Regulation of glucocorticoid receptor mRNA and heat shock protein 70 mRNA in the developing sheep brain

Fetal hypothalamo-pituitary-adrenal (HPA) activity increases dramatically at term in sheep, however, little is known about the regulation of glucocorticoid feedback in the developing brain. Heat shock protein 70 (hsp70) is closely associated with glucocorticoid actions within the cell. We hypothesized that there is a decrease in glucocorticoid negative feedback in the brain, near term, resulting from changes in the expression of glucocorticoid receptors (GR) and hsp70. Brains were removed at various stages of development. GR mRNA levels in the paraventricular nucleus (PVN) and cortex, and hsp70 mRNA in the PVN were determined by in situ hybridization. In the hippocampus, GR mRNA levels were measured by Northern analysis. In the PVN, GR mRNA was present by d60. GR mRNA levels reached a peak at d100-110, but then decreased significantly with progression of gestation, and were lowest at term. Hippocampal GR mRNA levels were highest on day 130 of gestation, decreasing to low levels at term. In the cerebral cortex, GR mRNA levels were expressed at high levels in all layers of the cortex by day 110 of gestation with levels decreasing to term. Hsp70 mRNA was present in both parvocellular and magnocellular regions of the PVN, and there was no significant change in late gestation. In conclusion, (1) The high levels of GR mRNA present in the PVN, hippocampus and cerebral cortex during fetal life are likely important in development of these structures at a time when circulating glucocorticoids are low. (2) Changes in GR mRNA levels in the PVN are not associated with alterations in the expression of hsp70. (3) The decrease in GR mRNA in the hippocampus and PVN in late gestation, at a time when fetal plasma cortisol is increasing, likely facilitates maintained hypothalamic drive to the pituitary corticotroph.     

Glucagon like peptide-1 (7-36) amide (GLP-1) nerve terminals densely innervate corticotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus

Glucagon like peptide-1 (7-36) amide (GLP-1), a potent regulator of glucose homeostasis, is also produced in the central nervous system and has been implicated in the control of hypothalamic-pituitary function and food intake. GLP-1 immunoreactive (IR) fibers and terminals are widely distributed in the septum, hypothalamus, thalamus and brainstem, likely originating from GLP-1-IR neuronal cell bodies from the nucleus of the solitary tract of the medulla oblongata. Central administration of GLP-1 increases plasma corticosterone levels and elicits c-fos expression in corticotropin releasing hormone (CRH) neurons of the hypothalamic paraventricular nucleus (PVN). To identify the endogenous neurocircuitry that may underlie this response, the present study determined whether there is an innervation of PVN CRH neurons by GLP-1-containing nerve terminals. GLP-1-IR fibers and nerve terminals were found in the parvocellular parts of the PVN, with highest concentrations in the anterior and medial parvocellular subdivisions. The magnocellular divisions of the PVN also showed moderate numbers of GLP-1-IR nerve fibers. Double immunolabelling revealed numerous GLP-1-IR nerve fibers in close apposition to approximately 65% of detectable CRH neurons in the medial parvocellular subdivision of the rat PVN. At the ultrastructural level, GLP-1-IR terminals were observed to establish synapses on both perikarya and dendrites of CRH neurons. These findings support the hypothesis that the GLP-1-induced activation of CRH neurons and the associated pituitary-adrenocortical activation may be accomplished by GLP-1's direct action on hypophysiotropic CRH neurons. Since central CRH is also thought to be an anorexigenic factor and GLP-1 neurons contain leptin receptors, activation of CRH neurons in the PVN by GLP-1 may contribute to the complex neuroendocrine and metabolic actions by the adipostatic hormone, leptin.     

Flurothyl-induced seizures in rats activate Fos in brainstem catecholaminergic neurons

