The immunocytochemical localization of neurons containing the 41 amino acid peptide corticotropin-releasing factor (CRF) in the rat brain is described. The detection of CRF-like immunoreactivity in neurons was facilitated by colchicine pretreatment of the rats and by silver intensification of the diaminobenzidine end-product. The presence of immunoreactive CRF in perikarya, neuronal processes, and terminals in all major subdivisions of the rat brain is demonstrated. Aggregates of CRF-immunoreactive perikarya are found in the paraventricular, supraoptic, medial and periventricular preoptic, and premammillary nuclei of the hypothalamus, the bed nuclei of the stria terminalis and of the anterior commissure, the medial septal nucleus, the nucleus accumbens, the central amygdaloid nucleus, the olfactory bulb, the locus ceruleus, the parabrachial nucleus, the superior and inferior colliculus, and the medial vestibular nucleus. A few scattered perikarya with CRF-like immunoreactivity are present along the paraventriculo-infundibular pathway, in the anterior hypothalamus, the cerebral cortex, the hippocampus, and the periaqueductal gray of the mesencephalon and pons. Processes with CRF-like immunoreactivity are present in all of the above areas as well as in the cerebellum. The densest accumulation of CRF-immunoreactive terminals is seen in the external zone of the median eminence, with some immunoreactive CRF also present in the internal zone. The widespread but selective distribution of neurons containing CRF-like immunoreactivity supports the neuroendocrine role of this peptide and suggests that CRF, similarly to other neuropeptides, may also function as a neuromodulator throughout the brain.

Neuroendocrine responses to psychosocial pressures have been well characterized. The defence reaction is followed by increased activity of the sympathetic nervous system. Essential hypertension might be induced by such mechanisms. The defeat reaction is characterized by increased activity along the corticotropin releasing factor-adrenocorticotropin hormone-cortisol axis, resulting in the inhibition of gonadotropin secretion. Such endocrine disturbances are followed by metabolic aberrations, and probably also by the accumulation of visceral fat. Subjects with abdominal fat accumulation (high waist/hip circumference ratio, WHR) have recently been found to exhibit a number of psychosocial handicaps, together with endocrine aberrations characteristic of the defence and, in particular, the defeat reaction, as well as the associated circulatory and metabolic aberrations. Such abnormalities, including the WHR itself, are established risk factors for cardiovascular disease and diabetes. It is postulated that increased WHR is a symptom of chronic hypothalamic arousal as a result of a defeat reaction to psychosocial pressures. This might lead to the development of disease via circulatory and metabolic derangements.

BACKGROUND

Diagnosis of adrenal insufficiency in critically ill patients has relied on random or cosyntropin-stimulated cortisol levels, and has not been corroborated by a more accurate diagnostic standard.

OBJECTIVE

We used the overnight metyrapone stimulation test to investigate the diagnostic value of the standard cosyntropin stimulation test, and the prevalence of sepsis-associated adrenal insufficiency.

METHODS

This was an inception cohort study.

RESULTS

In two consecutive septic cohorts (n = 61 and n = 40), in 44 patients without sepsis and in 32 healthy volunteers, we measured (1) serum cortisol before and after cosyntropin stimulation, albumin, and corticosteroid-binding globulin levels, and (2) serum corticotropin, cortisol, and 11beta-deoxycortisol levels before and after an overnight metyrapone stimulation. Adrenal insufficiency was defined by postmetyrapone serum 11beta-deoxycortisol levels below 7 microg/dl. More patients with sepsis (31/61 [59% of original cohort with sepsis] and 24/40 [60% of validation cohort with sepsis]) met criteria for adrenal insufficiency than patients without sepsis (3/44; 7%) (p < 0.001 for both comparisons). Baseline cortisol (< 10 microg/dl), Delta cortisol (< 9 microg/dl), and free cortisol (< 2 microg/dl) had a positive likelihood ratio equal to infinity, 8.46 (95% confidence interval, 1.19-60.25), and 9.50 (95% confidence interval, 1.05-9.54), respectively. The best predictor of adrenal insufficiency (as defined by metyrapone testing) was baseline cortisol of 10 microg/dl or less or Delta cortisol of less than 9 microg/dl. The best predictors of normal adrenal response were cosyntropin-stimulated cortisol of 44 microg/dl or greater and Delta cortisol of 16.8 microg/dl or greater.

