1. The circumventricular organs (CVO) are structures that permit polypeptide hypothalamic hormones to leave the brain without disrupting the blood-brain barrier (BBB) and permit substances that do not cross the BBB to trigger changes in brain function. 2. In mammals, CVO include only the median eminence and adjacent neurohypophysis, organum vasculosum lamina terminalis, subfornical organ and the area postrema. 3. The CVO are characterized by their small size, high permeability and fenestrated capillaries. The subcommissural organ is not highly permeable and does not have fenestrated capillaries, but new evidence indicates that it may be involved in the hypertension produced by aldosterone acting on the brain. 4. Feedback control of corticotropin-releasing hormone (CRH) secretion is exerted by free steroids diffusing into the brain, but substances such as cytokines and angiotensin II act on CVO to produce increases in CRH secretion. Gonadal steroids also diffuse into the brain to regulate gonadotrophin-releasing hormone secretion. Thyrotropin-releasing hormone secretion is regulated by thyroid hormones transported across cerebral capillaries. However, CVO may be involved in the negative feedback control of growth hormone and prolactin secretion.

Regions of the amygdala are involved in anticipation of negative events. Chronic anticipation of negative events leads to what we call allostatic load, or arousal pathology. Two hormones appear to be involved in arousal pathology; corticotropin-releasing hormone in the brain and glucocorticoids. We suggest that increases in corticotropin-releasing hormone, by stress or glucocorticoids, in the amygdala may have functional consequences for allostatic load. Whereas, corticotropin-releasing hormone in the parvocellular region of the paraventricular nucleus of the hypothalamus is decreased by glucocorticoids thereby under negative feedback and homeostatic control, the central nucleus of the amygdala is to some extent under positive feedback and is increased by glucocorticoids, and perhaps under allostatic control. The human and animal literature suggest that a variety of psychopathologies (e.g., melancholia) may be tied to neurohormonal signals activating regions of the amygdala.

The corticotropin releasing factor (CRF) family of ligands and their receptors coordinate endocrine, behavioral, autonomic, and metabolic responses to stress and play additional roles within the cardiovascular, gastrointestinal, and other systems. The actions of CRF and the related urocortins are mediated by activation of two receptors, CRF-R1 and CRF-R2, belonging to the B1 family of G protein-coupled receptors. The short-consensus-repeat fold (SCR) within the first extracellular domain (ECD1) of the CRF receptor(s) comprises the major ligand binding site and serves to dock a peptide ligand via its C-terminal segment, thus positioning the N-terminal segment to interact with the receptor's juxtamembrane domains to activate the receptor. Here we present the 3D NMR structure of ECD1 of CRF-R2beta in complex with astressin, a peptide antagonist. In the structure of the complex the C-terminal segment of astressin forms an amphipathic helix, whose entire hydrophobic face interacts with the short-consensus-repeat motif, covering a large intermolecular interface. In addition, the complex is characterized by intermolecular hydrogen bonds and a salt bridge. These interactions are quantitatively weighted by an analysis of the effects on the full-length receptor affinities using an Ala scan of CRF. These structural studies identify the major determinants for CRF ligand specificity and selectivity and support a two-step model for receptor activation. Furthermore, because of a proposed conservation of the fold for both the ECD1s and ligands, this structure can serve as a model for ligand recognition for the entire B1 receptor family.

