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1.
Korean J Pain ; 28(3): 185-92, 2015 Jul.
Article in English | MEDLINE | ID: mdl-26175878

ABSTRACT

BACKGROUND: Neuropathic pain is a global clinical problem; nevertheless, nerve injury treatment methods remain limited. Olanzapine has antinociceptive and anti-nueropathic properties; however, its preventive effects have not been assessed in nerve injury models. METHODS: We prepared a partial sciatic nerve ligation (Seltzer model) or sham-operated model in male Sprague-Dawley rats under isoflurane anesthesia. In a pre-treatment study, we administered olanzapine (10 mg/kg) intraperitoneally 1 h before nerve ligation. In post-treatment and dose-dependent studies, we injected 3 different doses of olanzapine intraperitoneally 1 h after nerve ligation. Mechanical allodynia was measured before and 7 days after surgery. Immunohistochemical analysis using anti-Iba-1 antibody was used to assess the effect of olanzapine at the spinal level. RESULTS: In the pre-treatment study, median withdrawal thresholds of the normal saline groups were significantly lower than those of the sham-operated groups; however, those of the olanzapine (10 mg/kg) and sham-operated groups were not different. In the post-treatment and dose-dependent studies, the median withdrawal thresholds of the olanzapine (2.5 mg/kg) and normal saline groups were not different; however, those of the olanzapine (10 and 50 mg/kg) groups were significantly higher than those of the normal saline groups. Olanzapine did not have a significant effect on the density of Iba-1 staining. CONCLUSIONS: Olanzapine attenuated mechanical allodynia dose-dependently in the Seltzer model. This anti-allodynic effect of olanzapine was observed even when injected 1 h after nerve ligation. This effect of olanzapine appeared to be unrelated to microglia activation in the ipsilateral dorsal horn of the lumbar spinal cord.

2.
Neurosci Res ; 96: 20-9, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25496933

ABSTRACT

The present study investigated the effect of acoustic stimulation on the activation of the hypothalamic-pituitary-adrenal (HPA) axis in rats submitted to acute restraint stress, through semi-quantitative histochemical analysis of expression of immediate early gene products (c-Fos, JunB and phosphorylated c-Jun) and arginine vasopressin (AVP) hnRNA in the paraventricular nucleus (PVN). Simultaneous presentation of white or pink noise with restraint resulted in a significant attenuation of stress-induced c-Fos and JunB expression in the dorsal body of dorsal medial parvicellular subdivision (mpdd) of the PVN, as compared with restraint without noise. However, this presentation did not change phosphorylation of c-Jun and the plasma corticosterone level. Moreover, white noise presentation during restraint led to a reduction in the number of c-Fos- or JunB-expressing corticotropin-releasing hormone (CRH) neurons and the number of neurons expressing AVP hnRNA in the mpdd. Dual-histochemical labeling revealed co-expression of c-Fos and JunB, as well as JunB and AVP hnRNA in mpdd neurons. These data suggest that acoustic stimuli have an attenuation effect on the restraint-induced activation of neuroendocrine CRH neurons, resulting in the reduction in AVP production as an adaptation of HPA axis to repeated stress.


Subject(s)
Arginine Vasopressin/metabolism , Genes, Immediate-Early/physiology , Noise , Paraventricular Hypothalamic Nucleus/metabolism , Stress, Psychological/metabolism , Animals , Corticosterone/blood , Male , Neurons/metabolism , Phosphorylation , Rats , Rats, Sprague-Dawley , Restraint, Physical
3.
Neurosci Res ; 72(1): 16-22, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21982845

ABSTRACT

The pineal gland secretes melatonin under circadian control via nocturnal noradrenergic stimulation, and expresses vesicular glutamate transporter (VGLUT) 1, VGLUT2 and a VGLUT1 splice variant (VGLUT1v). Although we previously reported that VGLUT2 mRNA level of rat pineal gland at postnatal day 21 is higher in the nighttime than in daytime, questions remained as to the time of postnatal onset of this phenomenon and a 24-h change in the mRNA or protein level at postnatal days. The day-night difference in VGLUT2 mRNA level was evident 14 days after birth. In the adult, VGLUT2 mRNA and protein levels increased in the dark phase, with the protein level showing a 6-h delay. The nocturnal elevation in VGLUT2 mRNA level diminished under the constant light condition but persisted under the constant dark condition. The present data suggest that VGLUT2 in the rat pineal gland is involved in some nocturnal glutamatergic function.