Autonomic changes accompany seizures in both animals and humans. While ictal autonomic dysfunction can be life-threatening, the participating neural networks involved are poorly understood. In this study we examined the activation of Fos following generalized seizures in brainstem structures known to mediate autonomic function. Adult female rats were sacrificed 2 h after flurothyl-induced seizures. Double-immunostaining for c-Fos and dopamine-beta-hydroxylase (DBH), and c-Fos and phenylethanol-N-methyl-transferase (PNMT) were performed in brainstem slices. Numbers of DBH-labeled neurons expressing Fos-like immunoreactivity (FLI) (DBH/Fos) and PNMT labeled neurons expressing FLI (PNMT/Fos) were counted in the noradrenergic (A1, A2, A5, A7) and adrenergic (C1, C2) cell groups localized in pons and medulla oblongata. Among the experimental animals, the highest degree of co-localization of DBH/Fos neurons was observed in the locus coeruleus (A6; 87.7%), and in the A1(72.8%) cell group located in the caudal ventrolateral medulla (VLM). No co-localization of DBH/Fos neurons was observed in control animals. The highest degree of co-localization of PNMT/Fos neurons was observed in the C1 adrenergic cell group (84.2%) located in the rostral VLM. Control animals showed very few (5.5%) PNMT/Fos co-localized neurons in the C1 adrenergic cell group. Our results indicate that flurothyl-induced generalized seizures in rats activate catecholaminergic neurons in the pons and medulla oblongata. Further studies are necessary to determine whether activation of brainstem catecholaminergic neurons contribute to the autonomic manifestations that frequently accompany epileptic seizures.     

Oxytocin innervation of caudal brainstem nuclei activated by cholecystokinin

The integration of 'long-term' adiposity signaling with the 'short-term' meal-related signal cholecystokinin (CCK) is proposed to involve descending hypothalamic projections to areas of the caudal brainstem (CBS) that regulate the amount of food consumed during a single meal. One such projection extends from cell bodies in the hypothalamic paraventricular nucleus (PVN) to the nucleus tractus solitarius (NTS), where cells that respond to peripheral CCK are concentrated. Candidate neuronal cell types that may comprise this PVN-NTS projection includes those expressing corticotropin-releasing hormone (CRH) or oxytocin. We therefore sought to determine whether oxytocin or CRH axons are preferentially located in close anatomical proximity to neurons of the NTS that are activated by peripheral administration of CCK, as determined by immunocytochemical staining for Fos protein. Rats received injections of either an anorexic dose of CCK (8 nmol/kg, i.p.) or vehicle and were perfused 2 h later with 4% paraformaldehyde. Immunocytochemistry was performed on cryostat sections (14 microm) of caudal brainstem, using a polyclonal antibody to Fos protein and either a monoclonal antibody to oxytocin or a polyclonal antibody to CRH. As expected, CCK administration significantly increased the numbers of Fos-positive neurons by 489% (p<0.01) and 400% (p<0.01), respectively, in the medial and gelatinosus subdivisions of the NTS. These same regions received dense oxytocin axon innervation, whereas CRH immunoreactivity was not as prevalent in these areas. In areas outside the NTS, such as the dorsal motor nucleus of the vagus (DMV), Fos activation was absent despite a dense oxytocin and CRH innervation. To investigate whether CCK-induced reductions of food intake require intact oxytocin signaling, we performed a separate study in which CCK injection was preceded by injection into the fourth ventricle of an oxytocin receptor antagonist [d(CH(2))(5), Tyr (Me)(2), Orn(8)]-vasotocin (OVT). This study showed CCK was 23% and 22% less effective at inhibiting food intake at 30 min (p<0.05) and 1 h (p<0.05) food intake, respectively, in the presence of OVT. Taken together, the data indicate that oxytocin axons within the descending pathway from the PVN to the NTS are anatomically positioned to interact with NTS neurons that respond to vagally mediated peripheral CCK signals such as those that occur following ingestion of a meal. These findings support the hypothesis that oxytocin exerts a tonic stimulatory effect on the response of key neurons within the NTS to CCK and further reduce meal size.     

Stimulating effect of HIV-1 coat protein gp120 on corticotropin-releasing hormone and arginine vasopressin in the rat hypothalamus: involvement of nitric oxide