Cotricotropin-releasing hormone (CRH) and related peptides are produced in skin that is dependent on species and anatomical location. Local peptide production is regulated by ultraviolet radiation (UVR), glucocorticoids and phase of the hair cycle. The skin also expresses the corresponding receptors (CRH-R1 and CRH-R2), with CRH-R1 being the major receptor in humans. CRH-R1 is expressed in epidermal and dermal compartments, and CRH-R2 predominantly in dermal structures. The gene coding for CRH-R1 generates multiple isoforms through a process modulated by UVR, cyclic adenosine monophosphate (cAMP) and phorbol 12-myristate 13-acetate. The phenotypic effects of CRH in human skin cells are largely mediated by CRH-R1alpha through increases in concentrations of cAMP, inositol triphosphate (IP3), or Ca2+ with subsequent activation of protein kinases A (PKA) and C (PKC) dependent pathways. CRH also modulates the activity of nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-kappaB), activator protein 1 (AP-1) and cAMP responsive element binding protein (CREB). The cellular functions affected by CRH depend on cell type and nutritional status and include modulation of differentiation program(s), proliferation, viability and immune activity. The accumulated evidence indicates that cutaneous CRH is also a component of a local structure organized similarly to the hypothalamo-pituitary-adrenal axis.

Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the opioid N/OFQ receptor (NOP), possesses marked functional anti-stress and anti-corticotropin-releasing factor (CRF) actions. We have shown that intracerebroventricular injection of N/OFQ reverses the hypophagic effect induced by stress or by CRF given intracerebroventricularly. To shed new light on the mechanisms involved in the anti-CRF action of N/OFQ, we investigated the ability of N/OFQ to prevent CRF-induced anorexia after microinjection studies into brain areas of potential interest in the control of feeding behavior and coexpressing NOP and CRF receptors. These areas include the bed nucleus of the stria terminalis (BNST), the central amygdala (CeA), the locus ceruleus (LC), the ventromedial hypothalamus (VMH), the paraventricular nucleus (PVN), and the dorsal raphe (DR). The results demonstrated that the anorectic effect of 0.04 nmol of CRF per rat (200 ng per rat) given intracerebroventricularly is reversed by pretreatment with 0.01-0.21 nmol of N/OFQ per rat (25-500 ng per rat) injected into the BNST but not into the CeA, LC, VMH, PVN, or DR. Microinjection of 0.01-0.02 nmol of CRF per site (50-100 ng per site) into the BNST but not into the CeA or the LC induced marked anorexia in food-deprived rats. Pretreatment with 0.01-0.21 nmol of N/OFQ per site (25-500 ng per site) into the BNST also blocked the anorectic action of 0.02 nmol of CRF per site (100 ng per site) given in the same area. Finally, intra-BNST microinjection of 0.01-0.21 nmol of N/OFQ per site (25-500 ng per site) did not modify food intake in either food-sated or food-deprived rats. These data demonstrate that the BNST is involved in the modulation of CRF-induced anorexia, which is prevented by activation of N/OFQ receptors.

In the course of a systematic genomic survey, 22 manic-depressive (bipolar) families were examined for linkage to 11 chromosome 18 pericentromeric marker loci, under dominant and recessive models. Overall logarithm of odds score analysis for the pedigree series was not significant under either model, but several families yielded logarithm of odds scores consistent with linkage under dominant or recessive models. Affected sibling pair analysis of these data yielded evidence for linkage (P < 0.001) at D18S21. Affected pedigree member analysis also suggests linkage, with multilocus results for five loci giving P < 0.0001 and P = 0.0007 for weighting functions f(p) = 1 and 1/square root p, respectively, where p is the allele frequency. These results imply a susceptibility gene in the pericentromeric region of chromosome 18, with a complex mode of inheritance. Two plausible candidate genes, a corticotropin receptor and the alpha subunit of a GTP binding protein, have been localized to this region.

