The involvement of brain oxytocin in the attenuated responsiveness of the hypothalamo-pituitary-adrenal axis and the oxytocin systems to external stressors found in pregnant and lactating rats has been studied, including both neuroendocrine and behavioural aspects. Intracerebroventricular infusion of an oxytocin receptor antagonist (0.75 microg/5 microl), but not of vehicle, elevated basal corticotropin and corticosterone secretion into blood of virgin female, but not of late pregnant or lactating rats. Oxytocin antagonist treatment further elevated the stress-induced (exposure to the elevated plus-maze or forced swimming) secretion of both corticotropin and corticosterone, but only in virgin and not in pregnant or lactating rats. Thus, corticotropin and corticosterone plasma concentrations remained attenuated in antagonist-treated pregnant and lactating animals. In contrast, infusion of the oxytocin antagonist significantly elevated the stress-induced secretion of oxytocin into blood in pregnant and lactating, but not in virgin, animals, indicating an autoinhibitory influence of intracerebral oxytocin on neurohypophysial oxytocin secretion induced by non-reproduction-related stimuli. Treatment with oxytocin antagonist 10 min prior to behavioural testing on the elevated plus-maze significantly reduced the anxiety-related behaviour in both pregnant and lactating rats, without exerting similar effects in virgin female rats. The results demonstrate a tonic inhibitory effect of endogenous oxytocin on corticotropin and, consequently, corticosterone secretion in virgin female rats, an effect which is absent in the peripartum period. In contrast, an anxiolytic action of endogenous oxytocin was detectable exclusively in pregnant and lactating rats. Therefore, we conclude that the actions of intracerebral oxytocin include independent effects on the responses of the hypothalamo-pituitary-adrenal axis and oxytocin systems to stressors and the anxiety-related behaviour which are modulated by the reproductive state of the animals.

In a previous study (Watts et al., '87) we reexamined the projections of the suprachiasmatic nucleus (SCh) with the PHA-L method and found that they could be divided conveniently into six groups of fibers. By far the densest projection ends just dorsal to the SCh in a comma-shaped region designated the "subparaventricular zone," although some fibers continue on through the paraventricular nucleus of the hypothalamus to end in the overlying midline thalamus, and others continue on to end in the dorsomedial nucleus, the region around the ventromedial nucleus, and the posterior hypothalamic area. Other relatively sparse projections from the SCh were also described to the preoptic region, lateral septal nucleus, parataenial and paraventricular nuclei of the thalamus, and ventral lateral geniculate nucleus. In addition, the same method was used to show that the subparaventricular zone projects in turn massively to these same regions, as well as back to the SCh itself and to the periaqueductal gray. The present series of experiments was designed to confirm these observations with retrograde tracer injections and to investigate the cellular and possible neurotransmitter organization of the major projections from the SCh and subparaventricular zone with a combined retrograde tracer-immunohistochemical method. For this, the distribution of neuronal cell bodies within the SCh that stain with antisera to vasopressin, vasoactive intestinal polypeptide (VIP), corticotropin-releasing factor, bombesin, substance P, neurotensin, somatostatin, thyrotropin-releasing hormone, and angiotensin II was described in detail first. Then the distribution of retrogradely labeled neurons that were also stained for one or another of these peptides was described after injections of true blue, or in some cases SITS, into the regions of the subparaventricular zone, the paraventricular and parataenial nuclei of the thalamus, the ventromedial nucleus, the dorsomedial nucleus, and the periaqueductal gray. The results confirm previous immunohistochemical and anterograde tracing studies and in addition indicate that cells in dorsal as well as ventral parts of the SCh project to each of the terminal fields examined, as do many cells in surrounding areas, including the subparaventricular zone. Our results also suggest that, at the very least, vasopressin-, VIP-, and neurotensin-stained cells in the SCh project to the subparaventricular zone, midline thalamus, and dorsomedial nucleus, and that the vasopressin and VIP-stained fiber systems are partially segregated at the level of the subparaventricular zone.(ABSTRACT TRUNCATED AT 400 WORDS)

Dieting to control body weight involves cycles of deprivation from palatable food that can promote compulsive eating. The present study shows that rats withdrawn from intermittent access to palatable food exhibit overeating of palatable food upon renewed access and an affective withdrawal-like state characterized by corticotropin-releasing factor-1 (CRF(1)) receptor antagonist-reversible behaviors, including hypophagia, motivational deficits to obtain less palatable food, and anxiogenic-like behavior. Withdrawal was accompanied by increased CRF expression and CRF(1) electrophysiological responsiveness in the central nucleus of the amygdala. We propose that recruitment of anti-reward extrahypothalamic CRF-CRF(1) systems during withdrawal from palatable food, analogous to abstinence from abused drugs, may promote compulsive selection of palatable food, undereating of healthier alternatives, and a negative emotional state when intake of palatable food is prevented.

