Many skin disorders, such as atopic dermatitis and psoriasis, worsen during stress and are associated with increased numbers and activation of mast cells which release vasoactive, nociceptive, and proinflammatory mediators. Nontraumatic acute psychological stress by immobilization has been shown to induce mast cell degranulation in the rat dura and colon. Moreover, intradermal injection of corticotropin-releasing hormone (CRH) or its analogue urocortin (10(-5)-10(-7) M) induced skin mast cell degranulation and increased vascular permeability. Here, we investigated the effect of acute immobilization stress on skin mast cell degranulation by light microscopy and electron microscopy. Immobilization for 30 min resulted (P < 0.05) in degranulation of 40.7 +/- 9.1% of skin mast cells compared to 22.2 +/- 7.3% in controls killed by CO(2) or 17.8 +/- 2.4% in controls killed by pentobarbital. Pretreatment intraperitoneally (ip) with antiserum to CRH for 60 min prior to stress reduced (P < 0.05) skin mast cell degranulation to 21.0 +/- 3. 3%. Pretreatment with the neurotensin (NT) receptor antagonist SR48692 reduced (P < 0.05) mast cell degranulation to 12.5 +/- 3.4%, which was significantly (P < 0.05) below control levels. In animals treated neonatally with capsaicin to deplete their sensory neurons of their neuropeptides, such as substance P (SP), mast cell degranulation due to immobilization stress was reduced to about 15%. This is the first time that stress has been shown to trigger skin mast cell degranulation, an action not only dependent on CRH, but apparently also involving NT and SP. These findings may have implications for the pathophysiology and possible therapy of neuroinflammatory skin disorders such as atopic dermatitis, neurogenic pruritus, or psoriasis, which are induced or exacerbated by stress.

BACKGROUND

Corticotropin-releasing factor (CRF) and noradrenaline (NA) have been shown in independent studies to mediate stress-induced reinstatement of drug seeking. To date, however, a functional interaction between the systems in reinstatement has not been demonstrated.

OBJECTIVE

The objectives of this study were to determine whether CRF and NA systems can interact to influence reinstatement responding and, if so, in what direction the interaction occurs.

METHODS

Rats were trained to self-administer cocaine (0.23 mg/kg per infusion) for 8-10 days. Subsequently, responding for drug was extinguished, and tests for reinstatement were conducted following: (1) pretreatment with the CRF receptor antagonist, D: -Phe CRF(12-41) [1 microg, intracerebroventricular (i.c.v.)], prior to i.c.v. injections of NA (10 microg; Experiment 1); (2) pretreatment with the alpha(2) adrenoceptor agonist, clonidine (40 microg/kg, i.p.), prior to i.c.v. injections of CRF (0.5 microg; Experiment 2); (3) pretreatment with D: -Phe (1, 5 microg, i.c.v.), prior to systemic injections of the alpha(2) adrenoceptor antagonist, yohimbine (1.25 mg/kg; Experiment 3A); or (4) pretreatment with clonidine (40 microg/kg, i.p.) prior to systemic injections of yohimbine (0.625 mg/kg, 1.25 mg/kg; Experiment 3B).

RESULTS

NA reliably induced reinstatement, an effect that was blocked by pretreatment with D: -Phe. In contrast, CRF-induced reinstatement was not attenuated by pretreatment with clonidine. Pretreatment with neither D: -Phe nor clonidine was effective in blocking yohimbine-induced reinstatement.

CONCLUSIONS

Together, the present findings suggest a functional interaction between NA and CRF systems in mediating stress-induced reinstatement of cocaine seeking, whereby activation of CRF receptors occurs subsequent to, and downstream of, the sites of action of NA.

The aim of this cohort study conducted in France in 1997-1998 was to investigate the effects of antenatal anxiety and depression on spontaneous preterm labor. A consecutive series of 634 pregnant women with singleton pregnancies was included. Anxiety and depression were assessed using self-administered questionnaires: Spielberger's State-Trait Anxiety Inventory and the Edinburgh depression scale. Depression scores were dichotomized with a cutoff value suggestive of major depression. The 75th percentile was used for anxiety scores. A logistic regression analysis, controlling for sociodemographic and biomedical factors and including interaction terms, revealed that depression was positively associated with the outcome among underweight women, defined as women with a prepregnancy body mass index below 19 (adjusted odds ratio (OR) = 6.9, 95% confidence interval (CI): 1.8, 26.2). A similar result was observed for trait anxiety in women with a history of preterm labor (adjusted OR = 4.8, 95% CI: 1.1, 20.4). The association was close to significance for state anxiety in women with vaginal bleeding (adjusted OR = 3.6, 95% CI: 0.9, 14.7). These findings show that anxiety and depression, when combined with specific biomedical factors, are associated with spontaneous preterm labor. A synergic action of psychological and biomedical factors on the secretion of placental corticotropin-releasing factor is hypothesized.