Subjects with human immunodeficiency virus type 1 (HIV-1) infection display increased activity of the hypothalamo-pituitary-adrenal (HPA) axis, which may play a role in both HIV-related neurodegenerative processes and disease progression. It has been speculated that the HIV coat protein gp120 may be responsible for these changes, and previous experimental evidence in both transgenic and nontransgenic mice supports this view. We speculated that one of the effects of gp120 in the CNS is to act within the hypothalamus to affect both corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP), the principal regulators of HPA axis. We therefore administered i.p. gp120 (100 ng/rat) or vehicle to male Wistar rats and then detected Fos protein (an index of neuronal activation), CRH, and AVP immunoreactivity in the cellular compartments of the hypothalamic paraventricular nucleus (PVN). In addition, we tested the direct effect of various concentrations of gp120 on the release of CRH and AVP from rat hypothalamic explants maintained in vitro. Any modulation of gp120 effects by nitric oxide (NO) pathways was also sought by coadministering i.p. to rats or adding to the hypothalamic preparations the NO synthase inhibitor N(G)-methyl-l-arginine (l-NMMA). Gp120 induced the expression of Fos protein in both the parvo- and the magnocellular PVN, which was significantly attenuated by l-NMMA 10(-6) nM/L (P < 0.001 vs gp120 alone). Double immunochemistry showed costaining for Fos protein and CRH or AVP in the PVN following gp120; the number of double-labeled CRH and AVP cells for Fos protein was markedly reduced (P < 0.001) by coadministration of l-NMMA 10(-6) nM/L. In the in vitro studies, addition of gp120 to the hypothalamic explants in the dose range of 10 pM-1 nM resulted in a clear stimulation of both CRH and AVP release (P < 0.05-0.001 compared to control); in the presence of l-NMMA at 10-fold higher concentrations the stimulatory effect of gp120 on the release of both peptides was completely lost. It would therefore appear that gp120 activates CRH and AVP-producing neurons in the hypothalamic PVN and stimulates the release of both peptides in vitro via NO-dependent mechanisms. These findings, in line with previous evidence, further suggest that the increased activity of the HPA axis associated with HIV infection may be of central origin, due to the effects of gp120 on hypothalamic CRH and AVP release.     

Serotonin increases the excitability of the hypothalamic paraventricular nucleus magnocellular neurons

Recent evidence that 5-hydroxytryptamine (5-HT or serotonin) enhances the release and the gene expression of vasopressin and oxytocin in the hypothalamic paraventricular nucleus (PVN) suggests that 5-HT can excite the PVN magnocellular neurons. The objective of this study was to examine the underlying mechanisms for such excitatory action in the electrophysiologically identified hypothalamic PVN magnocellular neurons in rats using whole-cell patch-clamp. We found that 5-HT weakly depolarizes 33.3% of PVN magnocellular neurons in the presence of tetrodotoxin. A minuscule inward current was produced by 5-HT in 48% of the cells, which was attenuated when the 5-HT(4) antagonist GR113808 or the 5-HT(7) antagonist SB269970 was added. In addition, 5-HT reduced the frequency of miniature inhibitory postsynaptic currents in a dose-dependent manner. This inhibition was mimicked by the 5-HT(1B) agonist CP93129, and reversed in the presence of 5-HT(1B) antagonists cyanopindolol and SB224289. Besides, 5-HT induced a biphasic effect on the frequency of miniature excitatory postsynaptic currents, comprising a transient inhibition and a delayed concentration-dependent excitation (onset latency approximately 5 min). The facilitation was mimicked by the 5-HT(2A/2C) agonist DOI and abolished in the presence of the 5-HT(2C) antagonist RS102221. Our findings reveal that 5-HT directly increases the excitability of the PVN magnocellular neurons via multiple receptor subtypes and mechanisms. This may help understanding the regulation of 5-HT-induced hormone release and feeding behavior in the PVN.     

5-HT1A Receptor Agonist Flesinoxan Enhances Fos Immunoreactivity in Rat Central Amygdala, Bed Nucleus of the Stria Terminalis and Hypothalamus

5-Hydroxytryptamine-1A (5-HT_1A) receptor agonists, including flesinoxan, reduce anxiety and activate the hypothalamus-pituitary-adrenal (HPA) axis under basal conditions. In order to investigate the underlying neural mechanisms we investigated immunoreactivity for the immediate early gene protein product Fos (Fos-ir) in rat brains 1 h after flesinoxan treatment (0.0, 0.3 or 3.0 mg/kg p.0.). Typically, 5-HT_1A receptor-containing brain areas, such as the dorsal raphe nuclei, hippocampus, septum, diagonal band and the cortical and basomedial amygdala, do not show Fos-ir. Apparently, binding of flesinoxan at the 5-HT_1A receptor does not directly lead to activation of c-fos in the cell, probably due to its negative coupling to adenylate cyclase. However, in typically non-5HT_lA receptor-containing brain areas Fos-ir is increased due to flesinoxan treatment, as in the paraventricular nucleus of the hypothalamus (PVN), the dorsolateral part of the bed nucleus of the stria terminalis (BNSTd1) and the central amygdala (CeA). Flesinoxan-treated rats also exhibited higher plasma corticosterone levels than vehicle-treated animals, which suggests the involvement of corticotropin-releasing hormone (CRH) or vasopressin in the hypothalamus. After double immunolabelling (Fos/CRH or Fos/vasopressin), every CRH neuron detected in the PVN also contained Fos. Moreover, a significant correlation existed between the number of Fos-ir neurons in the PVN and the plasma corticosterone level. Hardly any Fos/ vasopressin double labelling was visible in the PVN. Accordingly, flesinoxan exerts its activating effects on the HPA axis via CRH neurons in the PVN. These effects are transsynaptically mediated by other brain areas, such as the CeA and BNSTdl, which also show increased Fos-ir.     