Subject(s)
Circadian Rhythm/physiology , Pineal Gland/metabolism , Vesicular Glutamate Transport Protein 2/metabolism , Animals , Male , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Vesicular Glutamate Transport Protein 1/genetics , Vesicular Glutamate Transport Protein 1/metabolism , Vesicular Glutamate Transport Protein 2/genetics
4.
Neurosci Res ; 70(4): 376-82, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21609737

ABSTRACT

To study the development of glutamatergic neurons during the main olfactory bulb morphogenesis in rats, we examined the expression of vesicular glutamate transporters 1 (VGLUT1) and 2 (VGLUT2). On VGLUT1, expressions of mRNA and immunoreactivity were first detected in the mitral cell layer on embryonic day (E) 17.5 and E18.5, respectively, and persisted in the E20.5 olfactory bulb. Much earlier (on E12.5) than VGLUT1, expressions of VGLUT2 mRNA and/or immunoreactivity were found in the olfactory epithelium, migratory cells and telencephalon. On E14.5, the mRNA expression was also observed in the prospective bulbar region and vomeronasal organ, while immunoreactivity existed in migratory cells and growing fibers. Some fibers were observed in the deep telencephalic wall. From E16.5 onward, mRNA expression became gradually detectable in cells of the mitral cell layer with development. On E17.5, immunoreactivity was first found in fibers of the developing olfactory bulb and in some immature mitral cells from E18.5 to E20.5. The present study clarifies the expression of VGLUT2 precedent to VGLUT1 during olfactory bulb morphogenesis, suggesting differential contribution of the two VGLUT subtypes to glutamate-mediated embryonic events.


Subject(s)
Embryonic Development/physiology , Gene Expression Regulation, Developmental , Olfactory Bulb/embryology , Olfactory Bulb/metabolism , Vesicular Glutamate Transport Protein 1/biosynthesis , Vesicular Glutamate Transport Protein 2/biosynthesis , Amino Acid Sequence , Animals , Female , Glutamic Acid/physiology , Molecular Sequence Data , Olfactory Mucosa/embryology , Olfactory Mucosa/metabolism , Pregnancy , Rats , Rats, Sprague-Dawley , Time Factors
5.
Growth Horm IGF Res ; 21(1): 11-5, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21177131

ABSTRACT

Expression of the first exon variants of the rat growth hormone receptor mRNA was studied in the brain and the pituitary gland. Four of the five different variant mRNA previously identified in the liver were detected in the cerebral cortex by a conventional reverse-transcription polymerase chain reaction, and, unlike the data reported previously, a quantitative analysis revealed that approximately 90% of the total growth hormone receptor mRNA in the cerebral cortex is V1 form. The present results suggest that the V1 was also a dominant transcript in other brain areas and the pituitary gland, not only in adult but also in fetal and postnatal period. The growth hormone receptor expression in the brain was lower during fetal period than in adults, while in the pituitary gland, the expression is markedly higher in fetuses, suggesting a yet unknown role of growth hormone in the development of this organ.


Subject(s)
Brain/metabolism , Pituitary Gland/metabolism , Receptors, Somatotropin/genetics , Receptors, Somatotropin/metabolism , Age Factors , Animals , Embryo, Mammalian , Exons , Female , Fetus/metabolism , Male , Pregnancy , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Sex Characteristics , Tissue Distribution
6.
Anesth Analg ; 109(4): 1318-22, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19762763

ABSTRACT

BACKGROUND: Nociceptive behaviors might attenuate pain sensation. Phosphorylation of extracellular signal-regulated kinase (pERK) was recently reported to be induced by noxious stimuli in dorsal horn neurons. We investigated, in a formalin test, whether pERK of the dorsal horn is affected by licking. METHODS: Twenty-four adult male rats were divided into four groups: control, formalin test, restricted control, and restricted formalin test. Ten percent formalin was injected subcutaneously into the left rear paw of the formalin test and restricted formalin test groups. The control and formalin test group rats were kept in a clear plastic chamber, whereas the restricted control and restricted formalin test group rats were kept in a modified-restraint, pipe-shaped chamber. All rats were killed after 25 min. Twelve sections of the lumbar spinal cord were processed for p-ERK immunohistochemistry using the avidin-biotin peroxidase method. RESULTS: The number of p-ERK positive cells in the restricted formalin test group was significantly higher than in the other three groups in the ipsilateral-side superficial dorsal horn (P < 0.05). However, there was no significant difference between the formalin test group and the two control groups in pERK expression. CONCLUSION: Licking decreased pERK of the spinal cord of the formalin test group. The findings suggested that licking attenuated the pain of the formalin test.