Inescapable shock (IS) produces subsequent interference with escape behavior and increased fear conditioning that has been linked to increased activity and release of serotonin (5-HT) from neurons within the caudal dorsal raphe nucleus (DRN) both at the time of IS and later behavioral testing. Extrahypothalamic corticotropin-releasing hormone (CRH) has been implicated in many stress-related phenomena and has recently been shown to increase DRN 5-HT activity in the same caudal DRN area at which IS increases 5-HT activity. The current set of studies therefore examined the role of CRH in mediating the behavioral sequelae of IS. Intra-DRN microinjection of the nonselective CRH receptor antagonist d-Phe CRH (12-41) blocked the IS-induced behavioral changes when administered before IS but not when administered before later behavioral testing. Furthermore, intra-DRN administration of CRH in the absence of IS dose-dependently mimicked the effects of IS and interfered with escape behavior and increased fear conditioning 24 hr later. This effect was specific to injection of CRH into the caudal DRN and was not produced by microinjection into the rostral DRN. Intracerebroventricular CRH produced escape deficits and potentiated fear conditioning 24 hr later at only much higher doses, further confirming the site specificity of the effects. The potential role of the caudal DRN in states of anxiety is discussed.

Norepinephrine (NE), glutamate (Glu), and GABA have been identified as important neurotransmitters governing neuroendocrine mechanisms represented in the paraventricular nucleus of the hypothalamus (PVH). Microinjection studies were used to compare the efficacy of these transmitter mechanisms in stimulating PVH output neurons. Local administration of NE provoked an increase in plasma corticosterone levels and Fos induction in the both the parvocellular and magnocellular divisions of the nucleus. This treatment also stimulated a robust increase in corticotropin-releasing factor (CRF) heteronuclear (hn) RNA in the parvocellular PVH and a more subtle, although reliable, increase in arginine vasopressin (AVP) hnRNA in this same compartment. Local administration of the GABA(A) receptor antagonist bicuculline methiodide (BMI) resulted in increased plasma corticosterone and, in contrast to NE treatment, Fos induction limited primarily to the parvocellular PVH. BMI elicited marked increases in both CRH and AVP hnRNAs within the parvocellular division of the nucleus. Over a wide range of concentrations, Glu failed to produce reliable increases in corticosterone secretion and induced only weak activational responses limited primarily to non-neurosecretory regions of the PVH. Local Glu administration did, however, provoke Fos induction in identified GABAergic neurons immediately adjoining the PVH, suggesting that the muted response to Glu may be a consequence of concurrent activation of local inhibitory interneurons. These results support a differential involvement of adrenergic, glutamatergic and GABAergic mechanisms in regulating neurosecretory populations of the PVH and suggest that involvement of local circuit neurons must be carefully considered in the interpretation of microinjection studies.

Corticotropin-releasing factor (CRF) and its family of peptides are critical coordinators of homeostasis whose actions are mediated through their receptors, CRF receptor 1 (CRFR1) and CRFR2, found throughout the CNS and periphery. The phenotypes of mice deficient in either CRFR1 or CRFR2 demonstrate the critical role these receptors play. CRFR1-mutant mice have an impaired stress response and display decreased anxiety-like behavior, whereas CRFR2-mutant mice are hypersensitive to stress and display increased anxiety-like behavior. To further elucidate the roles of both CRF receptors and determine their interaction in behaviors, we have generated mice deficient in both CRFR1 and CRFR2. The behavioral phenotype of these mice demonstrates a novel role of the mother's genotype on development of pup anxiety. We have found that although the female double-mutant mice display anxiolytic-like behavior, the male double-mutant mice show significantly more anxiety-like behavior compared with the females. We have also determined that the dam's CRFR2 genotype affects the anxiety-like behavior of the male mice, such that a pup born to a heterozygous or mutant dam displays significantly more anxiety-like behavior regardless of that pup's genotype. Double-mutant mice also display an even greater impairment of their hypothalamic-pituitary-adrenal axis response to stress than that of the CRFR1-mutant mice. CRF mRNA levels are elevated in CRFR1- and double-mutant mice, and urocortin III and vasopressin mRNA levels are increased in CRFR2- and double-mutant mice. These results indicate that both CRFR1 and CRFR2 have critical roles in gene regulation and the maintenance of homeostasis in response to stress.