Glucocorticoids and serotonin alter glucocorticoid receptor (GR) but not mineralocorticoid receptor (MR) mRNA levels in fetal mouse hippocampal neurons, in vitro

Studies utilizing rats and guinea pigs have demonstrated that the hypothalamo-pituitary-adrenal (HPA) axis can be programmed by glucocorticoids during fetal life. Such programming is believed to occur, at least partially, at the level of hippocampal glucocorticoid receptors (GR) and mineralocorticoid receptors (MR). Studies have also demonstrated that serotonin up regulates GR levels within the developing hippocampus. However, the cell type in which these changes take place has not been determined. We hypothesized that dexamethasone, corticosterone and serotonin exposure modify GR and MR mRNA levels in fetal mouse hippocampal cultures, and that these effects are confined to neurons. Cultures were derived from CD1 mouse fetuses on day 18 of gestation (n=8 dams). Fetal hippocampi were dissected, then mechanically and chemically dispersed. Cultures were exposed to dexamethasone, corticosterone or serotonin (1-100 nM) for 4 days. Levels of GR and MR mRNA were examined by in situ hybridization and high-resolution silver emulsion autoradiography. Four days exposure to dexamethasone or corticosterone (10 or 100 nM) decreased levels of GR mRNA within neurons. There was no significant change in MR mRNA in either experiment. Exposure to serotonin (100 nM) significantly increased expression of GR mRNA in hippocampal neurons. MR mRNA levels were unaffected by serotonin treatment. Dexamethasone, corticosterone or serotonin exposure did not alter expression of GR mRNA within glial cells. We conclude that synthetic and endogenous glucocorticoids, as well as serotonin, can influence neuronal levels of GR mRNA during hippocampal development. However, whether these effects are permanent remains to be determined.     

Paraventricular nucleus of the human hypothalamus in primary hypertension: activation of corticotropin-releasing hormone neurons.

By using quantitative immunohistochemical and in situ hybridization techniques, we studied corticotropin-releasing hormone (CRH) -producing neurons of the hypothalamic paraventricular nucleus (PVN) in patients who suffered from primary hypertension and died due to acute cardiac failure. The control group consisted of individuals who had normal blood pressure and died of acute heart failure due to mechanical trauma. Both magno- and parvocellular populations of CRH neurons appeared to be more numerous in the PVN of hypertensive patients. Quantitative analysis showed approximately a twofold increase in the total number of CRH neurons and a more than fivefold increase in the amount of CRH mRNA in the hypertensive PVN compared with the control. It is suggested that synthesis of CRH in hypertensive PVN is enhanced. Increased activity of CRH-producing neurons in the PVN of hypertensive patients is proposed not only to entail hyperactivity of the hypothalamo-pituitary-adrenal axis, but also of the sympathetic nervous system and, thus, to be involved in the pathogenesis of hypertension.     

Differential regulation of parvocellular neuronal activity in the paraventricular nucleus of the hypothalamus following single vs. repeated episodes of water restriction-induced drinking

Acute activation of the hypothalamic-pituitary-adrenal (HPA) axis releases glucocorticoids to maintain homeostasis, whereas prolonged exposure to elevated glucocorticoids has deleterious effects. Due to the potential benefits of limiting stress-induced glucocorticoid secretion, the present study uses drinking in dehydrated rats as a model to delineate mechanisms mobilized to rapidly inhibit HPA activity during stress. Using Fos expression as an indicator of neuronal activation, the effect of a single or repeated episode of dehydration-induced drinking on the activity of magnocellular and parvocellular neurons in the paraventricular nucleus (PVN) of the hypothalamus was examined. Adult male rats underwent a single episode or repeated (six) episodes of water restriction and were sacrificed before or after drinking water in the AM. Plasma osmolality, vasopressin (AVP), adrenocorticotropic hormone (ACTH) and corticosterone were elevated by water restriction and reduced after drinking in both models. Fos expression was elevated in AVP-positive magnocellular PVN neurons and AVP- and corticotropin releasing hormone (CRH)-positive parvocellular PVN neurons after water restriction. Fos expression was reduced in magnocellular AVP neurons after both models of restriction-induced drinking. In contrast, Fos expression did not change in AVP and CRH parvocellular neurons after a single episode of restriction-induced drinking, but was reduced after repeated episodes of restriction-induced drinking. These data indicate that drinking-induced decreases in glucocorticoids in dehydrated rats involve multiple factors including reduction in magnocellular release of vasopressin and reduction in parvocellular neuronal activity. The differential inhibition of PVN parvocellular neurons after repeated rehydration may reflect a conditioned response to repeated stress reduction.     