Anxiety-like behaviors are integral features of withdrawal from chronic ethanol exposure. In the experiments in the current study, we tested the hypothesis that anxiety can be regulated independently of other withdrawal signs and thus may be responsive to selective pharmacological agents. For 17 days, rats were fed ethanol (8-12 g/kg/day) in a liquid diet. Between 5 and 6 h after cessation of ethanol treatment, rats were tested in either the social interaction or plus-maze test of anxiety-like behavior after treatment with drugs hypothesized to have anxiolytic action. SB242084, flumazenil, and CRA1000-antagonists for 5-hydroxytryptamine (serotonin) (5-HT) 2C (5-HT(2C)), benzodiazepine, and corticotropin-releasing factor type 1 (CRF(1)) receptors, respectively-attenuated decreased social interaction without concomitant effects on activity measures. In contrast, ifenprodil, MDL 72222, and zolpidem-antagonists for N-methyl-d-aspartate (NMDA) and 5-HT(3) receptors, and agonist for benzodiazepine type 1 receptors, respectively-did not share this effect. Results for SB242084, flumazenil, and ifenprodil in the elevated plus-maze test were comparable to those in the social interaction test. These results support the suggestion that multiple neuronal systems (CRF(1), 5-HT(2C), and benzodiazepine receptors) contribute to the ethanol withdrawal sign of decreased social interaction. Furthermore, the selective effects of pharmacological agents on social interaction seem to indicate that this behavior can be dissociated from other signs. Because anxiety may be a complicating factor in alcohol withdrawal and relapse, future studies of this type are needed to provide focus for the effort to define selective and novel antianxiety agents for these disorders.

Expression of mRNAs coding for the ACTH secretagogues corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) was examined in the hypothalamic paraventricular nucleus (PVN) of rats bearing hippocampal lesions. Either total hippocampectomy (HPX) or extirpation of the dorsal hippocampus (DHPX) precipitated a 4-fold increase in CRF mRNA expression relative to sham-operated controls (SHAM), as determined by semiquantitative in situ hybridization histochemistry. AVP mRNA was localized to individual parvocellular neurons of the medial parvocellular division of the PVN in only the HPX and DHPX groups, consistent with enhanced production of AVP message in this neuronal population subsequent to hippocampal damage. HPX did not affect AVP mRNA content in magnocellular divisions of PVN. Plasma beta-endorphin levels were significantly elevated in the HPX and DHPX groups relative to SHAM animals, indicating a chronic increase in release of proopiomelanocortin peptides from the anterior pituitary gland in response to hippocampal lesion. Circulating corticosterone levels were elevated in HPX rats as well. To control for effects of lesion size and location, additional animals received large ablations of cerebral cortex or cerebellum. In neither case was CRF or AVP mRNA significantly altered in the PVN. The results suggest that the hippocampus exercises a tonic inhibitory role on ACTH secretagogue production in neuroendocrine neurons promoting ACTH release.

We evaluated the effects of the lipophilic nonpeptide corticotropin-releasing hormone (CRH) type 1 receptor antagonist antalarmin on the behavioral, neuroendocrine, and autonomic components of the stress response in adult male rhesus macaques. After oral administration, significant antalarmin concentrations were detected in the systemic circulation and the cerebrospinal fluid by a mass spectrometry-gas chromatography assay developed specifically for this purpose. Pharmacokinetic and dose-response studies suggested that an oral dose of 20 mg/kg was optimal for behavioral and endocrine effects. We then administered this dose in a double-blind, placebo-controlled fashion to monkeys exposed to an intense social stressor: namely, placement of two unfamiliar males in adjacent cages separated only by a transparent Plexiglas screen. Antalarmin significantly inhibited a repertoire of behaviors associated with anxiety and fear such as body tremors, grimacing, teeth gnashing, urination, and defecation. In contrast, antalarmin increased exploratory and sexual behaviors that are normally suppressed during stress. Moreover, antalarmin significantly diminished the increases in cerebrospinal fluid CRH as well as the pituitary-adrenal, sympathetic, and adrenal medullary responses to stress. We conclude that CRH plays a broad role in the physiological responses to psychological stress in primates and that a CRH type 1 receptor antagonist may be of therapeutic value in human psychiatric, reproductive, and cardiovascular disorders associated with CRH system hyperactivity.