Metabotropic glutamate receptor (mGluR) subtypes (mGluR1 to mGluR8) act as important pre- and postsynaptic regulators of neurotransmission in the CNS. These receptors consist of two domains, an extracellular region containing the orthosteric agonist site and a transmembrane heptahelical domain involved in G protein activation and recognition of several recently synthesized pharmacological modulators. The presynaptic receptor mGluR7 shows the highest evolutionary conservation within the family, but no selective pharmacological tool was known. Here we characterize an mGluR7-selective agonist, N,N'-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082), which directly activates receptor signaling via an allosteric site in the transmembrane domain. At transfected mammalian cells expressing mGluR7, AMN082 potently inhibits cAMP accumulation and stimulates GTPgammaS binding (EC50-values, 64-290 nM) with agonist efficacies comparable with those of L-2-amino-4-phosphonobutyrate (L-AP4) and superior to those of L-glutamate. AMN082 (< or = 10 microM) failed to show appreciable activating or inhibitory effects at other mGluR subtypes and selected ionotropic GluRs. Chimeric receptor studies position the binding site of AMN082 in the transmembrane region of mGluR7, and we demonstrate that this allosteric agonist has little, if any, effect on the potency of orthosteric ligands. Here we provide evidence for full agonist activity mediated by the heptahelical domain of family 3 G protein-coupled receptors (which have mGluR-like structure) that may lead to drug development opportunities. Further, AMN082 is orally active, penetrates the blood-brain barrier, and elevates the plasma stress hormones corticosterone and corticotropin in an mGluR7-dependent fashion. Therefore, AMN082 is a valuable tool for unraveling the role of mGluR7 in stress-related CNS disorders.

The significance of melanotropic hormones as physiologic regulators of cutaneous pigmentation in humans is still controversial. Until recently, no direct effect for melanotropins could be demonstrated on human melanocytes. Here we present conclusive evidence that alpha-melanotropin (alpha-melanocyte-stimulating hormone, alpha-MSH) and the related hormone corticotropin (adrenocorticotropic hormone, ACTH) stimulate the proliferation and melanogenesis of human melanocytes maintained in culture in a growth medium lacking any AMP inducer. The minimal effective dose of either hormone is 0.1 nM. In time-course experiments, the increase in cell number and tyrosinase activity became evident after one treatment of the melanocytes with 100 nM alpha-MSH for 48 hr. The mitogenic effect gradually increased to 50-270% above control, depending on the individual melanocyte strain, with continuous treatment with 100 nM alpha-MSH for 8 days, whereas the melanogenic effect became maximal (70-450% increase above control) after 4 days of treatment. Western blot analysis of tyrosinase and the tyrosinase-related proteins TRP-1 and TRP-2 revealed that alpha-MSH increased the expression of those three melanogenic proteins. This was not accompanied by any change in their mRNA levels after brief (1.5-24 hr) or prolonged (6 days) treatment with 100 nM alpha-MSH, suggesting that the increased expression of these melanogenic proteins was due to posttranscriptional events. These results demonstrate both mitogenic and melanogenic effects of alpha-MSH and ACTH on human melanocytes. That both hormones are effective at subnanomolar concentrations, combined with the presence of melanotropin receptors on human melanocytes, strongly suggests that these melanotropins play a physiologic role in regulating human cutaneous pigmentation.

