The role of the anteriolateral bed nucleus of the stria terminalis in stress-induced nociception

Activation of the central amygdala (CeA) by corticosterone (CORT) induces somatic and colonic hypersensitivity through corticotrophin-releasing factor (CRF)-dependent mechanisms. However, the importance of the bed nucleus of the stria terminalis (BNST), part of the extended amygdala, on nociception remains unexplored. In the present study, we test the hypothesis that stimulation of the CeA by CORT induces somatic and colonic hypersensitivity through activation of the anteriolateral BNST (BNST(AL)). Animals were implanted with micropellets of CORT or cholesterol (CHOL) onto the CeA or the BNST(AL). Mechanical sensitivity was quantified using electronic von Frey filaments, and colonic nociception was measured by quantifying a visceromotor response to graded colorectal distension. In situ hybridization was used to determine mRNA levels for CRF, CRF(1), and CRF(2) receptors in the BNST(AL). In a second group, animals were implanted bilaterally with 1) CORT or CHOL micropellets onto the CeA; and 2) cannulas localized to the BNST(AL) to administer a CRF(1) receptor antagonist (CP376395). Animals implanted with CORT onto the CeA, but not the BNST(AL), exhibited increased expression of CRF mRNA and increased CRF(1)-to-CRF(2) receptor ratio in the BNST, as well as somatic and colonic hypersensitivity compared with CHOL controls. Infusion of CP376395 into the BNST(AL) inhibited somatic and colonic hypersensitivity in response to elevated amygdala CORT. Somatic and colonic hypersensitivity induced by elevated amygdala CORT is mediated via a CRF(1) receptor-dependent mechanism in the BNST(AL). The CeA through a descending pathway involving the BNST(AL) plays a pivotal role in somatic and colonic nociception.     

Urocortin 1 reduces food intake and ghrelin secretion via CRF(2) receptors

Although it is known that urocortin 1 (UCN) acts on both corticotropin-releasing factor receptors (CRF(1) and CRF(2)), the mechanisms underlying UCN-induced anorexia remain unclear. In contrast, ghrelin, the endogenous ligand for the growth hormone secretagogue receptor, stimulates food intake. In the present study, we examined the effects of CRF(1) and CRF(2) receptor antagonists (CRF(1)a and CRF(2)a) on ghrelin secretion and synthesis, c-fos mRNA expression in the caudal brain stem, and food intake following intracerebroventricular administration of UCN. Eight-week-old, male Sprague-Dawley rats were used after 24-h food deprivation. Acylated and des-acylated ghrelin levels were measured by enzyme-linked immunosorbent assay. The mRNA expressions of preproghrelin and c-fos were measured by real-time RT-PCR. The present study provided the following important insights into the mechanisms underlying the anorectic effects of UCN: 1) UCN increased acylated and des-acylated ghrelin levels in the gastric body and decreased their levels in the plasma; 2) UCN decreased preproghrelin mRNA levels in the gastric body; 3) UCN-induced reduction of plasma ghrelin and food intake were restored by CRF(2)a but not CRF(1)a; 4) UCN-induced increase of c-fos mRNA levels in the caudal brain stem containing the nucleus of the solitary tract (NTS) was inhibited by CRF(2)a; and 5) UCN-induced reduction of food intake was restored by exogenous ghrelin and rikkunshito, an endogenous ghrelin secretion regulator. Thus, UCN increases neuronal activation in the caudal brain stem containing NTS via CRF(2) receptors, which may be related to UCN-induced inhibition of both ghrelin secretion and food intake.     