Subject(s)
Behavior, Animal , Extracellular Signal-Regulated MAP Kinases/metabolism , Mechanotransduction, Cellular , Pain Threshold , Pain/physiopathology , Posterior Horn Cells/enzymology , Tongue/physiopathology , Animals , Disease Models, Animal , Down-Regulation , Formaldehyde , Immunohistochemistry , Lumbosacral Region , Male , Pain/chemically induced , Pain/enzymology , Pain Measurement , Phosphorylation , Proto-Oncogene Proteins c-fos/metabolism , Rats , Rats, Sprague-Dawley
7.
Endocrinology ; 150(2): 1061-8, 2009 Feb.
Article in English | MEDLINE | ID: mdl-18927214

ABSTRACT

The developmental process of prolactin (PRL) cells in the fetal pituitary gland was studied in mice. Although PRL cells were hardly detectable in the pituitary gland of intact fetuses, a treatment with 17beta-estradiol (E(2)) in vitro induced a number of PRL cells that varied drastically in number depending on the stage of gestation with a peak at embryonic d 15. This effect was specific to E(2), with epidermal growth factor, insulin, and forskolin failing to induce PRL cells. Although both estrogen receptor (ER)alpha and ERbeta were expressed in the fetal pituitary gland, the results from ER knockout models showed that only ERalpha mediates E(2) action on PRL cells. A few PRL cells were observed in ERalpha-deficient mice as well as in their control littermates, suggesting that estrogen is not required for the phenotype determination of PRL cells. Unexpectedly, the effect of E(2) on the induction of PRL cells in vitro was diminished after embryonic d 15. Present results suggest that the exposure of fetal PRL cells to glucocorticoids (GCs) results in a reduction of sensitivity to E(2). The mechanism underlying the down-regulation of estrogen sensitivity by GCs was found not to be down-regulation of ER levels, induction of annexin 1, a GC-inducible inhibitor of PRL secretion, or a decrease in the number of PRL precursors by apoptosis. The effect of GCs appeared within 2 h and did not require a de novo protein synthesis. GCs are considered to be involved in the mechanisms of silencing pituitary PRL in gestation possibly through a novel mechanism.


Subject(s)
Cell Differentiation/drug effects , Fetus/drug effects , Hormones/pharmacology , Pituitary Gland/embryology , Prolactin/metabolism , Animals , Cells, Cultured , Embryo, Mammalian , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Embryonic Stem Cells/physiology , Estradiol/pharmacology , Estrogen Receptor alpha/genetics , Estrogen Receptor beta/genetics , Female , Fetus/cytology , Fetus/metabolism , Glucocorticoids/pharmacology , Male , Mice , Mice, Inbred ICR , Mice, Knockout , Pituitary Gland/cytology , Pituitary Gland/drug effects , Pituitary Gland/metabolism , Pregnancy
8.
Growth Horm IGF Res ; 18(5): 379-388, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18329307

ABSTRACT

Recent studies have disclosed the molecular mechanisms responsible for the phenotype determination of the anterior pituitary cell types. However, as far as growth hormone (GH) cells are concerned, particular extra-cellular cues are required for the initiation of GH and GH-releasing hormone (GHRH)-receptor gene production in addition to the expression of the cell type specific transcription factor, pit-1. The glucocorticoids play a principal role in the functional maturation of nascent GH cells in the fetal pituitary glands in rodents, inducing GH and GHRH-receptor gene expression, and establish the GH secretory system regulated by the brain in late gestation. Research supporting this role for glucocorticoid in the development of GH cells is discussed.