Corticotropin-releasing factor (CRF) administered intracerebroventricularly (i.c.v.) activates noradrenergic locus coeruleus (LC) neurons of halothane-anesthetized and unanesthetized rats. This study used a technique for microinfusing CRF into the LC from calibrated micropipettes to characterize and quantify the effects of locally administered CRF on LC discharge in halothane-anesthetized rats. CRF (3-100 ng) microinfusion into the LC increased discharge rate in a dose-dependent manner from 28 +/- 8 to 105 +/- 26% above preinfusion discharge rates. The CRF dose-response curve generated by local microinfusion was parallel to, and shifted approximately 200-fold to the left, of that generated by i.c.v. administration. Intracoerulear microinfusion of the CRF antagonist, [DPhe12,Nle(21,38),CalphaMeLeu37]r/hCRF(12-41), greatly attenuated LC activation produced by a maximally effective dose of i.c.v. administered CRF, suggesting that these effects are primarily due to actions within the LC. In rats in which both LC discharge rate and norepinephrine levels in prefrontal cortex were measured by in vivo microdialysis, CRF microinfused into the LC increased both endpoints. Finally, LC activation produced by CRF (60 ng) microinfusion into the LC was associated with cortical electroencephalographic activation. Taken together with previous anatomical and electrophysiological evidence for endogenous CRF interactions in the LC, our results support the hypothesis that CRF serves as an excitatory neurotransmitter in the LC, and suggest that its actions on LC neurons are translated to enhanced norepinephrine release and an impact on cortical targets.

Glucocorticoids either inhibit or sensitize stress-induced activity in the hypothalamo-pituitary-adrenal (HPA) axis, depending on time after their administration, the concentration of the steroids, and whether there is a concurrent stressor input. When there are high glucocorticoids together with a chronic stressor, the steroids act in brain in a feed-forward fashion to recruit a stress-response network that biases ongoing autonomic, neuroendocrine, and behavioral outflow as well as responses to novel stressors. We review evidence for the role of glucocorticoids in activating the central stress-response network, and for mediation of this network by corticotropin-releasing factor (CRF). We briefly review the effects of CRF and its receptor antagonists on motor outflows in rodents, and examine the effects of glucocorticoids and CRF on monoaminergic neurons in brain. Corticosteroids stimulate behaviors that are mediated by dopaminergic mesolimbic "reward" pathways, and increase palatable feeding in rats. Moreover, in the absence of corticosteroids, the typical deficits in adrenalectomized rats are normalized by providing sucrose solutions to drink, suggesting that there is, in addition to the feed-forward action of glucocorticoids on brain, also a feedback action that is based on metabolic well being. Finally, we briefly discuss the problems with this network that normally serves to aid in responses to chronic stress, in our current overindulged, and underexercised society.

Stress exposure during pregnancy can 'programme' adult behaviour and hypothalamic-pituitary-adrenal (HPA) axis stress responsiveness. In the present study, we utilised an ethologically relevant social stressor to model the type of stress that pregnant women may experience. We investigated the effects of social defeat by a resident lactating rat over 5 days during the last week of pregnancy on the pregnant intruder rat HPA axis, and on HPA responsivity to stress and anxiety-related behaviour in the adult offspring of the socially-defeated intruder rats. HPA axis responses after social defeat were attenuated in the pregnant rats compared to virgin females. In the adult offspring, systemic interleukin (IL)-1beta or restraint increased adrenocorticotrophic hormone and corticosterone secretion in male and female control rats; however, in prenatally stressed (PNS) offspring, HPA responses were greatly enhanced and peak hormone responses to IL-1beta were greater in females versus males. Male PNS rats displayed increased anxiety behaviour on the elevated plus maze; however, despite marked changes in anxiety behaviour across the oestrous cycle, there were no differences between female control and PNS rats. Investigation of possible mechanisms showed mineralocorticoid mRNA levels were reduced in the hippocampus of male and female PNS offspring, whereas glucocorticoid receptor mRNA expression was modestly reduced in the CA2 hippocampal subfield in female PNS rats only. Corticotropin-releasing hormone mRNA and glucocorticoid receptor mRNA expression in the central amygdala was greater in PNS males and females compared to controls. The data obtained in the present study indicate that prenatal social stress differentially programmes anxiety behaviour and HPA axis responses to stress in male and female offspring. Attenuated glucocorticoid feedback mechanisms in the limbic system may underlie HPA axis hyper-reactivity to stress in PNS offspring.