There are well-replicated, independent lines of evidence supporting a role for corticotropin-releasing hormone (CRH) in the pathophysiology of depression. CRH receptor 1 (CRHR1), which we first mapped in the brain in 1994, has been implicated in the treatment of depression and anxiety. We studied the association of CRHR1 genotypes with the phenotype of antidepressant treatment response in 80 depressed Mexican-Americans in Los Angeles who completed a prospective randomized, placebo lead-in, double-blind treatment of fluoxetine or desipramine, with active treatment for 8 weeks. Subjects were included into the study if they had a diagnosis of depression without other confounding medical or psychiatric diagnoses or treatments. All patients were followed weekly and assessed for changes in the Hamilton rating scales for anxiety (HAM-A) and depression (HAM-D). Inclusion criteria in the study included a HAM-D of 18 or higher. Because CRHR1 affects both depression and anxiety. Patients were classified into a high-anxiety (HA) group if their HAM-A score was 18 or higher and in a low-anxiety (LA) group if their HAM-A score was less than 18. Utilizing the haplotype-tag single-nucleotide polymorphisms rs1876828, rs242939 and rs242941, we tested for haplotypic association between CRHR1 and 8-week response to daily antidepressant treatment. In the HA group (n=54), homozygosity for the GAG haplotype was associated with a relative 70% greater reduction in HAM-A scores compared to heterozygous (63.1+/-4.5 vs 37.1+/-6.9%, respectively, P=0.002). For HAM-D, GAG haplotype homozygosity was associated with a 31% greater reduction in scores after treatment compared to heterozygous (67.3+/-4.3 vs 51.2+/-6.0%, respectively, P=0.03). In those with lower-anxiety levels at screening, there were no associations between CRHR1 genotype and percent change in HAM-A or HAM-D. These findings of increased response to antidepressants in highly anxious patients homozygous for the GAG haplotype of CRHR1 need to be independently validated and replicated. Such work would support the hypotheses that response to antidepressant treatment is heterogeneous and that the CRHR1 gene and possibly other genes in stress-inflammatory pathways are involved in response to antidepressant treatment. These findings also suggest that variations in the CRHR1 gene may affect response to CRHR1 agonists or antagonists. All data are deposited in www.pharmgkb.org.

In a companion paper (Ju and Swanson; J. Comp. Neurol. 280:587-602, '89) we described a parcellation scheme for the bed nuclei of the stria terminalis (BST) that was based on cytoarchitectonic criteria. In the work reported here, antisera to the neuropeptides corticotropin-releasing hormone, neurotensin, galanin, substance P, and cholecystokinin were used to determine the extent to which immunostained neuronal cell bodies and presumed terminal fields are correlated with this cytoarchitectonic scheme in the adult male rat. The results confirm the validity of the cytoarchitectonic parcellation and provide additional chemoarchitectonic criteria for determining the (as yet still somewhat arbitrarily defined) border between the BST and the ventrally adjacent preoptic region, for distinguishing between the anterior and posterior divisions of the BST, and for identifying and distinguishing between the particular cell groups or nuclei within each division. The projections of each neuropeptide-containing cell group in various parts of the BST remain to be determined, as do the precise origins of the localized immunoreactive terminal fields identified here.

The stress system orchestrates body and brain responses to the environment. This action exerted by the mediators of the stress system has two modes of operation. The immediate response mode driven by corticotropin-releasing hormone (CRH) organises via CRH-1 receptors the behavioural, sympathetic and hypothalamic-pituitary-adrenal (HPA) responses to a stressor. In the other - slower - mode, which facilitates behavioural adaptation, the urocortins acting through CRH-2 receptors seem prominent. Corticosteroid hormones secreted by the adrenal cortex are implicated in both modes through their high affinity type 1 (mineralocorticoid receptors - MR) and lower affinity type 2 (glucocorticoid receptors - GR) receptors that are co-localised in limbic neural circuitry. Current data suggest that MR controls in specific afferents the threshold or sensitivity of the fast CRH-1 driven stress system mode and thus prevents disturbance of homeostasis, while GR facilitates its recovery by restraining in these very same circuits stress responses and by mobilising energy resources. In preparation for future events GR facilitates behavioural adaptation and promotes storage of energy. The balance in the two stress system modes is thought to be essential for cell homeostasis, mental performance and health. Imbalance induced by genetic modification or chronic stressors changes specific neural signalling pathways underlying psychic domains of cognition and emotion, anxiety and aggression. This Yin-Yang stress concept is fundamental for genomic strategies to understand the mechanistic underpinning of cortisol-induced stress-related disorders such as i.e. severe forms of depression and co-morbid diseases.