Urocortins and corticotropin releasing factor type 2 receptors in the hypothalamus and the cardiovascular system

In addition to urocortin (Ucn I), Ucn II and Ucn III were identified as endogenous ligands for corticotropin-releasing factor type 2 receptor (CRF2 receptor). CRF2 receptor is abundantly located in central hypothalamic ventromedial nucleus (VMH) and in peripheral cardiovascular system. In this mini-review, we focused on the roles of these urocortins and CRF2 receptor in the hypothalamus and the cardiovascular system. Ucn II mRNA was increased in the parvocellular part or the magnocellular part of the hypothalamic paraventricular nucleus (PVN) following immobilization stress or 3 days of water deprivation, respectively. Therefore, it is thought that Ucn II may modulate CRF and vasopressin synthesis in the PVN in a paracrine or autocrine fashion through PVN CRF2 receptor. The early and later phases of Ucn I-mediated feeding suppression may be CRF1 and CRF2 receptor-mediated events, respectively. Ucn II decreases food intake at a later phase, beyond 4 h post injection. A large dose of corticosterone increased plasma leptin and insulin levels as well as the levels of CRF2 receptor mRNA. Adrenalectomy, starvation, and immobilization each lowered plasma leptin and insulin levels and were associated with decrements in CRF2 receptor mRNA levels in the VMH. Peripheral injection of leptin increased VMH CRF2 receptor mRNA, as can induce reductions of food intake and body weight, indicating that circulating leptin is involved in the regulation of VMH CRF2 receptor mRNA expression. Therefore, it is also plausible that VMH CRF2 receptor transduces the anorexogenic effects of leptin as well as those of urocortins. The systemic administration of Ucn II decreases mean arterial pressure (arterial vascular tone) and causes tachycardia via vascular CRF2 receptor in rats, similar to the effects of Ucn I. Thus, CRF2 receptor seems to mediate cardioprotective effects of urocortins.     

The influence of CRF and alpha-helical CRF(9-41) on rat fear responses, c-Fos and CRF expression, and concentration of amino acids in brain structures

In the present study we have examined the influence of intracerebroventricullary administered CRF, and a non-selective CRF receptor antagonist, α-helical CRF_(9–41), on rat conditioned fear response, serum corticosterone, c-Fos and CRF expression, and concentration of amino acids (in vitro), in several brain structures. Pretreatment of rats with CRF in a dose of 1μg/rat, enhanced rat-freezing response, and further increased conditioned fear-elevated concentration of serum corticosterone. Moreover, exogenous CRF increased aversive context-induced expression of c-Fos in the parvocellular neurons of the paraventricular hypothalamic nucleus (pPVN), CA1 area of the hippocampus, and M1 area of the frontal cortex. A different pattern of behavioral and biochemical changes was present after pre-test administration of α-helical CRF_(9–41) (10μg/rat): a decrease in rat fear response and serum corticosterone concentration; an attenuation of fear-induced c-Fos expression in the dentate gyrus, CA1, Cg1, Cg2, and M1 areas of the frontal cortex; a complete reversal of the rise in the number of CRF immunoreactive complexes in the M2 cortical area, induced by conditioned fear. Moreover, α-helical CRF_(9–41) increased the concentration of GABA in the amygdala of fear-conditioned rats. Altogether, the present data confirm and extend previous data on the integrative role of CRF in the central, anxiety-related, behavioral and biochemical processes. The obtained results underline also the role of frontal cortex and amygdala in mediating the effects of CRF on the conditioned fear response.     

Differential distribution and regulation of OX1 and OX2 orexin/hypocretin receptor messenger RNA in the brain upon fasting