Hypothalamic paraventricular nucleus neurons regulate medullary catecholamine cell responses to restraint stress

Both physical and psychological stressors recruit catecholamine cells (CA) located in the ventrolateral medulla (VLM) and the nucleus of the solitary tract (NTS). In the case of physical stressors, this effect is initiated by signals that first access the central nervous system at or below the level of the medulla. For psychological stressors, however, CA cell recruitment depends on higher structures within the neuraxis. Indeed, we have recently provided evidence of a pivotal role for the medial amygdala (MeA) in this regard, although such a role must involve a relay, as MeA neurons do not project directly to the medulla. However, some of the MeA neurons that respond to psychological stress have been found to project to the hypothalamic paraventricular nucleus (PVN), a structure that provides significant input to the medulla. To determine whether the PVN might regulate medullary CA cell responses to psychological stress, animals were prepared with unilateral injections of the neurotoxin ibotenic acid into the PVN (Experiment 1), or with unilateral injections of the retrograde tracer wheat germ agglutinin-gold (WGA-Au) into the CA cell columns of the VLM or NTS (Experiment 2). Seven days later, animals were subjected to a psychological stressor (restraint; 15 minutes), and their brains were subsequently processed for Fos plus appropriate cytoplasmic markers (Experiment 1), or Fos plus WGA-Au (Experiment 2). PVN lesions significantly suppressed the stress-related induction of Fos in both VLM and NTS CA cells, whereas tracer deposits in the VLM or NTS retrogradely labeled substantial numbers of PVN cells that were also Fos-positive after stress. Considered in concert with previous results, these data suggest that the activation of medullary CA cells in response to psychological stress may involve a critical input from the PVN.     

Regulation of tyrosine hydroxylase levels and activity and Fos expression during opioid withdrawal in the hypothalamic PVN and medulla oblongata catecholaminergic cell groups innervating the PVN

Morphine withdrawal increases the hypothalamic-pituitary-adrenocortical (HPA) axis activity, which is dependent on an hyperactivity of noradrenergic pathways innervating the hypothalamic paraventricular nucleus (PVN). However, the possible adaptive changes that can occur in these pathways during morphine dependence are not known. We studied the alterations in tyrosine hydroxylase (TH; the rate-limiting enzyme in catecholamines biosynthesis) immunoreactivity levels and TH enzyme activity in the rat NTS-A2/VLM-A1 noradrenergic cell groups and in the PVN during morphine withdrawal. In the same paradigm, we measured Fos expression as a marker of neuronal activation. TH and Fos immunoreactivity was determined by quantitative Western blot analysis, combined with immunostaining for TH and Fos for immunohistochemical identification of active neurons during morphine withdrawal. Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by an injection of naloxone (5 mg/kg s.c.). Morphine withdrawal induced the expression of Fos in the PVN and NTS/VLM, which indicates an activation of neurons in these nuclei. TH immunoreactivity in the NTS/VLM was increased 90 min after morphine withdrawal, whereas there was a decrease in TH levels in the PVN at the same time point. Following withdrawal, Fos immunoreactivity was present in most of the TH-positive neurons of the A2 and A1 neurons. TH activity was measured in the PVN, a projection area of noradrenergic neurons arising from NTS-A2/VLM-A1. Morphine withdrawal was associated with an increase in the enzyme activity at different time points after naloxone-precipitated morphine withdrawal. The present results suggest that an increase in TH protein levels and TH enzyme activity might contribute to the enhanced noradrenergic activity in the PVN in response to morphine withdrawal.     