Urocortin was recently cloned from the rat midbrain. Urocortin is a member of the corticotropin releasing factor (CRF) peptide family and shows 45% sequence identity to CRF and 63% sequence identity to urotensin. It binds with a high affinity to CRF1 and CRF2 receptors, resulting in the stimulation of their adenylate cyclase activity. We used a polyclonal antibody against rat urocortin to define the distribution of urocortin-like immunoreactivity in the rat central nervous system. Several immunostained cell bodies were found in the supraoptic, paraventricular, and ventromedial hypothalamic nuclei. A large number of neurons with urocortin-like immunoreactivity were seen in the dorsolateral tegmental nucleus, in the linear and dorsal raphe nuclei, and in the substantia nigra. The most abundant immunoreactive (ir) perikarya were found in the Edinger-Westphal nucleus. Some neurons showed immunoreactivity in the interstitial nucleus of Cajal, the nucleus of Darkeschewitsch, and the periaqueductal gray. A dense immunoreactive fiber network was found in the lateral septal area. Some faintly stained axon terminals were observed among urocortin-ir perikarya in the supraoptic and paraventricular nuclei, in the central and periaqueductal gray, and in the Edinger-Westphal nucleus. No fibers with urocortin-ir were seen in the median eminence or the posterior pituitary. The distribution of urocortin-ir overlapped with the expression of the mRNA for the CRF2 receptor in several brain areas. These data support the hypothesis that this peptide is the endogenous ligand for the CRF2 receptor. Urocortin has been implicated in various endocrine responses, such as blood pressure regulation, as well as in higher cognitive functions.

BACKGROUND

Love has long been referred to as an addiction in literature and poetry. Scientists have often made comparisons between social attachment processes and drug addiction, and it has been suggested that the two may share a common neurobiological mechanism. Brain systems that evolved to govern attachments between parents and children and between monogamous partners may be the targets of drugs of abuse and serve as the basis for addiction processes.

OBJECTIVE

Here, we review research on drug addiction in parallel with research on social attachments, including parent-offspring attachments and social bonds between mating partners. This review focuses on the brain regions and neurochemicals with the greatest overlap between addiction and attachment and, in particular, the mesolimbic dopamine (DA) pathway.

RESULTS

Significant overlap exists between these two behavioral processes. In addition to conceptual overlap in symptomatology, there is a strong commonality between the two domains regarding the roles and sites of action of DA, opioids, and corticotropin-releasing factor. The neuropeptides oxytocin and vasopressin are hypothesized to integrate social information into attachment processes that is not present in drug addiction.

CONCLUSIONS

Social attachment may be understood as a behavioral addiction, whereby the subject becomes addicted to another individual and the cues that predict social reward. Understandings from both fields may enlighten future research on addiction and attachment processes.

We completed the mapping of a cutaneous CRH signaling system in two species with widely different determinants of skin functions, humans and mice. In human skin, the CRH receptor (CRH-R) 1 was expressed in all major cellular populations of epidermis, dermis, and subcutis with CRH-R1alpha being the most prevalent isoform. The CRH-R2 gene was expressed solely in hair follicle keratinocytes and papilla fibroblasts, whereas CRH-R2 antigen was localized predominantly in hair follicles, sebaceous and eccrine glands, muscle and blood vessels. In mouse skin, the CRH-R2 gene and protein were widely expressed in all cutaneous compartments and in cultured normal and malignant melanocytes. CRH-binding protein mRNA was present in dermal fibroblasts, melanoma cells, and sc fat of human skin and undetectable in mouse skin. The urocortin II gene was expressed equally in mouse and human skin. Taken together with our previous investigations, the present studies document the preferential expression of CRH-R1 in human skin, which mirrors CRH-R2 expression patterns in human and mouse skin. They are likely reflecting different functional activities of human and mouse skin. The adnexal location of CRH-R2 suggests a role for the receptor in hair growth. The differential interspecies CRH signaling expression pattern probably reflects adaptation to species-specific skin function determinants.

Polypeptide analogs of the known members of the corticotropin-releasing factor (CRF) family were synthesized and tested in vitro and in vivo for enhanced potency or competitive antagonism. Predictive methods and physicochemical measurements had suggested an internal secondary alpha-helical conformation spanning about 25 residues for at least three members of the CRF family. Maximization of alpha-helix-forming potential by amino acid substitutions from the native known sequences (rat/human and ovine CRF, sauvagine, and carp and sucker urotensin 1) led to the synthesis of an analog that was found to be more than twice as potent as either of the parent peptides in vitro. In contrast, certain amino-terminally shortened fragments, such as alpha-helical CRF or ovine CRF residues 8 to 41, 9 to 41, and 10 to 41, were found to be competitive inhibitors in vitro. Selected antagonists were examined and also found to be active in vivo.