Postnatal handling increases glucocorticoid receptor expression in the rat hippocampus, thus altering the regulation of hypothalamic synthesis of corticotropin-releasing hormone and the hypothalamic-pituitary-adrenal response to stress. The effect on glucocorticoid receptor gene expression represents one mechanism by which the early environment can exert a long-term effect on neural development. The handling effect on hippocampal glucocorticoid receptor expression is dependent on peripheral thyroid hormone release and the activation of ascending serotonergic pathways. In primary hippocampal cell cultures, serotonin (5-HT) increases glucocorticoid receptor expression, and this effect appears to be mediated by increased cAMP levels. In the current studies we examined the in vivo effects of handling on hippocampal cAMP-protein kinase A (PKA) activity. In 7-d-old rat pups, we found that (1) postnatal handling increased adenylyl cyclase activity and hippocampal cAMP levels, (2) the effect of handling on cAMP levels was completely blocked by treatment with either propylthiouracil (PTU), a thyroid hormone synthesis inhibitor, or the 5-HT receptor antagonist, ketanserin, and (3) handling also increased hippocampal PKA activity. We then examined the effects of handling on cAMP-inducible transcription factors. Handling rapidly increased levels of the mRNAs for nerve growth factor-inducible factor A (NGFI-A) (zif268, krox24) and activator protein-2 (AP-2) as well as for NGFI-A and AP-2 immunoreactivity throughout the hippocampus. Finally, we found that the effects of handling on NGFI-A and AP-2 expression were significantly reduced by concurrent treatment with either PTU or ketanserin, effects that paralleled those on cAMP formation. NGFI-A and AP-2 have been implicated in the regulation of glucocorticoid receptor expression during development. Thus, these findings suggest that postnatal handling might alter glucocorticoid receptor gene expression via cAMP-PKA pathways involving the activation of NGFI-A and AP-2.

Anorexia nervosa is mostly seen in adolescent females, although the gender-differentiation mechanism is unclear. Corticotropin-releasing factor (CRF), a key peptide for stress responses such as inhibition of food intake, increases in arousal and locomotor activity, and gonadal dysfunction, is thought to be involved in the pathophysiology of anorexia nervosa. CRF in the paraventricular nucleus of the hypothalamus (PVN) and CRF in the central nucleus of the amygdala (CeA) are involved in the regulation of stress responses, and gender differences in CRF mRNA expression in these regions in response to various stressors are controversial. We therefore examined CRF gene expression in the PVN and CeA as well as corticotropin (ACTH) and corticosterone secretion in response to a 60-min period of electric footshock (FS) or psychological stress (PS) induced by a communication box in both male and female rats in proestrus or diestrus in an effort to elucidate the mechanism underlying the gender difference in the activity of the hypothalamic-pituitary-adrenal (HPA) axis and the mechanism underlying the remarkable prevalence of anorexia nervosa in females. Female rats in proestrus showed higher basal plasma ACTH and CRF mRNA expression levels in the PVN and CeA than males. Females more rapidly showed higher plasma ACTH and corticosterone levels and a higher CRF mRNA expression level in the PVN in response to FS than males. Although females in both proestrus and diestrus showed significant increases in plasma ACTH and corticosterone and CRF mRNA expression in the PVN in response to PS, no significant responses of the HPA axis to PS were found in males. FS significantly increased CRF mRNA expression in the CeA in both females and males, with significantly higher peaks in females in proestrus than in males, while PS significantly increased CRF mRNA expression in the CeA only in males. These results suggest that gender affects differentially the function of the stress-related regions such as the PVN and CeA. The finding that CRF gene expression in the PVN responds to PS only in females may be a clue to elucidation of the neurobiological mechanism underlying the gender-differential prevalence of anorexia nervosa.