We have recently described the cloning and characterization of a novel corticotropin-releasing factor receptor subtype (CRF2) from rat brain that exists in two alternatively spliced forms, CRF2 alpha and CRF2 beta. These forms differ in their N-terminal coding sequence which results in the production of two distinct receptors of 411 and 431 amino acids, respectively. To assess whether these two forms might represent distinct targets for CRF action, RNase protection and in situ hybridization studies were performed using specific N-terminal cRNA probes. The results showed a differential distribution of the mRNAs for these two receptor forms in the rat. The mRNA for CRF2 alpha is found almost exclusively in the brain, particularly in the hypothalamus, lateral septum, and olfactory bulb, whereas the mRNA for CRF2 beta appears to be both in the brain and in the periphery, with the greatest abundance in the heart and skeletal muscle. Thus, the data suggest that these alternatively spliced forms of the CRF2 receptor may represent functionally distinct CRF receptors. In addition, it highlights the importance of probe specificity for in situ hybridization studies.

It is well established that adrenal stress hormone-induced activation of the basolateral complex of the amygdala (BLA) influences memory consolidation. The present experiments investigated the involvement of corticotropin-releasing hormone (CRH) in the BLA in modulating memory consolidation. Bilateral infusions of the CRH receptor antagonist [9-41]-alpha-helical CRH (0.3, 1.0, or 3.0 microg in 0.2 microl) administered into the BLA of male Sprague-Dawley rats immediately after aversively motivated inhibitory avoidance training produced dose-dependent impairment of 48-h retention performance. Because the CRH receptor antagonist infusions did not impair retention when administered into the BLA 3 h after training, the retention impairment selectively was due to time-dependent influences on memory consolidation. Furthermore, because immediate posttraining infusions of [9-41]-alpha-helical CRH into the adjacent central nucleus of the amygdala (CEA) were ineffective, the effect selectively involved the BLA. Immunocytochemistry showed that the aversive training stimulus of a single, brief footshock increased CRH levels in the CEA. These findings indicate that activation of CRH receptors in the BLA, likely by training-induced release of endogenous peptide originating from the CEA, participates in mediating stress effects on memory consolidation.

Neuroendocrine studies strongly suggest that dysregulation of the hypothalamic pituitary-adrenocortical (HPA) system plays a causal role in the development and course of depression. Whereas the initial mechanism resulting in HPA hyperdrive remains to be elucidated, evidence has emerged that corticosteroid receptor function is impaired in many patients with depression and in many healthy individuals at increased genetic risk for an depressive disorder. Assuming such impaired receptor function, then central secretion of CRH would be enhanced in many brain areas, which would account for a variety of depressive symptoms. As shown in rats and also in transgenic mice with impaired glucocorticoid receptor function, antidepressants enhance the signaling through corticosteroid receptors. This mechanism of action can be amplified through blocking central mechanisms that drive the HPA system. Animal experiments using antisense oligodeoxynucleotides directed against the mRNA of both CRH receptor subtypes identified the CRH1 receptor as the mediator of the anxiogenic effects of CRH. Studies in mouse mutants in which this receptor subtype had been deleted extended these findings as the animals were less anxious than wild-type mice when experimentally stressed. Thus, patients with clinical conditions that are causally related to HPA hyperactivity may profit from treatment with a CRH1 receptor antagonist.

Twenty-two 50-month-old rhesus monkeys were provided concurrent free access to an aspartame-sweetened 7% ethanol solution and an aspartame-sweetened vehicle before, during, and after social separation. Subjects had been reared for their first 6 months of life either without access to adults but with constant access to age mates (peer reared), a condition producing reduced exploration and increased fear-related behaviors, or as controls with their mothers; thereafter, all subjects received identical treatment. During home-cage periods, for 1 hr each day, 4 days a week, when the ethanol solution and vehicle were freely available, peer-reared subjects consumed significantly more alcohol than mother-reared subjects. When stress was increased via social separation, mother-reared animals increased their alcohol consumption to a level nearly as high as that of peer-reared monkeys. Average individual differences in alcohol consumption were markedly stable over time. In addition, there were strong positive correlations between alcohol consumption and distress behaviors. Biological indices of increased stress, such as plasma cortisol and corticotropin, were higher in peer-reared subjects. Within the peer- and mother-reared groups, these indices were positively correlated with alcohol consumption. The results suggest that early rearing experiences that predispose monkeys to increased fear-related behaviors produce excessive alcohol consumption under normal living conditions. Furthermore, a major challenge such as social separation increases alcohol consumption to levels producing intoxication even in monkeys not particularly vulnerable to stress.

Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor, was recently identified in the rat stomach. Previous studies have shown that ghrelin potently increases growth hormone release and food intake. We examined the effects of the gastric peptide ghrelin on anxiety-like behavior in association with the hypothalamic-pituitary-adrenal axis in mice. Both intra-third cerebroventricular and intraperitoneal administration of ghrelin potently and significantly induced anxiogenic activities in the elevated plus maze test. Ghrelin gene expression in the stomach was increased by tail pinch stress as well as by starvation stress. Administration of a corticotropin-releasing hormone (CRH) receptor antagonist significantly inhibited ghrelin-induced anxiogenic effects. Peripherally administered ghrelin significantly increased CRH mRNA, but not urocortin mRNA expression in the hypothalamus. Furthermore, intraperitoneal injection of ghrelin produced a significant dose- dependent increase in serum corticosterone levels. These findings suggest that ghrelin may have a role in mediating neuroendocrine and behavioral responses to stressors and that the stomach could play an important role, not only in the regulation of appetite, but also in the regulation of anxiety.

Stress from many sources, including pain, fever, and hypotension, activates the hypothalamic-pituitary-adrenal (HPA) axis with the sustained secretion of corticotropin and cortisol. Increased glucocorticoid action is an essential component of the stress response, and even minor degrees of adrenal insufficiency can be fatal in the stressed host. HPA dysfunction is a common and underdiagnosed disorder in the critically ill. We review the risk factors, pathophysiology, diagnostic approach, and management of HPA dysfunction in the critically ill.

BACKGROUND

Chronic cocaine abuse is associated with the development of anxiogenic states in humans. Corticotropin-releasing factor (CRF) is an endogenous neurotropic factor well known to modulate stress responses. It has been postulated that CRF is involved in the neurobiological mechanisms underlying the anxiety and/or stress responses associated with removal of cocaine after chronic administration.

OBJECTIVE

The present study investigated the role of endogenous CRF in mediating the "anxiety-like" effect 48 h after the cessation of saline or chronic cocaine treatment in rats, using the defensive burying paradigm and the elevated plus-maze.

METHODS

Rats received daily injections of cocaine (20 mg/kg IP, for 14 consecutive days) or vehicle. Forty-eight hours after the last injection, animals were tested in the plus-maze and then in the defensive burying paradigm. In a second experiment, intracerebroventricular (ICV) cannulae were implanted at the lateral ventricle. Animals were allowed a 1-week period for recovery before starting the chronic drug treatment. The defensive burying testing took place 48 h after cessation of the treatment. The CRF antagonist [DPhe12, Nle21,38, CalphaMe Leu37] r/h CRF(12-41), (also known as D-phe CRF(12-41)) (0.04, 0.2 and 1.0 microg/5 microl) was injected 5 min before the 15-min testing.

RESULTS

An "anxiogenic-like" effect following chronic cocaine treatment was demonstrated with the defensive burying paradigm, but not with the elevated plus-maze. This "anxiety-like" response was attenuated by ICV pretreatment with the CRF antagonist D-Phe CRF(12-41), with the highest dose of the CRF antagonist reversing the observed "anxiogenic-like" response.

CONCLUSIONS

These data suggest that brain CRF may be substantially involved in the development of "anxiety-like" responses related to cocaine withdrawal and could be important for future drug dependence treatments.

Systemic interleukin-1 (IL-1) activates the hypothalamo-pituitary-adrenal (HPA) axis, an effect exerted through increased synthesis and secretion of corticotropin-releasing factor (CRF) by parvicellular neurosecretory neurons. The site(s) and mechanism(s) through which circulating IL-1 may access central systems governing HPA axis output remain obscure. To identify potential cellular targets for blood-borne IL-1, we analyzed the distribution of mRNA encoding the rat type 1 IL-1 receptor (IL-1R1) in rat brain. Regional ribonuclease protection assays detected a single protected fragment corresponding to the membrane-bound form of the IL-1R1 mRNA in all areas analyzed. In situ hybridization revealed labeling predominantly over barrier-related cells, including the leptomeninges, non-tanycytic portions of the ependyma, the choroid plexus, and vascular endothelium. Low to moderate levels of the IL-1R1 mRNA were detected in just a few neuronal cell groups, including the basolateral nucleus of the amygdala, the arcuate nucleus of the hypothalamus, the trigeminal and hypoglossal motor nuclei, and the area postrema. No specific labeling for IL-1R1 mRNA was detected over neurons that respond to intravenous IL-1 beta by induction of transcription factor Fos, including hypophysiotropic CRF cells and brainstem catecholamine neurons. Injection of IL-1 beta did, however, provoke induction of mRNA encoding the immediate-early gene, NGFI-B, but not c-fos, in two major loci of IL-1R1 expression, vascular endothelial cells, and the area postrema. Intravenous injection of IL-1 beta acutely down-regulated IL-1R1 mRNA in perivascular cells, but not in neuronal cell groups. These results suggest the parenchymal sites of IL-1R1 expression in rat to be distinct from those reported previously in mouse. The common expression in both species of an IL-1R in non-neuronal elements highlights the possibility that IL-1-mediated activation of CRF neurons may result from cytokine-receptor interaction at vascular, and/or other barrier-related, sites to trigger release of secondary signalling molecules in a position to interact with components of HPA control circuitry.