Subject(s)
Fetus/metabolism , Growth Hormone/metabolism , Pituitary Gland, Anterior/embryology , Somatotrophs/metabolism , Animals , Cell Differentiation , Glucocorticoids/metabolism , Growth Hormone/genetics , Growth Hormone-Releasing Hormone/genetics , Growth Hormone-Releasing Hormone/metabolism , Humans , Models, Biological
9.
Neurosci Res ; 60(3): 259-65, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18164503

ABSTRACT

To study the effect of adrenal steroids on neuropeptide Y (NPY) synthesis in the hypothalamic-pituitary system, we examined NPY expression in rats treated with dexamethasone (a synthetic glucocorticoid) by in situ hybridization and immunohistochemistry. Rats were injected daily with dexamethasone (0.2mg/100g/day for 10 days, sc) or sesame oil (vehicle control), or non-injected (intact control). Relative staining area for corticotropin-releasing hormone or neurophysin II, a vasopressin carrier protein, was increased in the external zone of the median eminence in vehicle control, but was equivalent to that of intact control in the dexamethasone-injected group. Density of NPY-stained fiber varicosities was drastically increased in the external, but not the internal, zone of dexamethasone-injected group, coinciding with the increased NPY hybridization signal level in the arcuate nucleus. Dual-labeling experiments revealed no colocalization of NPY with hypophysiotropic or other peptides examined in single fibers of the median eminence. In the dexamethasone-injected group, expressions of NPY mRNA and peptide were detectable in a few pituitary cells, with some being corticotropes. These results suggest that NPY plays hormonal roles in the hypothalamic-pituitary-adrenal axis.


Subject(s)
Dexamethasone/pharmacology , Glucocorticoids/pharmacology , Hypothalamo-Hypophyseal System/drug effects , Hypothalamo-Hypophyseal System/physiology , Neuropeptide Y/genetics , Agouti-Related Protein/metabolism , Animals , Body Weight/drug effects , Corticotropin-Releasing Hormone/metabolism , Immunohistochemistry , In Situ Hybridization , Male , Median Eminence/drug effects , Median Eminence/physiology , Neuropeptide Y/metabolism , Neurophysins/metabolism , Pituitary Gland/drug effects , Pituitary Gland/physiology , RNA, Messenger/metabolism , Rats , Rats, Wistar , Up-Regulation/drug effects , Up-Regulation/physiology
10.
Neurosci Lett ; 432(2): 132-6, 2008 Feb 20.
Article in English | MEDLINE | ID: mdl-18222609

ABSTRACT

Although virtually all of serotonin (5-HT) neurons in the midbrain raphe nuclei of rats are known to express vesicular glutamate transporter 3 (VGLUT3), VGLUT3-positive 5-HT fibers have been identified only in the cerebral cortex and hippocampus. Thus, our understanding of forebrain sites where 5-HT-glutamate interaction may be potentially managed by such possible glutamatergic 5-HT fibers themselves, is still largely fragmentary from a morphological point of view. To address this issue, we analyzed the rat forebrain by immunohistochemistry and chemical lesion experiment of 5-HT neurons by intracerebroventricular injection of a neurotoxin, 5,7-dihydroxytriptamine. Contrary to expectation, the double-label immunofluorescence staining revealed that the incidence of VGLUT3-positive 5-HT fibers is generally low over the forebrain, demonstrating occasional fibers with one or two double-labeled varicosities. The most extreme example was the nucleus of the lateral olfactory tract (LOT), which seemed to be devoid of double-labeled fibers despite high densities of 5-HT fibers and VGLUT3-positive fibers. In sharp contrast, robust plexuses of VGLUT3-positive 5-HT fibers were found in the dorsal, but not ventral, part of the lateral septum. The lesion experiment carried out to explore whether VGLUT3 exists in 5-HT fibers showed that in lesioned rats VGLUT3-positive fibers almost completely disappear from the septal region but seemed unchanged in the LOT. The present study shows that midbrain raphe-derived 5-HT fibers can be classified into two subtypes depending on co-expression with VGLUT3 staining in the forebrain.


Subject(s)
Axons/metabolism , Prosencephalon/metabolism , Serotonin/metabolism , Vesicular Glutamate Transport Proteins/metabolism , 5,7-Dihydroxytryptamine , Animals , Axons/classification , Axons/ultrastructure , Brain Mapping , Denervation , Immunohistochemistry , Male , Neural Pathways/cytology , Neural Pathways/metabolism , Neurotoxins , Olfactory Pathways/cytology , Olfactory Pathways/metabolism , Prosencephalon/cytology , Raphe Nuclei/cytology , Raphe Nuclei/metabolism , Rats , Rats, Sprague-Dawley , Septal Nuclei/cytology , Septal Nuclei/metabolism , Synaptic Transmission
12.
Eur J Neurosci ; 26(3): 615-23, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17651422