The amygdala is a key limbic area involved in fear responses and pavlovian conditioning with the potential to directly respond to endocrine signals associated with fear or stress. To gain insights into the molecular mechanisms and subregional specificity of fear conditioning, we disrupted type II glucocorticoid receptors (GRs) in the central nucleus of the amygdala (CeA) by delivering lentiviral vectors containing Cre-recombinase into floxed-GR mice. GR deletion in the CeA (CeAGRKO mice) prevented conditioned fear behavior. In contrast, forebrain disruption of GRs excluding the CeA did not. The conditioned fear deficit in CeAGRKO mice was associated with decreases in cFos and corticotropin-releasing hormone (CRH) expression. Moreover, intracerebroventricular delivery of CRH rescued the conditioned fear deficit in CeAGRKO mice. We conclude that fear conditioning involves a neuroendocrine circuit by using GR activation in the CeA for acute CRH induction and long-lasting behavioral modulation.

In abstinent alcoholics, stress induces negative affect-a response linked to craving and relapse. In rats, repeated stresses at weekly intervals before 5-day ethanol diet sensitize withdrawal-induced anxiety-like behavior ("anxiety") that is blocked by a corticotrophin-releasing factor 1 (CRF-1)-receptor antagonist. Current experiments were performed to identify brain sites that support CRF involvement in stress sensitization of ethanol withdrawal-induced anxiety-like behavior. First, different doses of CRF microinjected weekly into the central amygdala (CeA) before ethanol exposure produced a dose-related sensitization of anxiety during ethanol withdrawal. Subsequently, CRF microinjection into the basolateral amygdala, dorsal raphe nucleus (DRN), or dorsal bed nucleus of the stria terminalis (d-BNST) also sensitized ethanol withdrawal-induced anxiety. In contrast, sensitization of ethanol withdrawal-induced anxiety was not observed after weekly CRF administration into the ventral-BNST, CA1-hippocampal region, or hypothalamic-paraventricular nucleus. Then, experiments documented the CRF receptor subtype responsible for CRF and stress sensitization of withdrawal-induced anxiety. Systemic administration of a CRF-1 receptor antagonist before CRF microinjection into the CeA, DRN, or d-BNST prevented CRF-induced sensitization of anxiety during ethanol withdrawal. Furthermore, repeated microinjections of urocortin-3, a CRF-2 receptor agonist, into the CRF-positive sites did not sensitize anxiety after withdrawal from ethanol. Finally, microinjection of a CRF-1 receptor antagonist into the CeA, DRN, or d-BNST before stress blocked sensitization of anxiety-like behavior induced by the repeated stress/ethanol withdrawal protocol. These results indicate that CRF released by stress acts on CRF-1 receptors within specific brain regions to produce a cumulative adaptation that sensitizes anxiety-like behavior during withdrawal from chronic ethanol exposure.

Stress-related psychiatric disorders such as anxiety and depression involve dysfunction of the serotonin [5-hydroxytryptamine (5-HT)] system. Previous studies have found that the stress neurohormone corticotropin-releasing factor (CRF) inhibits 5-HT neurons in the dorsal raphe nucleus (DRN) in vivo. The goals of the present study were to characterize the CRF receptor subtypes (CRF-R1 and -R2) and cellular mechanisms underlying CRF-5-HT interactions. Visualized whole-cell patch-clamp recording techniques in brain slices were used to measure spontaneous or evoked GABA synaptic activity in DRN neurons of rats and CRF effects on these measures. CRF-R1 and -R2-selective agonists were bath applied alone or in combination with receptor-selective antagonists. CRF increased presynaptic GABA release selectively onto 5-HT neurons, an effect mediated by the CRF-R1 receptor. CRF increased postsynaptic GABA receptor sensitivity selectively in 5-HT neurons, an effect to which both receptor subtypes contributed. CRF also had direct effects on DRN neurons, eliciting an inward current in 5-HT neurons mediated by the CRF-R2 receptor and in non-5-HT neurons mediated by the CRF-R1 receptor. These results indicate that CRF has direct membrane effects on 5-HT DRN neurons as well as indirect effects on GABAergic synaptic transmission that are mediated by distinct receptor subtypes. The inhibition of 5-HT DRN neurons by CRF in vivo may therefore be primarily an indirect effect via stimulation of inhibitory GABA synaptic transmission. These results regarding the cellular mechanisms underlying the complex interaction between CRF, 5-HT, and GABA systems could contribute to the development of novel treatments for stress-related psychiatric disorders.