METHODS

Cushing's syndrome is rare in children and adolescents. We analyzed the clinical presentation, diagnostic evaluation, and treatment of 59 patients with Cushing's syndrome between the ages of 4 and 20 years who were admitted to the National Institutes of Health during the period from 1982 to 1992. The cause of hypercortisolism was identified by low- and high-dose dexamethasone suppression tests, the ovine corticotropin-releasing hormone (CRH) stimulation test, imaging studies, and bilateral sampling of the inferior petrosal sinuses combined with administration of CRH.

RESULTS

Fifty patients had Cushing's disease, six had primary adrenal disease, and three had ectopic corticotropin secretion. The initial signs were excessive weight gain in 90 percent of the patients and growth retardation in 83 percent. Most patients (81 percent) had normal bone age at the time of diagnosis. Forty-seven percent had hypertension, whereas only 19 percent had mental or behavioral problems. The high-dose dexamethasone suppression test and the CRH stimulation test identified 68 and 80 percent, respectively, of the patients with Cushing's disease. Magnetic resonance imaging of the pituitary indicated the presence of tumor in 52 percent of the patients with pituitary adenomas. The maximal central-to-peripheral ratio of plasma corticotropin during sampling of the interior petrosal sinuses was>> or = 2.5 in all the patients with Cushing's disease and < 2.5 in those with ectopic corticotropin secretion. Remission of hypercortisolism was achieved in 48 of the 49 patients who underwent transsphenoidal surgery for Cushing's disease, in all 6 of the patients who underwent adrenalectomy for primary adrenal disease, and in the 2 patients in whom the ectopic source of corticotropin could be identified.

CONCLUSIONS

Weight gain and growth retardation are common clinical characteristics of Cushing's syndrome in children and adolescents. Diagnostic evaluation of such patients with CRH stimulation alone and combined with inferior petrosal sinus sampling and imaging studies is accurate, and therapy is usually successful.

Corticotropin-releasing factor (CRF) receptor-binding sites have been localized and quantified in the rat central nervous system (CNS) by autoradiography with an iodine-125-labeled analogue of ovine CRF substituted with norleucine and tyrosine at amino acid residues 21 and 32, respectively. High affinity and pharmacologically specific receptor-binding sites for CRF were found in discrete areas within the rat CNS. CRF receptors were highly concentrated in laminae 1 and 4 throughout the neocortex, the external plexiform layer of the olfactory bulb, the external layer of the median eminence, several cranial nerve nuclei in the brainstem including the facial, oculomotor, trochlear, vestibulocochlear, and trigeminal nuclei, the deep cerebellar nuclei, and the cerebellar cortex. Moderate concentrations of CRF receptors were present in the olfactory tubercle, caudate-putamen, claustrum, nucleus accumbens, nucleus of the diagonal band, basolateral nucleus of the amygdala, paraventricular nucleus of the hypothalamus, mammillary peduncle, inferior and superior olives, medullary reticular formation, inferior colliculus, and brainstem nuclei including tegmental, parabrachial, hypoglossal, pontine, cuneate, and gracilis nuclei, and in spinal cord. Lower densities of CRF binding were found in the bed nucleus of the stria terminalis, central and medial amygdaloid nuclei, and regions of the thalamus, hypothalamus, hippocampus, and brainstem. The distribution of CRF-binding sites generally correlates with the immunocytochemical distribution of CRF pathways and with the pharmacological sites of action of CRF. These data strongly support a physiological role for endogenous CRF in regulating and integrating functions in the CNS.