ABSTRACT

In the light of the various neurobiological effects of glutamate in brain development, although some embryonic cells are a probable source of glutamate involved in the development of precursor cells and/or immature neurons, little is known about when and where glutamate plays its crucial roles during corticogenesis. To investigate these roles, we focused on the developmental expression of vesicular glutamate transporter (VGLUT)1 and VGLUT2, which are regarded as the best markers for verifying glutamatergic neuron identity, especially the spatiotemporal distributions of their transcripts and proteins in the developing mouse cortex and hippocampus. In situ hybridization studies revealed that VGLUT1 mRNA is expressed in preplate and marginal zone cells at embryonic day (E)10 and in subplate cells by E13, whereas VGLUT2 mRNA is expressed in preplate and marginal zone cells at E10 and in cells of the subventricular zone by E13. Reverse transcriptase-polymerase chain reaction analysis detected full-length VGLUT1 and VGLUT2 gene transcripts in the embryonic brain. By dual labeling combined with immunostaining for microtubule-associated protein 2 (MAP2) or reelin, we showed that MAP2-positive preplate and marginal zone neurons and subplate neurons express VGLUT1, while reelin-positive preplate and marginal zone cells and MAP2-negative subventricular zone cells express VGLUT2. The present study is the first to provide morphologically reliable evidence showing that Cajal-Retzius cells and subplate neurons are glutamatergic, and that the two cells differentially express VGLUT1 and VGLUT2, respectively, as the specific transport system of glutamate in some events orchestrated by these cells during the cortical development of mice.


Subject(s)
Cerebral Cortex/embryology , Cerebral Cortex/metabolism , Glucose Transporter Type 2/genetics , Neurons/metabolism , Stem Cells/metabolism , Vesicular Glutamate Transport Protein 1/genetics , Animals , Biomarkers/metabolism , Cell Adhesion Molecules, Neuronal/metabolism , Cell Communication/physiology , Cell Differentiation/physiology , Cell Movement/physiology , Cell Proliferation , Cerebral Cortex/cytology , Extracellular Matrix Proteins/metabolism , Gene Expression Regulation, Developmental/physiology , Glutamic Acid/metabolism , Mice , Mice, Inbred ICR , Microtubule-Associated Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neurons/cytology , RNA, Messenger/metabolism , Reelin Protein , Serine Endopeptidases/metabolism , Signal Transduction/physiology , Stem Cells/cytology
13.
Neuroendocrinology ; 84(1): 31-41, 2006.
Article in English | MEDLINE | ID: mdl-17090972

ABSTRACT

The MtT/E and MtT/S cells have been established from a mammotrophic pituitary tumor, and postulated to be progenitor and premature growth hormone (GH) cells, respectively. The difference in the regulation of GH and GH-releasing hormone (GHRH) receptor gene transcription in relation to the developmental stage of GH cells were examined in these two cell lines. In MtT/S cells, triiodothyronine (T3), all-trans retinoic acid (RA) and 9-cis retinoic acid (9cRA) stimulated GH promoter activity but dexamethasone (DEX) did not. On the other hand, DEX stimulated GHRH-receptor promoter alone. T3, RA and 9cRA showed little effect alone but each of them augmented the effect of DEX when used together with DEX. In MtT/E cells, DEX, RA and 9cRA showed similar effect as observed in MtT/S cells on both GH and GHRH-receptor promoter activity. However, T3 neither stimulated GH promoter activity nor augmented the DEX-induced GHRH-receptor gene transcription in MtT/E cells. RT-PCR analyses revealed that both cell types expressed TRalpha1, TRbeta1 and TRalpha2, but expression of TRbeta2, a pituitary specific isoform of TR, was only detected in MtT/S cells. However, the deficiency of TRbeta2 for its own sake does not appear to be a reason why T3 action was not observed in MtT/E cells, because co-transfection of expression plasmids for TRbeta2 and RXRalpha failed in conferring on the cells an ability to respond to T3 by increased GH or GHRH-receptor promoter activity. These results suggest that the acquisition of mechanisms responsible for the regulation of GH or GHRH-receptor transcription by T3 may be involved in the process of functional development of GH cells.