Repeated antenatal glucocorticoid treatment decreases hypothalamic corticotropin releasing hormone mRNA but not corticosteroid receptor mRNA expression in the fetal guinea-pig brain

Approximately 10% of pregnant women are treated with synthetic glucocorticoids in late gestation, to promote fetal lung maturation. The effectiveness of this treatment has led to the use of repeated dose regimens, with little knowledge of the impact on neuroendocrine development. Animal studies have recently shown that repeated fetal glucocorticoid exposure can lead to permanent changes in hypothalamic-pituitary-adrenal (HPA) function in offspring. In this study, we hypothesized that such treatment modifies corticotropin releasing hormone (CRH), glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) systems in the developing limbic system and hypothalamus. Pregnant guinea-pigs were treated with dexamethasone, betamethasone or vehicle on days 40,41,50,51,60 and 61 of gestation (birth = 68 days). On day 62, guinea-pigs were killed and the fetuses rapidly removed. Glucocorticoid treatment resulted in a dose-dependent reduction in plasma cortisol concentrations in both male and female fetuses. There was also a significant reduction in CRH mRNA expression in the hypothalamic paraventricular nucleus. In contrast, exposure to glucocorticoid increased MR mRNA expression in the hippocampus (CA1/2 and CA3) and dentate gyrus of female fetuses. There was a small but significant increase in GR mRNA expression in limbic structures in male fetuses following treatment with 1 mg/kg dexamethasone. However, there was no significant effect of glucocorticoid exposure on hippocampal GR mRNA expression in female fetuses, or hypothalamic GR mRNA in either males or females. In conclusion, repeated maternal glucocorticoid treatment inhibits fetal HPA function. The fact that CRH mRNA levels were reduced indicates that synthetic glucocorticoids enter the fetal brain. By contrast, fetal glucocorticoid exposure does not downregulate GR mRNA, and increases MR mRNA expression. The latter likely reflects removal of circulating endogenous ligand (cortisol). These alterations may form the basis for permanently modified HPA activity in later life.     

侧脑室注射IL-1ra后脑缺血大鼠下丘脑室旁核CRH的表达

目的探讨白介素－１（ＩＬ－１）是否参与脑缺血大鼠下丘脑－垂体－肾上腺轴（ＨＰＡ）的激活。方法选用成年ＳＤ大鼠，分为１７组（Ａ～Ｑ组），Ａ组为正常对照组，Ｂ～Ｏ组大鼠脑缺血时间分别为１、３、６、２４、４８、７２ｈ及１、２、３、４、５、６、７、８周；Ｐ组的处理为：用微量注射泵将ＩＬ－１受体拮抗剂（ＩＬ－１ｒａ）注入大鼠侧脑室，之后建立脑缺血模型；Ｑ组为Ｐ组的对照组，用微量注射泵将生理盐水注入大鼠侧脑室，之后建立脑缺血模型。用免疫组化法显示各组大鼠下丘脑室旁核（ＰＶＮ）促肾上腺皮质激素释放激素（ＣＲＨ）的表达。结果正常大鼠ＰＶＮ有少量ＣＲＨ表达，脑缺血４８ｈ内ＰＶＮ无ＣＲＨ表达。从脑缺血７２ｈ起，ＰＶＮ之ＣＲＨ的表达开始增多，并且持续至脑缺血第８周。Ｐ组大鼠ＰＶＮ有较多ＣＲＨ表达，而Ｑ组大鼠ＰＶＮ无ＣＲＨ表达。结论ＩＬ－１可能通过促进下丘脑ＰＶＮ释放ＣＲＨ而参与脑缺血时下丘脑－垂体－肾上腺轴的激活。     

Centrally administered bombesin activates COX-containing spinally projecting neurons of the PVN in anesthetized rats

The paraventricular nucleus (PVN) of the hypothalamus has a heterogenous structure containing different types of output neurons that project to the median eminence, posterior pituitary, brain stem autonomic centers and sympathetic preganglionic neurons in the spinal cord. Presympathetic neurons in the PVN send mono- and poly-synaptic projections to the spinal cord. In the present study using urethane-anesthetized rats, we examined the effects of centrally administered bombesin (a homologue of the mammalian gastrin-releasing peptide) on the mono-synaptic spinally projecting PVN neurons pre-labeled with a retrograde tracer Fluoro-Gold (FG) injected into T8 level of the spinal cord, with regard to the immunoreactivity for cyclooxygenase (COX) isozymes (COX-1/COX-2) and Fos (a marker of neuronal activation). FG-labeled spinally projecting neurons were abundantly observed in the dorsal cap, ventral part and posterior part of the PVN. The immunoreactivity of each COX-1 and COX-2 was detected in FG-labeled spinally projecting PVN neurons in the vehicle (10 μl of saline/animal, i.c.v.)-treated group, while bombesin (1 nmol/animal, i.c.v.) had no effect on the number of these immunoreactive neurons for each COX isozyme with labeling of FG. On the other hand, the peptide significantly increased the number of double-immunoreactive neurons for Fos and COX-1/COX-2 with FG-labeling in the PVN (except triple-labeled neurons for FG, COX-2 and Fos in the dorsal cap of the PVN), as compared to those of vehicle-treated group. These results suggest that centrally administered bombesin activates spinally projecting PVN neurons containing COX-1 and COX-2 in rats.     