Maintenance of adequate levels of response of the hypothalamic-pituitary-adrenal axis during chronic stress is important for survival. Three basic patterns of response can be identified depending on the type of stress: (a) desensitization of ACTH responses to the sustained stimulus, but hyperresponsiveness to a novel stress despite elevated plasma glucocorticoid levels, as occurs in physical-psychological paradigms; (b) no desensitization of ACTH response to the repeated stimulus and hyperresponsiveness to a novel stress, as occurs during repeated painful stress and insulin hypoglycemia; and (c) small and transient increases in ACTH, but sustained elevations of plasma corticosterone and diminished ACTH responses. The level of response of the pituitary corticotroph is determined by differential regulation of the hypothalamic regulators, corticotropin-releasing hormone (CRH) and vasopressin (VP), and the sensitivity of the negative glucocorticoid feedback. While osmotic stimulation increases VP expression in magnocellular neurons of the paraventricular (PVN) and supraoptic nuclei of the hypothalamus, chronic stress paradigms with high pituitary responsiveness are associated with activation of CRH and CRH/VP parvicellular neurons of the PVN, predominantly of the VP-containing population. While moderate increase of CRH output is important for stimulation of POMC transcription, the increase of the VP:CRH secretion ratio appears to be important in maintaining the secretory capacity of the pituitary corticotroph during chronic stimulation. Decreased sensitivity of the glucocorticoid feedback, probably due to interaction of glucocorticoid receptors with transcription factors induced by CRH and VP, is critical for the maintenance of ACTH responses in the presence of elevated plasma glucocorticoid levels during chronic stress. Although both CRH and VP receptors are activated and undergo regulatory variations during chronic stress, only the changes in VP receptor levels are parallel to the changes in pituitary ACTH responsiveness. The inhibitory effect of chronic osmotic stimulation on ACTH secretion in spite of high circulating levels of VP is probably the result of diminished activity of parvicellular PVN neurons and downregulation of pituitary VP receptors. Although the exact interaction between regulatory factors and the molecular mechanisms controlling the sensitivity of the corticotroph during adaptation to chronic stress remain to be determined, it is clear that regulation of the proportional secretion of CRH and VP in the PVN, modulation of pituitary VP receptors, and the sensitivity to feedback inhibition play a critical role.

The regulation of corticotropin-releasing factor (CRF) mRNA expression in the rat brain by glucocorticoids and stress was examined by Northern blot analysis and in situ hybridization histochemistry. Rats either were exposed to a single electrical footshock session and killed 2, 4, 12, or 24 hr later (acute stress), or were subjected to the same regimen twice daily for 3 or 7 d and killed on the day following the last session (chronic stress). Rats placed in the experimental chamber but not administered shock comprised a "sham-handling" group. Chronic (7 d) intermittent footshock stress resulted in an 84 +/- 26% (P less than 0.05) increase in CRF mRNA levels in the whole hypothalamus as detected by Northern blot analysis and a 97 +/- 29% (P less than 0.05) increase in the paraventricular nucleus (PVN) as detected using in situ hybridization. No significant change in CRF mRNA levels was observed in the hypothalamus at any time up to 24 hr after a single exposure to footshock stress. A different pattern of results was obtained in other CRF-expressing cell groups. In Barrington's nucleus (a pontine micturition center), both acute and chronic stress produced significant increases in CRF mRNA, while in the olfactory bulb, both paradigms resulted in decreased levels. By Northern blot analysis, CRF mRNA in the olfactory bulb declined steadily, beginning at 4 hr after acute stress, and reached significance at 24 hr (69.2 +/- 1.9% of control, P less than 0.05). Levels from chronically (7 d) stressed animals declined to 54.1 +/- 5.1% of control value (P less than 0.05). Analysis of hybridization histochemical material revealed that both the number of positively hybridized cells and the number of silver grains per cell in the mitral and external plexiform layers of the bulb decreased following acute and chronic stress. However, CRF mRNA levels in the olfactory bulb were decreased to a comparable extent in the sham-handling group, suggesting that exposure to a novel environment can effect a decrease in CRF mRNA levels in the olfactory bulb. To provide comparisons with the effects of manipulation of glucocorticoid status, comparable analyses were carried out in separate groups of animals following adrenalectomy (ADX) with and without corticosteroid replacement. After ADX, CRF mRNA levels in the whole hypothalamus increased 60 +/- 5% (P less than 0.05) and were normalized following dexamethasone replacement. In contrast to the hypothalamus, no effects of steroid manipulation on CRF mRNA levels in the olfactory bulb, midbrain, cerebral cortex, or brain stem were detected.(ABSTRACT TRUNCATED AT 400 WORDS)