Subject(s)
Gene Expression Regulation , Promoter Regions, Genetic/genetics , Receptors, Neuropeptide/genetics , Receptors, Pituitary Hormone-Regulating Hormone/genetics , Receptors, Somatotropin/genetics , Animals , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , Gene Expression Regulation/drug effects , Gene Expression Regulation, Neoplastic , Growth Hormone/metabolism , Pituitary Neoplasms/genetics , Pituitary Neoplasms/metabolism , Pituitary Neoplasms/pathology , Rats , Receptors, Neuropeptide/metabolism , Receptors, Pituitary Hormone-Regulating Hormone/metabolism , Receptors, Somatotropin/metabolism , Tretinoin/analogs & derivatives , Tretinoin/pharmacology , Triiodothyronine/pharmacology
14.
J Anesth ; 20(4): 279-83, 2006.
Article in English | MEDLINE | ID: mdl-17072692

ABSTRACT

PURPOSE: In order to clarify the principal site for the antinociceptive effects of clonidine, we investigated the nociceptive behavior and neural activity (c-fos staining) of the dorsal horn (DH), locus ceruleus (LC), and A7 area after a formalin test in normal saline- or clonidine-injected rats. METHODS: Thirty-six rats were divided into 6 groups as follows: formalin test + saline (FS); formalin test + clonidine (1 mg.kg(-1)) (FC1); formalin test + clonidine (10 mg.kg(-1)) (FC10); saline (S); clonidine (1 mg.kg(-1)) (C1); and clonidine (10 mg.kg(-1)) (C10). Normal saline or clonidine was injected intraperitoneally 30 min before the formalin test. In the FS, FC1, and FC10 groups, 10% formalin was injected into the left rear paw. All rats were killed 2.5 h after normal saline or clonidine injection. Sections of the lumbar spinal cord, LC, and A7 area were processed for c-fos immunohistochemistry using the avidin-biotin peroxidase complex method. To evaluate the sedative effects of clonidine, we investigated the loss of righting reflex (LORR) for 90 min in 6 other rats as follows: clonidine (1 mg.kg(-1)) (n = 3) and clonidine (10 mg.kg(-1)) (n = 3). RESULTS: The FC10 group showed fewer nociceptive behaviors and higher c-fos expression in the DH, but not in the A7 area, as well as lower c-fos expression in the LC than rats in the FS and FC1 groups (P < 0.05). The C10 group showed lower c-fos expression in the LC than that of rats in the S and C1 groups (P < 0.05). No rats exhibited LORR. CONCLUSION: The antinociceptive effects of clonidine might be mediated primarily by neural activity in the DH.


Subject(s)
Adrenergic alpha-Agonists/pharmacology , Analgesics/pharmacology , Clonidine/pharmacology , Locus Coeruleus/drug effects , Posterior Horn Cells/drug effects , Animals , Locus Coeruleus/metabolism , Male , Pain Measurement , Posterior Horn Cells/metabolism , Rats , Rats, Sprague-Dawley
15.
J Neurochem ; 99(1): 142-53, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16987242

ABSTRACT

Three distinct subtypes of vesicular glutamate transporters (VGLUTs) have been identified to date that are expressed basically in a cell type-specific manner. We have found a splice variant of VGLUT1 mRNA that is expressed almost exclusively in photosensitive tissues, i.e. the retina and the pineal gland. The variant mRNA, termed VGLUT1v, contains an additional 75 base pair sequence derived from part of a second intron (designated as exon IIa) between exons 2 and 3. The variant accounted for approximately 70% and 25%of VGLUT1 mRNA in the adult retina and pineal gland, respectively. The expression of VGLUT1v was developmentally regulated in both tissues. Organ culture showed that expression of the variant in the retina increased in association with the development of rod cells, suggesting that VGLUT1v is expressed in rod cells. In situ hybridization with variant-specific probes showed expression of VGLUT1v in the inner segment layer of photoreceptor cells. On the other hand, variant expression did not parallel the development of rhodopsin-positive cells in the pineal gland. As rod cells and pinealocytes are known to release glutamate continuously at ribbon synapses, it is possible that the variant has some functional advantage over the wild-type transporter in such a specialized manner of glutamate release.