To further understand the functions of the orexin/hypocretin system, we examined the expression and regulation of the orexin/hypocretin receptor (OX1R and OX2R) mRNA in the brain by using quantitative in situ hybridization. Expression of OX1R and OX2R mRNA exhibited distinct distribution patterns. Within the hypothalamus, expression for the OX1R mRNA was largely restricted in the ventromedial (VMH) and dorsomedial hypothalamic nuclei, while high levels of OX2R mRNA were contained in the paraventricular nucleus, VMH, and arcuate nucleus as well as in mammilary nuclei. In the amygdala, OX1R mRNA was expressed throughout the amygdaloid complex with robust labeling in the medial nucleus, while OX2R mRNA was only present in the posterior cortical nucleus of amygdala. High levels of OX2R mRNA were also observed in the ventral tegmental area. Moreover, both OX1R and OX2R mRNA were observed in the hippocampus, some thalamic nuclei, and subthalamic nuclei. Furthermore, we analyzed the effect of fasting on levels of OX1R and OX2R mRNA in the hypothalamic and amygdaloid subregions. After 20 h of fasting, levels of OX1R mRNA were significantly increased in the VMH and the medial division of amygdala. An initial decrease (14 h) and a subsequent increase (20 h) in OX1R mRNA levels after fasting were observed in the dorsomedial hypothalamic nucleus and lateral division of amygdala. Levels of OX2R mRNA were augmented in the arcuate nucleus, but remained unchanged in the dorsomedial hypothalamic nucleus, paraventricular hypothalamic nucleus, and amygdala following fasting. The time-dependent and region-specific regulatory patterns of OX1R and OX2R suggest that they may participate in distinct neural circuits under the condition of food deprivation.     

CRF receptor type 1 mediates continual hypoxia-induced CRF peptide and CRF mRNA expression increase in hypothalamic PVN of rats

We demonstrated previously that hypoxia activated CRF and CRF mRNA in PVN, and CRF receptor 1 (CRFR1) mRNA in rat pituitary. The aim of the study is to test whether the hypoxia-activated CRF and CRF mRNA is associated with triggering CRFR1. Rats were exposed to hypobaric hypoxia at altitude of 2 and 5 km. CRF and CRF mRNA were assayed by immunostaining and in situ hybridization. CRFR1 mRNA was assayed by RT-PCR. Results showed that 5 km continual hypoxia increased CRF and CRF mRNA in PVN, CRFR1 mRNA in pituitary, and plasma corticosterone. The hypoxia-increased CRF, CRF mRNA, CRFR1 mRNA, and corticosterone were blocked by CRFR1 antagonist (CP-154,526), suggesting that CRFR1 in PVN and pituitary are responsible for the hypoxia-increased CRF and CRF mRNA in PVN.     

Age-specific threats induce CRF expression in the paraventricular nucleus of the hypothalamus and hippocampus of young rats

Young animals respond to threatening stimuli in an age-specific way. Their endocrine and behavioral responses reflect the potential threat of the situation at a given age. The aim of the present study was to determine whether corticotropin-releasing factor (CRF) is involved in the endocrine and behavioral responses to threat and their developmental changes in young rats. Preweaning 14-day-old and postweaning 26-day-old rats were exposed to two age-specific threats, cat odor and an adult male rat. The acute behavioral response was determined during exposure. After exposure, the time courses of the corticosterone response and of CRF expression in the paraventricular nucleus of the hypothalamus (PVN) and in extrahypothalamic areas were assessed. Preweaning rats became immobile when exposed to cat odor or the male rat, whereas postweaning rats became immobile to cat odor only. Male exposure increased serum corticosterone levels in 14-day-old rats, but cat odor failed to increase levels at either age. Exposure induced elevation of CRF mRNA levels in the PVN that paralleled changes in corticosterone levels. CRF may thus play a role in endocrine regulation and its developmental changes during early life. Neither cat odor nor the adult male altered CRF mRNA levels in the bed nucleus of the stria terminalis (BNST) or the amygdala, but both stimuli increased levels in the hippocampus. Hippocampal CRF mRNA expression levels did not parallel cat odor or male-induced immobility, indicating that CRF is not involved in this response in young rats but may be involved in aspects of learning and memory.     