Tobacco dependence is a chronic disorder that is characterized by relapse after periods of abstinence. It has been hypothesized that the activation of brain stress systems mediates stress-induced relapse to smoking. The aim of these experiments was to investigate the role of corticotropin-releasing factor (CRF) and norepinephrine in stress-induced reinstatement of extinguished nicotine-seeking behavior. Rats were allowed to self-administer nicotine under a fixed-ratio 5 schedule for 14 days and then nicotine-seeking behavior was extinguished by substituting saline for nicotine. In experiment 1, footshocks reinstated extinguished nicotine-seeking behavior. In experiment 2, there was a trend for the CRF(1/2) receptor antagonist D-Phe CRF((12-41)) (5, 25microg, icv) to decrease stress-induced reinstatement of nicotine-seeking behavior. Footshock-induced reinstatement of nicotine-seeking behavior was observed only in a subset of stress-responsive rats (71%). D-Phe CRF((12-41)) significantly attenuated stress-induced reinstatement of nicotine-seeking behavior in this subset of rats. In experiment 3, the alpha2-adrenergic receptor agonist clonidine (20, 40microg/kg, sc) attenuated footshock-induced reinstatement of nicotine-seeking behavior. In experiment 4, the effects of D-Phe CRF((12-41)) and clonidine on responding for chocolate-flavored food pellets was investigated in order to determine if these compounds have sedative effects. D-Phe CRF((12-41)) did not affect responding for food pellets. Clonidine slightly, but significantly, decreased responding for food pellets. Clonidine decreased responding for food to a lesser degree than it decreased stress-induced reinstatement of nicotine-seeking behavior. These data provide support for the hypothesis that an increased activity of brain CRF and norepinephrine systems mediates stress-induced relapse to nicotine-seeking behavior.

New insights into our understanding of drug abuse and addiction have revealed that the desire to use drugs and the process of addiction depend on effects on brain function. Drugs of abuse have been hypothesized to produce their rewarding effects by neuropharmacological actions on a common brain reward circuit called the extended amygdala. The extended amygdala involves the mesolimbic dopamine system and specific subregions of the basal forebrain, such as the shell of the nucleus accumbens, the bed nucleus of the stria terminalis, and the central nucleus of the amygdala. The psychomotor stimulants cocaine and amphetamine activate the mesolimbic dopamine system; opiates activate opioid peptide receptors within and independent of the mesolimbic dopamine system. Sedative hypnotics alter multiple neurotransmitter systems in this circuitry, including: 1) gamma aminobutyric acid; 2) dopamine; 3) serotonin; 4) glutamate; and 5) opioid peptides. Nicotine and tetrahydrocannabinol both activate mesolimbic dopamine function and possibly opioid peptide systems in this circuitry. Repeated and prolonged drug abuse leads to compulsive use, and the mechanism for this transition involves, at the behavioral level, a progressive dysregulation of brain reward circuitry and a recruitment of brain stress systems such as corticotropin-releasing factor. The molecular mechanisms of signal transduction in these systems are a likely target for residual changes in that they convey allostatic changes in reward set point, which lead to vulnerability to relapse.

In vivo studies suggest that the stress-related neuropeptide corticotropin-releasing factor (CRF) modulates serotonergic neurotransmission. To investigate the underlying mechanisms for this interaction, the present study examined the effects of CRF in vitro on dorsal raphe neurons that displayed electrophysiological and pharmacological properties consistent with a serotonergic phenotype. In the presence of either 1 or 2 mm Ca(2+), perfusion of ovine CRF or rat/human CRF rapidly and reversibly increased firing rates of a subpopulation (19 of 70, 27%) of serotonergic neurons predominantly located in the ventral portion of the dorsal raphe nucleus. For a given responsive neuron, the excitatory effects of CRF were reproducible, and there was no tachyphylaxis. Excitatory effects were dose-dependent (over the range of 0.1-1.6 micrometer) and were completely absent after exposure to the competitive CRF receptor antagonists alpha-helical CRF(9-41) or rat/human [d-Phe(12), Nle(21, 38), alpha-Me-Leu(37)]-CRF(12-41). Both the proportion of responsive neurons and the magnitude of excitatory responses to CRF in the ventral portion of the caudal dorsal raphe nucleus were markedly potentiated in slices prepared from animals previously exposed to isolation and daily restraint stress for 5 d. Immunohistochemical staining of the recorded slices revealed close associations between CRF-immunoreactive varicose axons and tryptophan hydroxylase-immunoreactive neurons in the area of the recordings, providing anatomical evidence for potential direct actions of CRF on serotonergic neurons. The electrophysiological properties and the distribution of responsive neurons within the dorsal raphe nucleus are consistent with the hypothesis that endogenous CRF activates a topographically organized mesolimbocortical serotonergic system.