CONCLUSIONS

In sepsis, adrenal insufficiency is likely when baseline cortisol levels are less than 10 microg/dl or delta cortisol is less than 9 microg/dl, and unlikely when cosyntropin-stimulated cortisol level is 44 microg/dl or greater or Delta cortisol is 16.8 microg/dl or greater.

BACKGROUND

Retrospective studies in humans have identified characteristics that promote stress resistance, including childhood exposure to moderately stressful events (ie, stress inoculation).

OBJECTIVE

Because of limited opportunities for prospective studies in children, we tested whether exposure to moderate stress early in life produces later stress resistance in a primate model.

METHODS

Twenty squirrel monkeys were randomized to intermittent stress inoculation (IS; n = 11) or a nonstress control condition (NS; n = 9) from postnatal weeks 17 to 27. At postnatal week 35, each mother-offspring dyad underwent testing in a moderately stressful novel environment for inferential measures of offspring anxiety (ie, maternal clinging, mother-offspring interactions, object exploration, and food consumption) and stress hormone concentrations (corticotropin [ACTH] and cortisol). At postnatal week 50, after acclimation to an initially stressful wire-mesh box attached to the home cage, independent young monkeys underwent testing for inferential measures of anxiety (ie, voluntary exploration and play) in the box.

RESULTS

In the novel environment test, IS compared with NS offspring demonstrated diminished anxiety as measured by decreased maternal clinging (P =.02), enhanced exploratory behavior (P =.005), and increased food consumption (P =.02). Mothers of IS offspring accommodated offspring-initiated exploration (P =.009) and served as a secure base more often compared with NS mothers (P =.047). Compared with NS offspring, IS offspring had lower basal plasma ACTH (P =.001) and cortisol (P =.001) concentrations and lower corticotropin (P =.04) and cortisol (P =.03) concentrations after stress. In the subsequent home-cage wire-box test, IS offspring demonstrated enhanced exploratory (P<.001) and play (P =.008) behaviors compared with NS offspring.

CONCLUSIONS

These results provide the first prospective evidence that moderately stressful early experiences strengthen socioemotional and neuroendocrine resistance to subsequent stressors. This preclinical model offers essential opportunities to improve our understanding and enhance prevention of human stress-related psychiatric disorders by elucidating the etiology and neurobiology of stress resistance.

mRNA was isolated from cultures of AtT-20/D-16v tumor cells and translated in a mRNA-dependent reticulocyte cell-free system. The corticotropin (ACTH) product was purified by a double-antibody immunoprecipitation procedure using antisera specific for the alpha(1-24) sequence of ACTH. The product is shown by sodium dodecyl sulfate/gel electrophoresis and gel filtration on guanidine-HCl columns to be homogeneous with an apparent molecular weight (Mr) of 28,500. A product with the same molecular weight is synthesized when membrane-bound polysomes from D-16v cells are allowed to complete their nascent chains in a reticulocyte cell-free system. Mr 31,000 ACTH isolated from tumor cells has been separated into three proteins of different apparent Mr:29,000, 32,000, and 34,000. The cell-free product contains the same lysine-, methionine-, and phenylalanine-labeled tryptic peptides as the Mr 29,000 ACTH synthesized in the tumor cells. Tryptic peptide analysis also reveals the presence of the alpha(1-39) sequence in the Mr 28,500 cell-free product and suggests that there is only one copy of this sequence in the Mr 28,500 molecule.