Modulation of Ca2+ influx by corticotropin-releasing factor (CRF) family of peptides via CRF receptors in rat pancreatic beta-cells

The actions of the corticotropin-releasing factor (CRF) family of peptides are mediated by the seven transmembrane-domain G-protein-coupled receptors, the CRF receptors type 1 (CRF1 receptor) and type 2 (CRF2 receptor). In a previous study, we reported that CRF, an endogenous ligand for CRF1 receptor, modulated Ca2+ influx in rat pancreatic beta-cells. In addition to CRF, other additional members of the family, urocortins, have been identified in mammals. Urocortin 1 (UCN 1), a peptide of the CRF family, binds both CRF1 receptor and CRF2 receptor with equal affinities. Urocortin 3 (UCN 3), a highly selective ligand for CRF2 receptor with little affinity for CRF1 receptor, has been shown in rat pancreatic beta-cells. The present study focused on the effects of the CRF family peptides on intracellular Ca2+ ([Ca2+]i) concentration via CRF receptors in rat pancreatic beta-cells. Microfluorimetric experiments showed that CRF (0.2 nM) and UCN 1 (0.2 nM) elevated [Ca2+]i levels. Both CRF and UCN 1 effects were attenuated by astressin, a non-selective CRF receptor antagonist. Antisauvagine-30, a selective CRF2 receptor antagonist, appeared to enhance the UCN 1 effect on the elevation of [Ca2+]i. The CRF effect on the elevation of [Ca2+]i was inhibited by the addition of UCN 3. Taken together, the activation of CRF2 receptor antagonizes the CRF1 receptor-stimulated Ca2+ influx.     

N-methyl-D-aspartate (NMDA)-mediated corticotropin-releasing factor (CRF) release in cultured rat amygdala neurons

Corticotropin-releasing factor (CRF) plays an important role in the activation of centrally mediated responses to stress. The amygdala, a limbic structure involved in the stress response, has a significant number of CRF cell bodies and CRF receptors. Activation of glutamatergic projections to the amygdala has been implicated in the stress response. Few studies have evaluated neurotransmitter-stimulated CRF release in the amygdala. We measured the effects of glutamate (0.1-1000 microM) and N-methyl-D-aspartate (NMDA, 0.1-1000 microM) on CRF release from the amygdala using primary neuronal cultures from embryonic rat brains (E18-19). Experiments were performed after the cultures grew for 17-20 days. CRF was measured using radioimmunoassay. The excitatory amino acid neurotransmitters, glutamate and NMDA, stimulated CRF release in a concentration-dependent manner. The apparent EC50 values for glutamate and NMDA were 17.5 microM and 12 microM, respectively. Consistent with a NMDA receptor-driven event, glutamate-stimulated CRF release was blocked by the NMDA antagonist, 2-amino-5-phosphonovaleric acid (AP-5, 1-100 microM) and antagonized by the addition of 1.2 mM MgCl2 to the incubation medium. These results implicate an inhibition of CRF release in the amygdala as a possible mechanism for the reported anxiolytic effects of NMDA antagonists.     

阿片受体样受体ORL1基因在大鼠脑内的表达

我们采用地主辛标记的寡聚核苷酸探针和原位杂产技术研究了新发现的阿片受体样受体ＯＲＬ１基因在正常大鼠脑仙的表达和分布。发现ＯＲＬ１基因在大鼠脑内广泛表达。尤其在大脑皮层、丘脑、下丘脑、杏仁核、海马、隔核、缰核、导水管周围灰质、中缝核群及蓝斑等区域。提示ＯＲＬ１除参与痛觉调制外,还可能参与脑内多项生理功能的调控。     

The effects of corticotropin-releasing factor and the urocortins on hypothalamic gamma-amino butyric acid release--the impacts on the hypothalamic-pituitary-adrenal axis