Introduction of a socially naive male rat into the home territory of a resident counterpart results in agonistic interactions, leading to the rapid social defeat of the intruder. Exposure to the aggressive resident produces a stress-response profile consisting of neuroendocrine activation and coping behaviors such as submission. The present studies examined the dependence of these adaptive responses on endogenous brain Corticotropin-Releasing Factor (CRF), a peptide hormone known to coordinate neuronally mediated- and pituitary-adrenal responses to stress. The Elevated Plus-Maze was employed as an animal model of emotionality in which stressors reduce subsequent exploration of open maze arms without walls in favor of enclosed maze arms. A CRF antagonist, alpha-hel CRF9-41, administered intracerebroventricularly (5 and 25 micrograms i.c.v.) immediately post-stress and 5 min prior to maze testing reversed the heightened emotionality produced by the resident exposure stressor. This action paralleled that of an anxiolytic dose of the short-acting benzodiazepine, midazolam (1.5 mg/kg i.p.). Intra-amygdaloid administration of lower doses of the CRF antagonist (125, 250 and 500 ng i.c.) also reversed, dose-dependently, the effect of exposure to an aggressive resident without altering the behavior of unstressed control animals. Further, the enhanced release of ACTH and corticosterone following social conflict was not modified over the short term by the intra-amygdaloid dose of CRF antagonist (250 ng i.c.) which was effective in reversing stress-induced hyper-emotionality. These results suggest that limbic system CRF substrates exert an anxiogenic effect on the exploratory behavior of socially defeated rats via a pituitary-adrenal-independent mechanism.

A possible interaction between synthetic ovine corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) was tested in anesthetized and in freely moving rats. In animals whose endogenous CRF release was blocked by chlorpromazine-morphine-nembutal, AVP elicited a significantly lower maximum ACTH response than did CRF, whereas the concomitant injection of both peptides resulted in a marked potentiation of CRF-induced ACTH secretion. In freely moving rats, AVP was more potent than CRF (on an equimolar basis) in elevating plasma ACTH levels. Since immunoneutralization of endogenous CRF by the administration of anti-CRF serum significantly reduced ACTH release due to injected AVP in these animals, we suggest that at least part of the AVP-induced ACTH secretion observed in nonanesthetized rats may be due to a potentiation of endogenous CRF by exogenously administered AVP. These data support previous reports of an in vitro synergism between CRF and vasopressin, and emphasize the complex role played by the interaction of these two peptides on ACTH release in vivo.

Hypothalamic nuclei, including the anterior periventricular (aPV), paraventricular (PVN), and supraoptic (SON) nuclei strongly express the homeobox gene Orthopedia (Otp) during embryogenesis. Targeted inactivation of Otp in the mouse results in the loss of these nuclei in the homozygous null neonates. The Otp null hypothalamus fails to secrete neuropeptides somatostatin, arginine vasopressin, oxytocin, corticotropin-releasing hormone, and thyrotropin-releasing hormone in an appropriate spatial and temporal fashion, and leads to the death of Otp null pups shortly after birth. Failure to produce these neuropeptide hormones is evident prior to E15.5, indicating a failure in terminal differentiation of the aPV/PVN/SON neurons. Absence of elevated apoptotic activity, but reduced cell proliferation together with the ectopic activation of Six3 expression in the presumptive PVN, indicates a critical role for Otp in terminal differentiation and maturation of these neuroendocrine cell lineages. Otp employs distinct regulatory mechanisms to modulate the expression of specific molecular markers in the developing hypothalamus. At early embryonic stages, expression of Sim2 is immediately downregulated as a result of the absence of Otp, indicating a potential role for Otp as an upstream regulator of Sim2. In contrast, the regulation of Brn4 which is also expressed in the SON and PVN is independent of Otp function. Hence no strong evidence links Otp and Brn4 in the same regulatory pathway. The involvement of Otp and Sim1 in specifying specific hypothalamic neurosecretory cell lineages is shown to operate via distinct signaling pathways that partially overlap with Brn2.