Subject(s)
Gene Expression Regulation, Developmental , Pineal Gland/physiology , Retina/physiology , Vesicular Glutamate Transport Protein 1/genetics , Alternative Splicing , Animals , Brain/physiology , DNA/genetics , DNA/isolation & purification , DNA Primers , Genetic Variation , Organ Specificity , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Reverse Transcriptase Polymerase Chain Reaction
16.
J Comp Neurol ; 498(5): 581-92, 2006 Oct 10.
Article in English | MEDLINE | ID: mdl-16917821

ABSTRACT

Vesicular glutamate transporters (VGLUT1, -2, and -3) mediate the accumulation of transmitter glutamate into synaptic vesicles in glutamatergic neurons. VGLUT1 and VGLUT2 are more reliable glutamatergic neuron markers, since VGLUT3 also exists in other neuron types. To study whether the dopaminergic neuron uses glutamate as a cotransmitter, we analyzed VGLUTs expression in dopamine neurons of adult male rats by in situ hybridization and immunohistochemistry. In the ventral midbrain, in situ hybridization analysis revealed no VGLUT1 mRNA expression, a widespread but discrete pattern of VGLUT2 mRNA expression, and a highly limited expression of VGLUT3 mRNA. Reverse-transcriptase polymerase chain reaction analysis detected full-length VGLUT2 gene transcripts in the ventral midbrain. Using in situ hybridization combined with tyrosine hydroxylase (TH) immunostaining, only VGLUT2 signals were detectable in some TH-labeled neurons of A10 dopamine neuron groups, with the highest incidence (20%) in the rostral linear nucleus of the ventral tegmental area. In the forebrain, VGLUT2 signals were demonstrated in half of the A11 TH-labeled neurons in the hypothalamus. Double-label immunostaining for VGLUT2 and vesicular monoamine transporter 2 or TH showed that double-labeled varicosities are rarely observed in any target regions examined of A10 and A11 dopamine neuron groups. These results indicate that VGLUT2 is expressed in subsets of A10 and A11 dopamine neurons, which might release dopamine and glutamate separately from different varicosities in the majority of their single axons.


Subject(s)
Dopamine/metabolism , Gene Expression/physiology , Hypothalamus/cytology , Mesencephalon/cytology , Neurons/metabolism , Vesicular Glutamate Transport Protein 2/metabolism , Animals , Blotting, Northern/methods , Cell Count/methods , Immunohistochemistry/methods , In Situ Hybridization/methods , Male , Mesencephalon/metabolism , Neurons/classification , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction/methods , Tyrosine 3-Monooxygenase/metabolism , Vesicular Monoamine Transport Proteins/metabolism
17.
Neurosci Res ; 56(1): 124-7, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16842872

ABSTRACT

A second vesicular glutamate transporter (VGLUT2) is detected in magnocellular neurons in the rat hypothalamus. The present study revealed what phenotype of neurons express VGLUT2 mRNA by the histological method. We found that most vasopressin (VP) neurons and several oxytocin (OT) neurons express VGLUT2 mRNA. VGLUT2 gene expression in VP and OT neurons is enhanced with osmotic challenges. In the neurohypophysis, VGLUT2-staining in OT terminals was reduced with osmotic stimulation. These results indicate that VGLUT2 is principally expressed in VP neurons and also in some OT neurons and that VGLUT2 in VP and OT neurons is involved in osmotic regulation.


Subject(s)
Neurons/chemistry , Neurons/metabolism , Paraventricular Hypothalamic Nucleus/cytology , Supraoptic Nucleus/cytology , Vasopressins/metabolism , Vesicular Glutamate Transport Protein 2/metabolism , Animals , Cell Shape , Dehydration , Male , Neurons/cytology , Osmolar Concentration , Oxytocin/metabolism , Paraventricular Hypothalamic Nucleus/metabolism , Rats , Rats, Sprague-Dawley , Salts/administration & dosage , Supraoptic Nucleus/metabolism , Up-Regulation , Vesicular Glutamate Transport Protein 2/genetics , Water Deprivation
18.
Neurosci Lett ; 403(1-2): 20-3, 2006 Jul 31.
Article in English | MEDLINE | ID: mdl-16701947