Hypothalamic oxytocin in the developing ovine fetus: interaction with pituitary-adrenocortical function

Oxytocin (OT) stimulates corticotroph function in adult sheep, however, there is little information on OT synthesis and its potential involvement in hypothalamo-pituitary-adrenal (HPA) function in the fetus. The objectives of this study were to examine developmental changes in hypothalamic OT synthesis and to investigate the actions of OT on fetal corticotroph function. Hypothalami were removed at various stages of pre- and post-natal development. OT mRNA levels were measured using in situ hybridization. For in vitro studies, fetal pituitaries were removed on days 129 and 138 of gestation. Anterior pituitary cells were dispersed and cells were treated with different concentrations and combinations of OT, corticotrophin-releasing hormone (CRH), vasopressin (AVP) and cortisol. OT mRNA was present in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) by day 60 of gestation, and levels significantly increased at term. OT mRNA was present in parvocellular and magnocellular fields of the PVN. In vitro, OT stimulated adrenocorticotropin (ACTH) output in a dose-dependent fashion, but had no effect on cellular pro-opiomelanocortin (POMC) mRNA levels. There was no significant difference in corticotroph responsiveness to secretagogues between cells harvested at gestation day 129 or gestation day 138. Simultaneous exposure to CRH and OT stimulated increases in ACTH output that were significantly greater than for OT or CRH alone. However, no similar synergistic interaction existed between OT and AVP. Cortisol attenuated OT-stimulated ACTH output. In conclusion, hypothalamic OT mRNA increases at term and OT can stimulate ACTH output from fetal corticotrophs. Together, these data indicate that OT may be involved in the regulation of ACTH secretion in fetal sheep in late gestation.     

Involvement of Medullary Catecholamine Cells in Neuroendocrine Responses to Systemic Cholecystokinin

Systemic administration of cholecystokinin (CCK) stimulates neurosecretory oxytocin (OT) and tuberoinfundibular corticotrophin releasing factor (CRF) cells of the hypothalamus. Data from previous studies suggest that A2 noradrenergic neurons of the dorsomedial medulla contribute to the OT  cell response, but the role of other medullary catecholamine cells remains unclear. Using c- fos expression as a marker for cellular activity, we have found that CCK (100  μg/kg, ip) activates substantial populations of tyrosine hydroxylase and phenyl-N-methyl-transferase immunoreactive cells in the medulla, consistent with recruitment of overlapped noradrenergic and adrenergic cell populations in both the ventrolateral and dorsomedial medulla. In the ventrolateral medulla there was a particularly prominent activation of C1 adrenergic neurons at the level of the obex. To directly test the contribution of VLM catecholamine cells to hypothalamic neuroendocrine cell responses to CCK, animals were prepared with unilateral VLM lesions corresponding to those areas that had displayed the most marked response to CCK. VLM lesioned animals treated with CCK displayed a significant although small reduction in paraventricular nucleus (PVN) OT  cell c- fos expression ipsilateral to the lesion, but no change in the responses of supraoptic nucleus OT  cells or in cells of the medial parvocellular PVN, many of which are CRF cells. These findings indicate that VLM catecholamine cells make little contribution to hypothalamic neuroendocrine cell responses to CCK and thus serve to further highlight the role of dorsomedial catecholamine cells. However, it is now apparent that, in addition to A2 noradrenergic cells, CCK treatment also recruits C2 adrenergic cells of the dorsomedial medulla, many of which have previously been shown to project to the PVN.     