Chronic stress early in postnatal life influences hormonal and behavioural responses to stress persistently, but the mechanisms and molecular cascades that are involved in this process have not been clarified. To approach these issues, a chronic stress paradigm for the neonatal rat, using limited bedding material to alter the cage environment, was devised. In 9-day-old rats subjected to this chronic stress for 1 week, significant and striking changes in the expression and release patterns of key molecules that govern the neuroendocrine stress responses were observed. The presence of sustained stress was evident from enhanced activation of peripheral elements of the neuroendocrine stress response, i.e. increased basal plasma corticosterone concentrations, high adrenal weight and decreased body weight. Central regulatory elements of the neuroendocrine stress response were perturbed, including reduced expression of hypothalamic corticotropin-releasing hormone that, surprisingly, was accompanied by reduced glucocorticoid receptor expression. Thus, the effects of chronic sustained stress in the neonatal rat on the hypothalamic-pituitary-adrenal axis included substantial changes in the expression and activity of major regulators of this axis. Importantly, the changes induced by this chronic stress differed substantially from those related to acute or recurrent stress, providing a novel model for studying the long-term effects of chronic, early life stress on neuroendocrine functions throughout life.

BACKGROUND

Drinking in the dark (DID) procedures have recently been developed to induce high levels of ethanol drinking in C57BL/6J mice, which result in blood ethanol concentrations (BECs) reaching levels that have measurable affects on physiology and/or behavior. The present experiments determined whether the increased ethanol drinking caused by DID procedures can be attenuated by pretreatment with CP-154,526; a corticotropin releasing factor type-1 (CRF1) receptor antagonist.

METHODS

In Experiment 1, male C57BL/6J mice received ethanol (20% v/v) in place of water for 4 hours, beginning with 3 hours into the dark cycle. On the fourth day, mice were given an intraperitoneal injection of one of the 4 doses of CP-154,526 (0, 1, 3, 10 mg/kg) 30 minutes before receiving their ethanol bottle. In Experiment 2, C57BL/6J mice had 2 hours of access to the 20% ethanol solution, beginning with 3 hours into the dark cycle on days 1 to 3, and 4 hours of access to the ethanol bottle on day 4 of DID procedures. Mice were given an intraperitoneal injection of one of the 4 doses of CP-154,526 (0, 1, 3, 10 mg/kg) 30 minutes before receiving their ethanol bottle on day 4. Tail blood samples were collected immediately after the 4-hour ethanol access period on the fourth day of each experiment. Additional control experiments assessed the effects of CP-154,526 on 4-hour consumption of a 10% (w/v) sucrose solution and open-field locomotor activity.

RESULTS

In Experiment 1, the vehicle-treated group consumed approximately 4.0 g/kg/4 h of ethanol and achieved BECs of approximately 30 mg%. Furthermore, pretreatment with the CRF1 receptor antagonist did not alter ethanol consumption. On the other hand, procedures used in Experiment 2 resulted in vehicle-treated mice consuming approximately 6.0 g/kg/4 h of ethanol with BECs of about 80 mg%. Additionally, the 10 mg/kg dose of CP-154,526 significantly reduced ethanol consumption and BECs to approximately 3.0 g/kg/4 h and 27 mg%, respectively, relative to vehicle-treated mice. Importantly, the 10 mg/kg dose of the CRF1R antagonist did not significantly alter 4-hour sucrose consumption or locomotor activity.

CONCLUSIONS

These data indicate that CRF1R signaling modulates high, but not moderate, levels of ethanol drinking associated with DID procedures.

Inflammatory bowel disease (IBD) is a chronic, relapsing condition involving complex interactions between genes and the environment. The mechanisms triggering the initial attack and relapses, however, are not well understood. In the past several years the enteric nervous system (ENS) has been implicated in the pathophysiology of IBD. Both the ENS and the central nervous system (CNS) can amplify or modulate aspects of intestinal inflammation through secretion of neuropeptides that serve as a link between the ENS and CNS. Neuropeptides are defined as any peptide released from the nervous system that serves as an intercellular signaling molecule. Neuropeptides thought to play a potentially key role in IBD include substance P, corticotropin-releasing hormone, neurotensin, vasoactive intestinal peptide, mu-opioid receptor agonists, and galanin. This review focuses on the role of these neuropeptides in the pathophysiology of IBD and discusses the cell types and mechanisms involved in this process. The available evidence that neuropeptide blockade may be considered a therapeutic approach in both Crohn's disease and ulcerative colitis will also be discussed.