Epidemiological studies have implicated stress (psychosocial and physical) as a trigger of first onset or exacerbation of irritable bowel syndrome (IBS) symptoms of which visceral pain is an integrant landmark. A number of experimental acute or chronic exteroceptive or interoceptive stressors induce visceral hyperalgesia in rodents although recent evidence also points to stress-related visceral analgesia as established in the somatic pain field. Underlying mechanisms of stress-related visceral hypersensitivity may involve a combination of sensitization of primary afferents, central sensitization in response to input from the viscera and dysregulation of descending pathways that modulate spinal nociceptive transmission or analgesic response. Biochemical coding of stress involves the recruitment of corticotropin releasing factor (CRF) signaling pathways. Experimental studies established that activation of brain and peripheral CRF receptor subtype 1 plays a primary role in the development of stress-related delayed visceral hyperalgesia while subtype 2 activation induces analgesic response. In line with stress pathways playing a role in IBS, non-pharmacologic and pharmacologic treatment modalities aimed at reducing stress perception using a broad range of evidence-based mind-body interventions and centrally-targeted medications to reduce anxiety impact on brain patterns activated by visceral stimuli and dampen visceral pain.

Though much is known about the cellular and molecular components of the circadian clock, output pathways that couple clock cells to overt behaviors have not been identified. We conducted a screen for circadian-relevant neurons in the Drosophila brain and report here that cells of the pars intercerebralis (PI), a functional homolog of the mammalian hypothalamus, comprise an important component of the circadian output pathway for rest:activity rhythms. GFP reconstitution across synaptic partners (GRASP) analysis demonstrates that PI cells are connected to the clock through a polysynaptic circuit extending from pacemaker cells to PI neurons. Molecular profiling of relevant PI cells identified the corticotropin-releasing factor (CRF) homolog, DH44, as a circadian output molecule that is specifically expressed by PI neurons and is required for normal rest:activity rhythms. Notably, selective activation or ablation of just six DH44+ PI cells causes arrhythmicity. These findings delineate a circuit through which clock cells can modulate locomotor rhythms.

Drug addiction is a chronically relapsing disorder characterized by loss of control over intake and dysregulation of stress-related brain emotional systems. Since the discovery by Wylie Vale and his colleagues of corticotropin-releasing factor (CRF) and the structurally-related urocortins, CRF systems have emerged as mediators of the body's response to stress. Relatedly, CRF systems have a prominent role in driving addiction via actions in the central extended amygdala, producing anxiety-like behavior, reward deficits, excessive, compulsive-like drug self-administration and stress-induced reinstatement of drug seeking. CRF neuron activation in the medial prefrontal cortex may also contribute to the loss of control. Polymorphisms in CRF system molecules are associated with drug use phenotypes in humans, often in interaction with stress history. Drug discovery efforts have yielded brain-penetrant CRF1 antagonists with activity in preclinical models of addiction. The results support the hypothesis that brain CRF-CRF1 systems contribute to the etiology and maintenance of addiction.