The discovery of a 41-amino acid peptide with potent corticotropin-releasing factor properties has prompted a search for neurons that contain this substance and potentially utilize it in intercellular communication. The present study utilized immunohistochemical methods and an antiserum directed against a synthetic replica of ovine corticotropin-releasing factor. The rat hypothalamus was found to contain striking immunoreactive groups of neuronal perikarya within the paraventricular, periventricular, and anterior hypothalamic nuclei, some of which are likely to project to the external layer of the median eminence and thereby comprise a hypophysiotropic system. Certain other hypothalamic nuclei, as well as many other regions of the central nervous system, were found to contain corticotropin-releasing factor-immunoreactive neurons. Among the most prominent of these were neurons in the bed nucleus of stria terminalis, the central nucleus of the amygdala, the region of the dorsal raphe, locus ceruleus, the external cuneate nucleus, and the medullary reticular formation. Thus, corticotropin-releasing factor, like many other neurohormones and peptides, may participate in neuroendocrine regulation as well as play a role as a neurotransmitter-like substance in numerous extrahypothalamic circuits.

We previously reported that corticosterone (CORT) increased corticotropin-releasing hormone (CRH) mRNA in the central nucleus of the amygdala (CEA), while reducing it in the paraventricular nucleus (PVN) of the hypothalamus by using in situ hybridization histochemistry. The bed nucleus of the stria terminalis (BNST) is closely related to the amygdala, and it is also a source of extrahypothalamic CRH; therefore, we examined CRH mRNA changes in the BNST following systemic treatment with CORT in adrenally-intact rats. Effects of adrenalectomy on CRH mRNA in the BNST, PVN and CEA were also examined. In addition, CRH content in these nuclei and in the median eminence (ME) were determined by micropunch dissection technique combined with CRH radioimmunoassay in CORT pellet implanted rats. Subcutaneous injections of high CORT (5 mg/day, over 14 days) increased CRH mRNA in the dorsal part of the lateral BNST (BSTLD) at 2, 4 and 8 days, although the low dose of CORT (1 mg/kg/day) had no significant effects. By contrast, in the ventral part of the BNST (BSTV) neither the high nor low dose of CORT altered CRH mRNA levels. In a second experiment, a slowly-releasing CORT pellet (200 mg, 60-day release) produced an elevation of CRH mRNA at both 1 and 2 weeks or at 1 week in the BSTLD or in the BSTV, respectively. These results show that glucocorticoids can facilitate CRH mRNA expression in the BSTLD in the same manner as seen in the CEA, and that CRH mRNA in the BSTLD can respond to CORT more than in the BSTV. In a third experiment, bilateral adrenalectomy, however, did not affect CRH mRNA in the BNST although there was a modest decrease in the CEA and a robust increase in the PVN. Finally, in CORT pellet (200 mg, for 2 weeks) implanted rats, CRH content in the ME significantly decreased and modestly increased in the CEA compared with control rats, whereas it did not change in the PVN and BNST. Taken together, these results suggest that (1) CRH in the BNST and the CEA may share some common functions in neuroendocrine and behavioral changes, but that (2) mechanisms of CRH synthesis or its releasing sites may be different in the BNST and CEA.

The amygdala plays a pivotal role in the generation of appropriate responses to emotional stimuli. In the case of emotional stressors, these responses include activation of the hypothalamic-pituitary-adrenal (HPA) axis. This effect is generally held to depend upon the central nucleus of the amygdala, but recent evidence suggests a role for the medial nucleus. In the present study, c-fos expression, amygdala lesion and retrograde tracing experiments were performed on adult rats in order to re-evaluate the role of the central as opposed to the medial amygdala in generating neuroendocrine responses to an emotional stressor. Brief restraint (15 min) was used as a representative emotional stressor and was found to elicit c-fos expression much more strongly in the medial than central nucleus of the amygdala; relatively few Fos-positive cells were seen in other amygdala nuclei. Subsequent experiments showed that ibotenic acid lesions of the medial amygdala, but not the central amygdala, greatly reduced restraint-induced activation of cells of the medial paraventricular nucleus, the site of the tuberoinfundibular corticotropin-releasing factor cells that constitute the apex of the HPA axis. Medial amygdala lesions also reduced the activation of supraoptic and paraventricular nucleus oxytocinergic neurosecretory cells that commonly accompanies stress-induced HPA axis activation in rodents. To assess whether the role of the medial amygdala in the control of neuroendocrine cell responses to emotional stress might involve a direct projection to such cells, retrograde tracing of amygdala projections to the paraventricular nucleus was performed in combination with Fos immunolabelling. This showed that although some medial amygdala cells activated by exposure to an emotional stressor project directly to the paraventricular nucleus, the number is very small. These findings provide the first direct evidence that it is the medial rather than the central amygdala that is critical to hypothalamic neuroendocrine cell responses during an emotional response, and also provide the first evidence that the amygdala governs oxytocin as well as HPA axis responses to an emotional stressor.

