Junko Imaki

Expression of Corticotropin-Releasing Hormone Type 1 Receptor in Paraventricular Nucleus after Acute Stress

We have previously proposed the existence of ultrashort loop-positive feedback regulation of corticotropin-releasing hormone (CRH) in the hypothalamus. To gain a better understanding of this effect, we performed double-label in situ hybridization to identify the neurons in the paraventricular nucleus (PVN) that express CRH type 1 receptor (CRH-R1) following stress. We also conducted immunohistochemistry to determine whether CRH-R1 mRNA was translated to CRH-R1 protein in the PVN. Thirty-minute restraint stress given to male Wistar rats increased c-fos mRNA expression primarily in the CRH-producing neurons of the parvocellular PVN. Small numbers of vasopressin and oxytoxin-producing cells were also labeled by c-fos probes. Approximately 70% of CRH-R1 positive neurons exhibited CRH mRNA 2 h after the beginning of stress, while only a small percentage of the vasopressin and oxytocin-producing cells coexpressed CRH-R1 mRNA. CRH-R1 immunoreactivity, which was detected in the perikarya and fibers of PVN neurons, appeared to increase in response to stress, though this was not statistically significant. Pretreatment with a selective CRH-R1 antagonist, CP-154,526, significantly attenuated stress-induced corticotropin (ACTH) secretion as well as c-fos mRNA expression in the PVN. These results demonstrate that acute stress increases neuronal activation and CRH-R1 mRNA expression primarily in CRH-producing neurons of the parvocellular PVN, that CRH-R1 message is translated to CRH-R1 protein, and that PVN neurons are activated at least in part through CRH-R1 under acute stress. The data further support the possibility of feedback regulation of CRH itself in CRH-producing neurons.

Cellular localization of prolactin-releasing peptide messenger RNA in the rat brain.

Prolactin-releasing peptide (PrRP), a novel peptide identified as the endogenous ligand for an orphan receptor isolated from the pituitary, is a potent stimulator of prolactin release. To get a clue of the functional roles of the peptide, we performed in situ hybridization histochemistry for PrRP mRNA to define the cellular localization of PrRP-producing cells in the brain of the cycling adult female rat during diestrus. The PrRP mRNA-containing cells were located in the caudal part of the dorsomedial nucleus of the hypothalamus. In the brainstem, the cells were found in the caudal part of the solitary tract nucleus and in the caudal ventrolateral medulla (ventrolateral intermediate reticular field). Specific signals for PrRP mRNA were not detected in other brain regions. Although PrRP is a candidate for being a hypophysiotropic specific releasing factor, the discrete distribution of PrRP in the extrahypothalamic area suggests that the peptide has other physiological functions in the central nervous system.

Prolactin secretion in response to prolactin-releasing peptide and the expression of the prolactin-releasing peptide gene in the medulla oblongata are estrogen dependent in rats

Prolactin-releasing peptide (PrRP), recently isolated from bovine hypothalamus as an endogenous ligand to a seven transmembrane-domain orphan receptor, is a candidate specific prolactin-releasing factor. The prolactin-releasing activity of the peptide and the expression of the PrRP gene were examined in vivo in relation to estrogen status. Plasma prolactin levels increased significantly with a peak at 5 min after the administration of 50 microg/kg PrRP in female rats in estrus under urethane anesthesia as compared with those in vehicle-treated control rats, but not in female rats in diestrus or proestrus or in male rats. In ovariectomized rats treated with supraphysiological concentration of estrogen, a dose-dependent increase of prolactin secretion in response to 2-50 microg/kg PrRP was observed. However, the peak values induced by 50 microg/kg PrRP were much less than those induced by 2 microg/kg thyrotropin-releasing hormone (TRH). PrRP mRNA levels in the medulla oblongata were decreased by ovariectomy and increased by estrogen treatment. The data indicate that estrogen is prerequisite to the stimulatory effect of PrRP on the secretion of prolactin and to the increase of PrRP mRNA levels in the medulla oblongata. The weak in vivo potency of PrRP on prolactin secretion relative to TRH suggests that PrRP differs from the classical hypophysiotropic hypothalamic releasing hormones.

A prostaglandin E2 receptor subtype EP1 receptor antagonist (ONO-8711) reduces hyperalgesia, allodynia, and c-fos gene expression in rats with chronic nerve constriction.