Disruptions in homeostasis (ie, stress) place demands on the body that are met by the activation of 2 systems, the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS). Stressor-induced activation of the HPA axis and the SNS results in a series of neural and endocrine adaptations known as the "stress response" or "stress cascade." The stress cascade is responsible for allowing the body to make the necessary physiological and metabolic changes required to cope with the demands of a homeostatic challenge. Here we discuss the key elements of the HPA axis and the neuroendocrine response to stress. A challenge to homeostasis (a stressor) initiates the release of corticotropin-releasing hormone (CRH) from the hypothalamus, which in turn results in release of adrenocortiotropin hormone (ACTH) into general circulation. ACTH then acts on the adrenal cortex resulting in release of a species-specific glucocorticoid into blood. Glucocorticoids act in a negative feedback fashion to terminate the release of CRH. The body strives to maintain glucocorticoid levels within certain boundaries and interference at any level of the axis will influence the other components via feedback loops. Over- or underproduction of cortisol can result in the devastating diseases of Cushing's and Addison's, respectively, but less severe dysregulation of the HPA axis can still have adverse health consequences. These include the deposition of visceral fat as well as cardiovascular disease (eg, atherosclerosis). Thus, chronic stress with its physical and psychological ramifications remains a persistent clinical problem for which new pharmacological treatment strategies are aggressively sought. To date, treatments have been based on the existing knowledge concerning the brain areas and neurobiological substrates that subserve the stress response. Thus, the CRH blocker, antalarmin, is being investigated as a treatment for chronic stress because it prevents CRH from having its ultimate effect-a protracted release of glucocorticoids. New therapeutic strategies will depend on the discovery of novel therapeutic targets at the cellular and intracellular level. Advances in molecular biology provide the tools and new opportunities for identifying these therapeutic targets.

A concatenation of findings from preclinical and clinical studies support a preeminent function for the corticotropin-releasing factor (CRF) system in mediating the physiological response to external stressors and in the pathophysiology of anxiety and depression. Recently, human genetic studies have provided considerable support to several long-standing hypotheses of mood and anxiety disorders, including the CRF hypothesis. These data, reviewed in this report, are congruent with the hypothesis that this system is of paramount importance in mediating stress-related psychopathology. More specifically, variants in the gene encoding the CRF(1) receptor interact with adverse environmental factors to predict risk for stress-related psychiatric disorders. In-depth characterization of these variants will likely be important in furthering our understanding of the long-term consequences of adverse experience.

The neurosteroid tetrahydroprogesterone (5 alpha-pregnan-3 alpha-ol-20-one, allopregnanolone, THP), has been previously shown to counteract the anxiogenic effects of corticotropin-releasing hormone (CRH) and to interfere with noradrenergic and corticosteroid-mediated regulation of CRH release and gene transcription. Those observations indicated that, besides its sedative and analgesic activity, THP may also affect the neuroendocrine response to stress in a mode resembling that of corticosteroids. To examine this possibility, we compared the ability of THP, its precursor progesterone (P4), and the glucocorticoids dexamethasone (DEX) and corticosterone (CORT) to influence the pituitary-adrenal response to acute emotional stress and the adrenalectomy-induced increase in the gene transcription of the stress-related peptide arginine vasopressin (AVP) and of corticosteroid receptors (MR and GR) in the brain. Pretreatment of rats with a single dose of THP or P4 (50 micrograms/kg) significantly attenuated the elevation of plasma adrenocorticotropin (ACTH) and serum corticosterone after emotional stress; both steroids were, however, less potent than a similar dose of DEX. Administration of 1 mg of THP, CORT, or P4 to adrenalectomized (ADX) rats attenuated the increase in AVP mRNA levels in the ventromedial subdivision of the hypothalamic paraventricular nucleus (PVN), as compared with vehicle-treated ADX rats. However, whereas CORT and P4 influenced the ADX-induced increase in the transcription of both types of corticosteroid receptors in the hippocampus, these were unaffected by THP. In contrast to the glucocorticoids, THP and P4 failed to decrease plasma ACTH levels in rats deprived of endogenous steroids. These results demonstrate that the neurosteroid THP and its precursor P4 resemble glucocorticoids in their suppression of the pituitary-adrenal response to emotional stress; however, THP influences the transcription of glucocorticoid-responsive genes in brain structures involved in the regulation of the hypothalamo-pituitary-adrenal system in a fashion that is quite distinct from that obtained with glucocorticoids.