Acute audiogenic stress-induced activation of CRH neurons in the hypothalamic paraventricular nucleus and catecholaminergic neurons in the medulla oblongata

Strong c-fos expression was induced in neuronal cells of several brain nuclei and the auditory cortex by a short duration auditory stimulation (white noise) in rats. By double immunostaining, Fos-immunoreactive cell nuclei appeared in corticotropin-releasing hormone (CRH)-containing neurons in the hypothalamic paraventricular nucleus, but not in CRH neurons elsewhere in the brain including the central nucleus of the amygdala. Among brain catecholaminergic neurons, only cells in the medulla oblongata (in the A1/C1and A2/C2 cell groups) established double immunostaining for Fos and tyrosine hydroxylase. Sound stimulus in rats with unilateral tympanotomy and plugging the airways resulted in side differences of Fos immunoreactivity in neurons of the auditory pathways and the auditory cortex, but the effect was bilateral in hypothalamic and amygdaloid nuclei. The present data provide evidence for the participation of CRH-synthesizing neurons in hypothalamus and medullary catecholaminergic neurons in the central organization of responses to audiogenic stress stimuli.     

Changing patterns of Fos expression in brainstem catecholaminergic neurons during the rat oestrous cycle

Brainstem catecholaminergic neurons are believed to play an important role in the activation of luteinising hormone-releasing hormone (LHRH) neurons on the afternoon of proestrus which results in the luteinising hormone (LH) surge. To examine the respective roles of brainstem A1 and A2 neurons and the adjoining C1 and C2 adrenergic cells at this time, we have examined the patterns of Fos-immunoreactivity within tyrosine hydroxylase (TH) and phenylethanolamine-N-methyl transferase (PNMT) neurons during diestrus and proestrus. Initial studies demonstrated that the LH surge commenced at approximately 15:00 h in proestrous animals and that peak plasma levels of LH were observed between 16:00 and 17:00 h. Groups of cycling female rats (n = 6) were then perfused between 09:00 and 11:00 (diestrus early) and 18:00 to 19:30 h (diestrus late) on diestrus and at the same times on proestrus (proestrus early and proestrus late). Double-labelling immunocytochemistry revealed little Fos expressions by adrenergic neurons of the C1 or C2 cell groups and this did not change significantly between any of the experimental groups. Analysis of the A2 region was divided into rostral, middle and caudal divisions and all regions showed a significant (P < 0.01) increase in the number of Fos-expressing TH neurons (up to 35% of TH cells) in proestrus early animals compared with diestrus and proestrus late rats. In the A1 region, a significant increase in the number of TH neurons expressing Fos ( 33%) was detected in both proestrus early (P < 0.05) and diestrus early (P < 0.01) rats compared with animals perfused in the late afternoon ( 12%). These results indicate that TH-immunoreactive neurons in both A1 and A2 cell groups are activated on the morning of proestrus prior to the LH surge whilst the C1 and C2 adrenergic neurons express little Fos throughout. The morning increase in Fos-expression by TH neurons within the A1 region on both diestrus and proestrus indicates a circadian pattern of activation for A1 noradrenergic cells and suggests different roles for the A1 and A2 cell groups in regulating the activity of LHRH neurons on proestrus.     

Glucocorticoids down-regulate lipopolysaccharide-induced de novo production of neurotensin mRNA in the rat hypothalamic, paraventricular, corticotrophin-releasing hormone neurons

OBJECTIVE: Intraperitoneal injection of the endotoxin lipopolysaccharide (LPS) produces inflammation accompanied by activation of the immune system and the secretion of cytokines. Cytokines stimulate the hypothalamo-pituitary-adrenal (HPA) axis to release the anti-inflammatory corticosterone which controls its own production by acting on the HPA axis. Upstream in the HPA axis are neuroendocrine corticotrophin-releasing hormone (CRH) neurons located in the paraventricular nucleus (PVN), whose multipeptidergic phenotype changes during inflammation: while CRH mRNA is up-regulated in these conditions, neurotensin (NT) mRNA expression is induced de novo. The negative feedback control of glucocorticoids on CRH production is well documented; however, their action on NT production in the PVN of the hypothalamus is poorly documented. The aim of this study was to determine if glucocorticoids modulate the de novo production of NT during inflammation. METHODS: Using quantitative in situ hybridization histochemistry, we examined whether the absence (adrenalectomy) or excess (corticosterone implants) of glucocorticoids modulate de novo production of NT mRNA in the PVN during inflammation induced by LPS treatment. RESULTS: A relatively low dose of LPS (50 microg/kg) that is not efficient to induce NT mRNA production in the PVN becomes efficient after adrenalectomy. Moreover, corticosterone excess reduces LPS-induced production of NT mRNA in the PVN. CONCLUSION: Glucocorticoids exert a negative control on NT mRNA production in the PVN of the hypothalamus, and this effect requires that NT mRNA production be triggered, such as during inflammation.