Chronic constriction injury (CCI) of the sciatic nerve in rats induces persistent mechanical hyperalgesia and allodynia. CCI is widely known as a model of neuropathic pain, and many studies using this model have been reported. Recently, c-fos has been used as a neural marker of pain, and various studies have assessed the relationship between hyperalgesia and c-fos expression in the lumbar spinal cord. In this study, we examined the role of a prostaglandin E2 receptor subtype EP1 receptor antagonist (ONO-8711) in a rat CCI model. EP1 receptor antagonist (EP1-ra) oral administration from day 8 to day 14 significantly reduced hyperalgesia and allodynia in the three pain tests on day 15. EP1-ra treatment from day 8 to 14 also reduced c-fos-positive cells in laminae I-II, III-IV, and V-X compared with saline treatment. A single dose of EP1-ra treatment on day 8 significantly reduced hyperalgesia and allodynia at 1 h and 2 h after administration, but the efficacy was not observed at 24 h. We conclude that EP1-ra treatment may be useful for hyperalgesia and allodynia and that EP1 receptor mechanisms are involved in the maintenance of c-fos gene expression induced by nerve injury.

Hyperresponsiveness to Palatable and Aversive Taste Stimuli in Genetically Obese (Bombesin Receptor Subtype-3–Deficient) Mice

Taste preference in obese mice was examined using genetically obese (bombesin receptor subtype-3: BRS-3 deficient) animals. Preference for either sodium saccharin (0.2%). sodium chloride (0.9%), citric acid (0.1%), or quinine sulfate (0.002%) solution was examined using a two-bottle test situation, and BRS-3 deficient mice not only showed a stronger preference for saccharin solution, but also a stronger aversive response to quinine solution, relative to wild-type littermates. Furthermore, a conditioned taste-aversion test measured the consumption of sodium saccharin (0.2%) and sodium chloride (0.9%) solutions after intraperitoneal injection of LiCl (0.3 M, 1 mg/kg), and BRS-3-deficient mice exhibited stronger aversion to both solutions than did control animals. In situ hybridization demonstrated that the BRS-3 gene is expressed in the parabrachial nucleus, the medial and central nuclei of the amygdala, and the hypothalamic nuclei such as paraventricular nucleus, all of which are known to be involved in taste perception. These results suggest that expression of the BRS-3 gene in these nuclei is important for the modulation of taste preference, as well as the development of obesity.

Expression of Corticotropin Releasing Factor (CRF), Urocortin and CRF Type 1 Receptors in Hypothalamic-Hypophyseal Systems Under Osmotic Stimulation

The expression of corticotropin releasing factor (CRF) and urocortin in hypothalamic magnocellular neurones increases in response to osmotic challenge. To gain a better understanding of the physiological roles of CRF and urocortin in fluid homeostasis, CRF, urocortin and CRF type 1 receptor (CRFR‐1) gene expression was examined in the hypothalamic‐hypophyseal system usingin situ and double‐label in situ hybridization following chronic salt loading. CRFR‐1 expression was further examined by immunohistochemistry and receptor binding. Ingestion of hypertonic saline by Sprague‐Dawley rats for 7 days induced CRF mRNA exclusively in the oxytocin neurones of the magnocellular paraventricular nucleus (PVN) and the supraoptic nucleus (SON), but induced CRFR‐1 mRNA in both oxytocin and vasopressin‐containing magnocellular neurones. Hypertonic saline treatment also increased urocortin mRNA expression in the PVN and the SON. In the SON, urocortin was localized to vasopressin and oxytocin neurones but was rarely seen in CRF‐positive cells. Changes in CRFR‐1 mRNA expression in magnocellular neurones by hypertonic saline treatment were accompanied by changes in CRFR‐1 protein levels and receptor binding. Hypertonic saline treatment increased CRFR‐1‐like immunoreactivity in the magnocellular PVN and SON, and decreased it in the parvocellular PVN. CRF receptor binding in the PVN and SON was also increased in response to osmotic stimulation. Finally, hypertonic saline treatment increased CRFR‐1 mRNA, CRFR‐1‐like immunoreactivity and CRF receptor binding in the intermediate pituitary. These results demonstrate that the increase in the expression of CRF and urocortin message in magnocellular neurones induced by salt loading is accompanied by an increase in CRF receptor levels and binding in the hypothalamus and intermediate pituitary. Thus, CRF and urocortin may exert modulatory effects locally within magnocellular neurones as well as at the pituitary gland in response to osmotic stimulation.