Multiple neurochemical pathways are involved in mediating craving and relapse to alcohol. Opioidergic and glutamatergic systems have a key role in alcoholism, as demonstrated by the clinically effective compounds naltrexone and acamprosate acting through these systems. The dopaminergic system has long featured in alcoholism research; hitherto disappointing results from clinical studies could improve following the discovery that dopamine D3 receptor antagonism produces consistent and robust results in preclinical studies. Corticotropin-releasing factor signalling and the endocannabinoid system integrate stress-related events and thereby mediate relapse behaviour. Overall, these new targets have yielded several compounds that are undergoing clinical testing. However, the heterogeneity in treatment response makes it necessary to characterize genetic and protein markers and endophenotypes for individualized pharmacotherapy.

BACKGROUND

Hypocretin (Hcrt), an arousal- and feeding-associated peptide, is expressed in lateral hypothalamic neurons that project to the ventral tegmental area (VTA). Intra-VTA Hcrt reinstates morphine-conditioned place preferences, and intracerebroventricular and intra-VTA corticotropin-releasing factor (CRF) reinstate cocaine seeking. Each is presumed to act, at least in part, through actions local to the VTA. Here, we examined the possibility that VTA perfusion of Hcrt reinstates cocaine seeking and, if so, whether it does so through the VTA mechanism that is implicated in reinstatement by CRF.

METHODS

Rats were trained to lever-press for intravenous cocaine (2 weeks) and then underwent extinction training (saline substituted for cocaine: 3 weeks). Reinstatement behavior was tested and VTA dialysates were collected and assayed for glutamate or dopamine following footshock or perfusion of Hcrt or CRF, with or without Hcrt or CRF antagonists, into the VTA.

RESULTS

Ventral tegmental area perfusion of Hcrt-1 or footshock stress reinstated cocaine seeking and caused release of VTA glutamate and dopamine. The effects of Hcrt-1 were blocked by a selective Hcrt-1 antagonist, but not a CRF antagonist, and were not mimicked by Hcrt-2. The Hcrt-1 antagonist did not block CRF-dependent footshock-induced reinstatement or glutamate or dopamine release. The behavioral and neurochemical effects of Hcrt-1 were attenuated but not blocked by kynurenic acid, an ionotropic glutamate antagonist that blocks footshock-induced reinstatement and glutamate release.

CONCLUSIONS

While Hcrt and CRF are known to interact in some area of the brain, in the VTA proper they appear to have largely independent actions on the mesolimbic dopamine mechanisms of cocaine seeking.

In order to assess the effect of interleukin-6 on the hypothalamo-pituitary-adrenal axis, we administered recombinant human interleukin-6 to conscious, freely-moving rats. The intravenous injection of interleukin-6 significantly increased the plasma level of adrenocorticotropic hormone 30 min after the injection in a dose-related manner. Immunoneutralization of corticotropin-releasing hormone blocked the stimulatory effects of interleukin-6 on adrenocorticotropic hormone secretion. These observations suggest that interleukin-6 stimulates the secretion of adrenocorticotropic hormone through the corticotropin-releasing hormone and is possibly involved in the interaction between the neuroendocrine and immune system.

Corticotropin-releasing factor (CRF), when administered directly into the CNS, can have activating properties on behaviour and can enhance behavioural responses to stress. CRF injected intraventricularly produces a dose-dependent increase in locomotor activity and increased responsiveness to an acoustic startle stimulus. However, this profile of activation changes to enhanced suppression of behaviour in stressful situations and includes increased freezing, increased conditioned suppression, increased conflict, decreased feeding and decreased behaviour in a novel open field. These effects of CRF are independent of the pituitary-adrenal axis and can be reversed by the CRF antagonist alpha-helical CRF(9-41). More importantly, the CRF antagonist can also reverse many behavioural responses to stressors. alpha-Helical CRF(9-41) reverses stress-induced fighting behaviour, stress-induced freezing, stress-induced suppression of feeding, stress-induced decreases in exploration of an elevated plus maze, fear-potentiated startle and the development of conditioned suppression. Intracerebral microinjections suggest that the amygdala may be an important site for the anti-stress effects of alpha-helical CRF(9-41). These results suggest that endogenous CRF systems in the CNS may have a role in mediating behavioural responses to stress and further suggest that CRF in the brain may function as a fundamental behavioural activating system. This CRF system may be particularly important in situations where an organism must mobilize not only the pituitary-adrenal system but also the CNS in response to environmental challenge.

