rats after estrogen treatment. The number of ER-IR neurons in the OVX+E2 rats was consistent with that of the intact female rats, but was significantly increased in the OVX rats. As shown by a double-labeling immunocytochemical method, over 80% of the CGRP-IR neurons at the lumbar level showed ER immunoreactivity in the female, OVX and OVX+E2 rats, compared to only about 46% in the male rats. These results indicate that there is a gender difference in CGRP expression in the rat DRG, and that this CGRP expression might be downregulated by estrogen (at least in part) through its receptor.

Steroid hormones and their receptors in the brain.

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.

Immunohistochemical and Enzyme Histochemical Localization of Peptidergic, Aminergic and Cholinergic Nerve Fibers in the Rat Seminal Vesicle

The distribution of the autonomic nervous system in the rat seminal vesicle was studied with immuno- and enzyme-histochemistry. In the smooth muscle layer, a large number of neuropeptide Y (NPY)- and tyrosine hydroxylase (TH)-immunoreactive nerve fibers were observed, while few fibers were distributed in the mucosal layer. A small number of substance P-immunoreactive fibers were present only in the smooth muscular layer, but vasoactive intestinal polypeptide (VIP)-immunoreactive fibers were abundantly distributed in both layers. In the mucosal layer, the VIP-fibers ran attached to blood vessels and encircled the glandular cavities. Acetylcholinesterase-positive fibers were also observed in the mucosal and muscular layers. Electron-microscopic studies revealed that NPY- and TH-containing nerve terminals were in apposition to smooth muscle cells, and VIP-fibers terminated at blood vessels. These results suggest different modes of action of NPY-, TH- and VIP-containing nerve fibers in the seminal vesicle.

Estrogen receptor-immunoreactive neurons contain calcitonin gene-related peptide, methionine-enkephalin or tyrosine hydroxylase in the female rat preoptic area

We have shown in our previous studies that estrogen treatment selectively influences calcitonin gene-related peptide (CGRP)-, methionine-enkephalin (Met-Enk)- and tyrosine hydroxylase (TH)-immunoreactive (IR) intensities in the neurons of the periventricular preoptic nucleus (PPN) and the medial preoptic area (MPA) of the female rat. In the present study, we examined whether estrogen receptor (ER)-IR neurons in the PPN and MPA contain CGRP, Met-Enk, or TH using a double-labeling immunohistochemical method and investigated changes in the number of double-labeling cells upon treatment with estrogen. Brain sections of ovariectomized rats and ovariectomized and estrogen-treated rat were stained using the avidin-biotin-peroxidase complex method followed by the peroxidase-anti-peroxidase method. The sections were first incubated with an anti-ER antibody in conjunction with nickel diaminobenzidine which produces a dark blue reaction product in the nucleus. Subsequently, CGRP, Met-Enk or TH antisera were applied to these sections and the resulting brown diaminobenzidine reaction product in the cytoplasm was examined. Neurons that were double-labeled for ER and CGRP, Met-Enk or TH were investigated in the PPN and MPA. The number of doubly labeled ER/CGRP- and ER/TH-IR neurons was large, whereas the number of ER/Met-Enk-IR neurons was small. These results suggest that ER in the PPN and MPA may be more closely related to the mechanism of changes in CGRP- and TH-IR intensities upon estrogen treatment than that in Met-Enk-IR intensity.

Steroid hormone effects on gene expression, neuronal structure, and differentiation.

Steroid hormones modify several functions of the nervous system by altering expression of particular genes that are relevant for cell-to-cell communication, neuronal structure, and differentiation. There are many regions of the brain showing structural differences between male and female. These sexual dimorphisms are primarily based on cytological features of the nervous system brought about by the organizational action of sex steroid hormones. Glucocorticoids also have a great influence on neuronal survival and differentiation. First, in this paper the estrogen effect on the phenotypic changes of neurons in the preoptic area in vivo was described, particularly in the neuronal system of methionine-enkephalin and calcitonin gene-related peptide. Second, the tissue culture system for the preoptic area was developed and the estrogen effect on process extension associated with cytoskeletal changes was examined. Third, glucocorticoid suppression of the differentiation of adrenal chromaffin cells and transmitter/neuropeptide plasticity was studied in the dissociated culture system. The results suggest that steroids modulate neurotransmitters and neuropeptides expression along with the alteration of cell structures in a different manner in a tissue-specific pattern.