The expression of SCDGF/PDGF-C/fallotein and SCDGF-B/PDGF-D in the rat central nervous system.

We examined the expression patterns of the two homologous genes, spinal cord-derived growth factor (SCDGF)/platelet-derived growth factor (PDGF)-C/fallotein and SCDGF-B/PDGF-D in the rat central nervous system. In the spinal cord, SCDGF/PDGF-C/fallotein was expressed in the floor plate at embryonic day (E) 11 and also in the ventricular zone at E16 but not in adult. However, SCDGF-B/PDGF-D was prominently expressed in the adult motoneurons, although faint expression was observed in the ventral ventricular zone at E16. Also in the brain, the expression of SCDGF/PDGF-C/fallotein was more remarkable at E16 than at adult. It was highly expressed in the cortex, pontine area and choroid plexus at E16. Contrary to SCDGF/PDGF-C/fallotein, SCDGF-B/PDGF-D expression was notable in several nuclei at adult.

Proliferation in the posterior region of the lens of c-maf-/- mice

Purpose. To examine the involvement of the c-maf gene in the proliferation of the lens cells. Methods. Eyes of the E13 and E18 stages of the wild-type and c-maf-/- mice were analyzed by BrdU incorporation assay, TUNEL assay and immunocytochemistry using a anti-P27(KIP1) and a anti-P57(KIP2) antibody. Results. In the E13 and E18 c-maf mutant lens, BrdU-positive cells were detected at the posterior region of the lens. Cell-cycle inhibitor P27(KIP1) and P57(KIP2) were expressed in the equatorial and posterior region of the lens of both wild-type and c-maf-/- lenses. Conclusion. These results suggest that the expression of c-maf is required for differentiation and cell cycle arrest of lens fiber cells. It is also suggested that P27(KIP1) and P57(KIP2) were not involved in the continued proliferation of posterior region of the c-maf-/- lens.

CREB-induced transcriptional activation depends on mGluR6 in rod bipolar cells

To investigate the molecular mechanisms of stimuli-induced transcriptional activation in neuronal cells, we have investigated the light-induced gene expression in the neural retina of rats. The immunoreactivity for phosphorylated cAMP responsive element binding protein (PCREB-IR) was expressed in the outer half of the inner nuclear layer (INL) and the ganglion cell layer (GCL) after 5 min exposure to steady light also in mice. In addition to these cells, PCREB-IR was also detected in the inner border of the INL after 5 min exposure to flashing light. Both steady and flashing lights induced c-fos mRNA in the same types of cells as the PCREB-IR-positive cells. Majority of PCREB immunoreactive nuclei in the outer half of the INL were also immunopositive for anti-protein kinase C alpha (PKC alpha), a marker of rod bipolar cells, while CaM kinase IV immunoreactivity was not detected in these cells. PCREB-IR and c-fos gene expression in the PKC alpha positive rod bipolar cells were lost in mice lacking metabotropic glutamate receptor 6 (mGluR6). Thus, we propose that the transcriptional response of CREB to light stimulation in rod bipolar cells is regulated via mGluR6.

Differential expression of c-fos mRNA in the rat neocortex by in situ hybridization

The c-fos mRNA expression pattern in rat neocortex, was determined in the rat kept in a 12:12 light/dark cycle, in constant dark, or in constant light by in situ hybridization. At the beginning of the light period, c-fos mRNA was induced both in the neocortex and suprachiasmatic nucleus (SCN). Transiently increased c-fos mRNA expression was detected from 0830 to 0900 and soon declined to basal levels. Immediately prior to the beginning of the dark period, c-fos mRNA expression also increased and remained elevated in the neocortex following the dark period. In the constant dark group, c-fos mRNA expression showed no transient elevation at the beginning of the light period. On the other hand, c-fos mRNA expression in the constant light group increased during their subjective dark period as well as normal light/dark cycle. These results demonstrate a circadian pattern of c-fos mRNA expression in the neocortex which is similar to that observed previously in the inner and outer nuclear layers of the retina.