Chronic intermittent access to alcohol leads to the escalation of alcohol intake, similar to binge drinking in humans. Converging lines of evidence suggest that impairment of medial prefrontal cortex (mPFC) cognitive function and overactivation of the central nucleus of the amygdala (CeA) are key factors that lead to excessive drinking in dependence. However, the role of the mPFC and CeA in the escalation of alcohol intake in rats with a history of binge drinking without dependence is currently unknown. To address this issue, we examined FBJ murine osteosarcoma viral oncogene homolog (Fos) expression in the mPFC, CeA, hippocampus, and nucleus accumbens and evaluated working memory and anxiety-like behavior in rats given continuous (24 h/d for 7 d/wk) or intermittent (3 d/wk) access to alcohol (20% vol/vol) using a two-bottle choice paradigm. The results showed that abstinence from alcohol in rats with a history of escalation of alcohol intake specifically recruited GABA and corticotropin-releasing factor (CRF) neurons in the mPFC and produced working memory impairments associated with excessive alcohol drinking during acute (24-72 h) but not protracted (16 -68 d) abstinence. Moreover, abstinence from alcohol was associated with a functional disconnection of the mPFC and CeA but not mPFC and nucleus accumbens. These results show that recruitment of a subset of GABA and CRF neurons in the mPFC during withdrawal and disconnection of the PFC-CeA pathway may be critical for impaired executive control over motivated behavior, suggesting that dysregulation of mPFC interneurons may be an early index of neuroadaptation in alcohol dependence.

OBJECTIVE

Stress often worsens the symptoms of irritable bowel syndrome (IBS). We hypothesised that this might be explained by altered neuroendocrine and visceral sensory responses to stress in IBS patients.

METHODS

Eighteen IBS patients and 22 control subjects were assessed using rectal balloon distensions before, during, and after mental stress. Ten controls and nine patients were studied in supplementary sessions. Rectal sensitivity (thresholds and intensity-visual analogue scale (VAS)) and perceived stress and arousal (VAS) were determined. Plasma levels of corticotropin releasing factor (CRF), adrenocorticotropic hormone (ACTH), cortisol, noradrenaline, and adrenaline were analysed at baseline, immediately after stress, and after the last distension. Heart rate was recorded continuously.

RESULTS

Thresholds were increased during stress in control subjects (p<0.01) but not in IBS patients. Both groups showed lower thresholds after stress (p<0.05). Repeated distensions without stress did not affect thresholds. Both groups showed increased heart rate (p<0.001) and VAS ratings for stress and arousal (p<0.05) during stress. Patients demonstrated higher ratings for stress but lower for arousal than controls. Basal CRF levels were lower in patients (p<0.05) and increased significantly during stress in patients (p<0.01) but not in controls. Patients also responded with higher levels of ACTH during stress (p<0.05) and had higher basal levels of noradrenaline than controls (p<0.01). Controls, but not patients, showed increased levels of adrenaline and noradrenaline in response to stress (p<0.05).

CONCLUSIONS

Stress induced exaggeration of the neuroendocrine response and visceral perceptual alterations during and after stress may explain some of the stress related gastrointestinal symptoms in IBS.

Fear (i.e., decreased percentage time spent on open-arm exploration) in the elevated plus-maze can be potentiated by prior inescapable stressor exposure, but not by escapable stress. The use of fear-potentiated plus-maze behaviour has several advantages as compared to more traditional animal models of anxiety. (a) In contrast to the traditional (spontaneous) elevated plus-maze, which measures innate fear of open spaces, fear-potentiated plus-maze behaviour reflects an enhanced anxiety state (allostatic state). This "state anxiety" can be defined as an unpleasant emotional arousal in face of threatening demands or dangers. A cognitive appraisal of threat is a prerequisite for the experience of this type of emotion. (b) Depending on the stressor used (e.g., fear of shock, predator odour, swim stress, restraint, social defeat, predator stress (cat)), this enhanced anxiety state can last from 90 min to 3 weeks. Stress effects are more severe when rats are isolated in comparison to group housing. (c) Drugs can be administered in the absence of the original stressor and after stressor exposure. As a consequence, retrieval mechanisms are not affected by drug treatment. (d) Fear-potentiated plus-maze behaviour is sensitive to proven/putative anxiolytics and anxiogenics which act via mechanisms related to the benzodiazepine-gamma-aminobutyric acid receptor, but it is also sensitive to corticotropin-releasing receptor antagonists and glucocorticoid receptor antagonists and serotonin receptor agonists/antagonists complex (high predictive validity). (e) Fear-potentiated plus-maze behaviour is very robust, and experiments can easily be replicated in other labs. (f) Fear-potentiated plus-maze behaviour can be measured both in males and females. (g) Neural mechanisms involved in contextual fear conditioning, fear potentiation and state anxiety can be studied.Thus, fear-potentiated plus-maze behaviour may be a valuable measure in the understanding of neural mechanisms involved in the development of anxiety disorders and in the search for novel anxiolytics. Finally, the involvement of corticotropin-releasing factor and corticosteroid-corticotropin-releasing factor interactions in the production of fear-potentiated plus-maze behaviour are discussed.