Corticotropin-releasing factor (CRF) and the urocortins (UCNs) are structurally and pharmacologically related neuropeptides which regulate the endocrine, autonomic, emotional and behavioral responses to stress. CRF and UCN1 activate both CRF receptors (CRFR1 and CRFR2) with CRF binding preferentially to CRFR1 and UCN1 binding equipotently to both receptors. UCN2 and UCN3 activate selectively CRFR2. Previously an in vitro study demonstrated that superfusion of both CRF and UCN1 elevated the GABA release elicited by electrical stimulation from rat amygdala, through activation of CRF1 receptors. In the present experiments, the same in vitro settings were used to study the actions of CRF and the urocortins on hypothalamic GABA release. CRF and UCN1 administered in equimolar doses increased significantly the GABA release induced by electrical stimulation from rat hypothalamus. The increasing effects of CRF and UCN1 were inhibited considerably by the selective CRFR1 antagonist antalarmin, but were not influenced by the selective CRFR2 antagonist astressin 2B. UCN2 and UCN3 were ineffective. We conclude that CRF1 receptor agonists induce the release of GABA in the hypothalamus as well as previously the amygdala. We speculate that CRF-induced GABA release may act as a double-edged sword: amygdalar GABA may disinhibit the hypothalamic CRF release, leading to activation of the hypothalamic-pituitary-adrenal axis, whereas hypothalamic GABA may inhibit the hypothalamic CRF release, terminating this activation.     

The role of corticotropin-releasing factor receptors in stress and anxiety

Corticotropin releasing factor (CRF) is a critical integratorof the hypothalamic-pituitary-adrenal (HPA) axis in responseto stress. CRF and its related molecule urocortin (UCN) bindCRF receptor 1 (CRFR1) and CRFR2 with distinct affinities. Micedeficient for CRFR1 or CRFR2 were generated in order to determinethe physiological role of these receptors. While CRFR1-mutantmice show a depleted stress response and display anxiolytic-likebehavior, CRFR2-mutant mice are hypersensitive to stress anddisplay anxiogenic-like behavior. Both CRFR1- and CRFR2-mutantmice show normal basal feeding and weight gain, but CRFR2-mutantmice exhibit decreased food intake following a stress of fooddeprivation. While CRFR2-mutant mice display increased levelsof CRF mRNA in the central nucleus of the amygdala (cAmyg) butnot in the paraventricular nucleus of the hypothalamus (PVN),the CRFR1-mutant mice express high levels of CRF in the PVNbut normal levels in the cAmyg. CRFR2-mutant mice also displayincreased levels of Ucn mRNA and protein in the edinger westphalnucleus (EW) as well as an increased number of cells expressingUcn. The levels of these CRF-receptor ligands reflect the stateof the receptor-deficient mice. These results demonstrate apossible modulatory function of CRFR2 in response to CRFR1 stimulationof the HPA axis or anxiety.     

Role of corticotropin-releasing factor receptor-1 in opiate withdrawal

Previous studies indicate that corticotropin-releasing factor (CRF) contributes to the anxiety-like and aversive states associated with drug-induced withdrawal. The present study extends this work by analyzing the CRF receptor subtype involved in withdrawal responses. First, the influence of a selective CRF receptor-1 (CRF-R1) antagonist, CP-154,526, on opiate withdrawal behavior was examined. Pretreatment with the CRF-R1 antagonist significantly attenuated several behavioral signs of naltrexone-induced morphine withdrawal, including writhing, chewing, weight loss, lacrimation, salivation, and irritability, measured during the first hour of withdrawal. Next the expression of CRF-R1 was determined as a second measure of the involvement of this receptor in opiate withdrawal. Naltrexone-induced morphine withdrawal resulted in down-regulation of CRF-R1 mRNA in several brain regions, including the frontal cortex, parietal cortex, striatum, nucleus accumbens, and amygdala, but not in the hypothalamus or periaqueductal gray. Expression of CRF-R2, the other major CRF receptor subtype, was not down-regulated significantly by withdrawal in any of the regions examined, although morphine alone significantly increased levels of this receptor subtype. Taken together, the behavioral and receptor regulation findings indicate that CRF-R1 is the primary mediator of the actions of the CRF system on opiate withdrawal, although it is possible that CRF-R2 contributes to the response.