ABSTRACT

The purpose of this report is to explore the mechanisms of hypercapnia-induced antinociception. We carried out three experiments, the first to confirm whether moderate hypercapnia induces anesthetic effects, the second to determine whether naloxone reverses the anesthetic effects, and the third to evaluate whether beta-endorphin is related to the anesthetic effects. In a pre-test, we determined the optimal CO(2) concentration in a chamber which would cause moderate hypercapnia in rats. Eighteen rats were divided into control, hypercapnia, and hypercapnia plus naloxone groups in experiment 1. The naloxone group rats were injected with naloxone (10 mg/kg) intraperitoneally before gas inhalation. After 60 min gas inhalation, 10% formalin was injected into the left rear paw of all rats, and nociceptive behaviors were observed for 1 h. In experiment 2, 11 rats were divided into control and hypercapnia groups. The brain was removed and fixed under pentobarbital anesthesia. Sections were immunostained for c-Fos and beta-endorphin (ACTH) with the ABC method. All neurons double-labeled for c-Fos and beta-endorphin (ACTH) in the arcuate nucleus were counted by blinded investigators. Moderate hypercapnia (PaCO(2) 83+/-7 mmHg) reduced nociceptive behavior in the formalin test and naloxone pre-treatment attenuated this phenomenon. However, beta-endorphin-producing neurons were not activated by CO(2) inhalation. Endogenous opioids are related to moderate, hypercapnia-induced anesthetic effects, but, beta-endorphin-producing neurons in the hypothalamus were not activated by the CO(2) inhalation stress.


Subject(s)
Anesthetics, Inhalation/pharmacology , Carbon Dioxide/pharmacology , Pain/physiopathology , Receptors, Opioid/physiology , beta-Endorphin/biosynthesis , Animals , Hypothalamus/drug effects , Hypothalamus/metabolism , Male , Naloxone/pharmacology , Narcotic Antagonists , Neurons/drug effects , Neurons/metabolism , Pain/metabolism , Pain Measurement , Pain Threshold , Proto-Oncogene Proteins c-fos/metabolism , Rats , Rats, Sprague-Dawley
19.
Anesth Analg ; 102(3): 811-4, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16492833

ABSTRACT

We investigated whether c-fos expression in the dorsal horn is affected by licking in the formalin test. Thirty adult Sprague-Dawley rats were divided into 5 groups of 6 rats each: a free condition control (Free Cont) group, formalin test under free condition (Free F-test) group, scrub stimulation under free condition (Free Scrub) group, restrained condition control (Restricted Cont) group, and formalin test under restrained condition (Restricted F-test) group. Animals in the three free condition groups and two restricted groups were put in a clear plastic chamber and a restraining chamber, respectively. Ten percent formalin was injected into the left rear paw in the Free and Restricted F-test groups. Animals in the Free Scrub group were scrubbed on the left rear paw with a wet cotton swab. The Free Cont, Restricted Cont, and Free Scrub groups showed little c-fos expression. The number of c-fos positive cells in the ipsilateral surface dorsal horn of the Restricted F-test group was significantly less than that of the Free F-test group (P < 0.05). The results indicated that the licking action increased c-fos expression of the lumbar dorsal horn in the formalin test.


Subject(s)
Behavior, Animal/physiology , Gene Expression Regulation/physiology , Genes, fos/physiology , Pain Measurement/methods , Posterior Horn Cells/metabolism , Animals , Male , Rats , Rats, Sprague-Dawley , Restraint, Physical/physiology
20.
J Mol Endocrinol ; 35(3): 477-88, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16326834

ABSTRACT

Glucocorticoids are involved in the regulation of the rat growth hormone-releasing hormone (GHRH) receptor gene expression, but they act only in the presence of the pituitary specific transcription factor, pit-1. In this study, the role of pit-1 in the glucocorticoid stimulation of the GHRH-receptor gene transcription was examined. The results suggest the presence of a silencer element in the promoter and it is postulated that pit-1 permits glucocorticoid action through suppressing the inhibitory effect of an as yet unknown factor that binds to this element. The present results also suggest that the synergistic activation of the rat GHRH-receptor gene transcription depends on the proper distance between the proximal glucocorticoid response element and the pit-1 binding site.


Subject(s)
Glucocorticoids/pharmacology , Receptors, Neuropeptide/genetics , Receptors, Pituitary Hormone-Regulating Hormone/genetics , Transcription Factor Pit-1/metabolism , Animals , Base Sequence , Binding Sites/genetics , COS Cells , Cell Line , Chlorocebus aethiops , DNA/genetics , DNA/metabolism , Dexamethasone/pharmacology , Genes, Reporter , Luciferases/genetics , Plasmids/genetics , Promoter Regions, Genetic , Rats , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Sequence Deletion , Silencer Elements, Transcriptional , Transcription Factor Pit-1/genetics , Transcription, Genetic/drug effects , Transfection
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