Juvenile social experience regulates central neuropeptides relevant to emotional and social behaviors

Stressful social experiences during early-life can increase the risk of developing neuropsychiatric disorders associated with anxiety, mood, and personality. Early neglect also alters peripheral arginine vasopression (AVP) and oxytocin (OXT). We hypothesized that a lack of social stimuli should adversely affect developmental AVP and OXT systems. To test this idea, we examined changes of central AVP- and OXT-immunoreactive (ir) cell number as well as its related behaviors in socially isolated rats. Animals were weaned at 23 days of age, divided into group- or isolation-reared conditions, and maintained for at least 2 weeks. At 38-48 days of age, animals were sacrificed for immunohistochemistry, or used for two behavioral tests: elevated plus-maze test and social recognition test. The results from immunohistochemistry showed that isolation-reared males have decreased AVP-ir cells in the paraventricular nucleus hypothalamus (PVH), medial parvicellular part, ventral zone, and that isolation-reared females have decreased OXT-ir cells in the PVH, medial parvicellular part, dorsal zone, when compared with group-reared counterparts. The results from behavioral assessment showed that isolation-reared animals have difficulty with social recognition, and that isolation-reared males, but not females, have anxiogenic profile. The present study demonstrates that post-weaning social isolation results in decrease of male AVP-ir cells and female OXT-ir cells in the PVH parvocellular divisions, and supports the idea that juvenile social environment may play a critical role in neuronal and behavioral development.

A mental retardation gene, motopsin/neurotrypsin/prss12, modulates hippocampal function and social interaction

Motopsin is a mosaic serine protease secreted from neuronal cells in various brain regions, including the hippocampus. The loss of motopsin function causes nonsyndromic mental retardation in humans and impairs long‐term memory formation in Drosophila. To understand motopsin’s function in the mammalian brain, motopsin knockout (KO) mice were generated. Motopsin KO mice did not have significant deficits in memory formation, as tested using the Morris water maze, passive avoidance and Y‐maze tests. A social recognition test showed that the motopsin KO mice had the ability to recognize two stimulator mice, suggesting normal social memory. In a social novelty test, motopsin KO mice spent a longer time investigating a familiar mouse than wild‐type (WT) mice did. In a resident–intruder test, motopsin KO mice showed prolonged social interaction as compared with WT mice. Consistent with the behavioral deficit, spine density was significantly decreased on apical dendrites, but not on basal dendrites, of hippocampal pyramidal neurons of motopsin KO mice. In contrast, pyramidal neurons at the cingulate cortex showed normal spine density. Spatial learning and social interaction induced the phosphorylation of cAMP‐responsive element‐binding protein (CREB) in hippocampal neurons of WT mice, whereas the phosphorylation of CREB was markedly decreased in mutant mouse brains. Our results indicate that an extracellular protease, motopsin, preferentially affects social behaviors, and modulates the functions of hippocampal neurons.

The distributions of apoptotic cells in the medial preoptic areas of male and female neonatal rats

The medial preoptic area (MPA) of the hypothalamus of the rat contains two sexually dimorphic nuclei, the periventricular preoptic nucleus (PVpo) and the medial preoptic nucleus (MPN). To examine the relationship between sexual dimorphism and neuronal death, we examined the number of apoptotic cells in the subdivisions of the MPA in neonatal rats of postnatal days 1 (P1), 4 (P4), 7 (P7) and 14 (P14). Apoptotic cells in these areas were classified according to their progression into three stages. P1 and P4 rats contained many apoptotic cells in the subfield along the third ventricle, including the PVpo, and their number was significantly larger in P1 males: in particular, the number of early-stage cells was larger in males than females. The number of apoptotic cells in the MPN was increased in P4 and P7 rats, although no significant sexual differences were seen in the total number or in the number of each progressive stage of apoptotic cells. In P14 rats, very few apoptotic cells were seen in the MPA. Our data revealed that the distribution of apoptotic cells in the MPA of developing rats depends on the sexuality, subdivision of the area and postnatal period.