The neuropeptides orexin-A and orexin-B are produced in neurons of the lateral hypothalamic area and have been implicated to be involved in the regulation of food/water intake and sleep-wake control. The orexins act at two different G-protein-coupled orexin receptors (OX-R1 and OX-R2) that are derived from separate genes and expressed differentially throughout the central nervous system. In the present study, we have used a polyclonal antipeptide antiserum to analyse in detail the distribution of OX-R1-immunoreactive neurons in the rat hypothalamus. In order to identify the chemical mediators of orexin action in the hypothalamus, the OX-R1-containing neurons were characterized with regard to the content of peptides shown previously to affect ingestive and drinking behaviour. Neurons containing OX-R1 immunoreactivity were widely distributed in the hypothalamus with cell bodies located in the suprachiasmatic, periventricular, paraventricular (both magno- and parvocellular division), supraoptic, arcuate, ventromedial, dorsomedial and tuberomammillary nuclei and the lateral hypothalamic area. In magnocellular neurons of the paraventricular and supraoptic nuclei, OX-R1 immunoreactivity was seen in both vasopressin- and oxytocin-containing neurons. OX-R1 immunoreactivity was demonstrated in vasopressin and vasoactive intestinal polypeptide (VIP) neurons of the suprachiasmatic nucleus, in somatostatin neurons of the periventricular nucleus and in corticotropin-releasing hormone (CRH) neurons of the parvocellular paraventricular nucleus. In the arcuate nucleus, OX-R1 immunoreactivity was present in neuropeptide Y (NPY) and agouti-related peptide (AGRP) neurons of the ventromedial part as well as in proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) neurons of the ventrolateral division. In the lateral hypothalamic area, OX-R1 immunoreactivity was demonstrated in melanin-concentrating hormone (MCH)- and orexin-containing neurons. In the hypothalamic tuberomammillary nucleus, OX-R1-immunoreactivity was shown in many histamine-containing neurons. The results support the idea that orexins have important actions on hypothalamic neurons that control food intake and fluid balance, but also that orexins may regulate other neuroendocrine systems.

The corticotropin-releasing factor (CRF) receptors (CRF1 and CRF2) are crucial mediators of physiological and behavioral responses to stress. In animals, CRF1 appears to primarily mediate CRF-induced anxiety-like responses, but the role of CRF2 during stress is still unclear. Here we report the effects of CRF1 and CRF2 on the magnitude and plasticity of defensive startle responses in mice. Startle plasticity is measured by inhibition of startle by sensory stimuli, i.e., prepulse inhibition (PPI), and is disrupted in patients with panic or posttraumatic stress disorders in which CRF neurotransmission may be overactive. Pharmacological blockade of CRF1 reversed both CRF-induced increases in startle and CRF-induced deficits in PPI. CRF2 blockade attenuated high-dose but not low-dose CRF-induced increases in startle and reduced PPI. Conversely, activation of CRF2 enhanced PPI. CRF had no effect on startle and increased PPI in CRF1 knock-out mice. These data indicate that CRF receptors act in concert to increase the magnitude of defensive startle yet in opposition to regulate the flexibility of startle. These data support a new model of respective CRF receptor roles in stress-related behavior such that, although both receptors enhance the magnitude of defensive responses, CRF1 receptors contravene, whereas CRF2 receptors enhance, the impact of sensory information on defensive behavior. We hypothesize that excessive CRF1 activation combined with reduced CRF2 signaling may contribute to information processing deficits seen in panic and posttraumatic stress disorder patients and support CRF1-specific pharmacotherapy.