Corticotropin-releasing factor (CRF) plays a major role in coordinating the endocrine, autonomic, behavioral and immune responses to stress through actions in the brain and the periphery. CRF receptors identified in brain, pituitary and spleen have comparable kinetic and pharmacological characteristics, guanine nucleotide sensitivity and adenylate cyclase-stimulating activity. Differences were observed in the molecular mass of the CRF receptor complex between the brain (58,000 Da) and the pituitary and spleen (75,000 Da), which appeared to be due to differential glycosylation of the receptor proteins. The recently cloned CRF receptor in the pituitary and the brain (designated as CRF1) encodes a 415 amino acid protein comprising seven putative membrane-spanning domains and is structurally related to the calcitonin/vasoactive intestinal peptide/growth hormone-releasing hormone subfamily of G-protein-coupled receptors. A second member of the CRF receptor family encoding a 411 amino acid rat brain protein with approximately 70% homology to CRF1 has recently been identified (designated as CRF2); there exists an additional splice variant of the CRF2 receptor with a different N-terminal domain encoding a protein of 431 amino acids. In autoradiographic studies, CRF receptors were localized in highest densities in the anterior and intermediate lobes of the pituitary gland, olfactory bulb, cerebral cortex, amygdala, cerebellum and the macrophage-enriched zones and red pulp regions of the spleen. CRF can modulate the number of CRF receptors in a reciprocal manner. For example, stress and adrenalectomy increase hypothalamic CRF secretion which, in turn, down-regulates CRF receptors in the anterior pituitary. CRF receptors in the brain and pituitary are also altered as a consequence of the development and aging processes. In addition to a physiological role for CRF in integrating the responses of the brain, endocrine and immune systems to physiological, psychological and immunological stimuli, recent clinical data implicate CRF in the etiology and pathophysiology of various endocrine, psychiatric, neurologic and inflammatory illnesses. Hypersecretion of CRF in the brain may contribute to the symptomatology seen in neuropsychiatric disorders, such as depression, anxiety-related disorders and anorexia nervosa. Furthermore, overproduction of CRF at peripheral inflammatory sites, such as synovial joints may contribute to autoimmune diseases such as rheumatoid arthritis. In contrast, deficits in brain CRF are apparent in neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease and Huntington's disease, as they relate to dysfunction of CRF neurons in the brain areas affected in the particular disorder. Strategies directed at developing CRF-related agents may hold promise for novel therapies for the treatment of these various disorders.

Neuropeptides modulate neuronal function in hippocampus, but the organization of hippocampal sites of peptide release and actions is not fully understood. The stress-associated neuropeptide corticotropin releasing hormone (CRH) is expressed in inhibitory interneurons of rodent hippocampus, yet physiological and pharmacological data indicate that it excites pyramidal cells. Here we aimed to delineate the structural elements underlying the actions of CRH, and determine whether stress influenced hippocampal principal cells also via actions of this endogenous peptide. In hippocampal pyramidal cell layers, CRH was located exclusively in a subset of GABAergic somata, axons and boutons, whereas the principal receptor mediating the peptide's actions, CRH receptor 1 (CRF1), resided mainly on dendritic spines of pyramidal cells. Acute 'psychological' stress led to activation of principal neurons that expressed CRH receptors, as measured by rapid phosphorylation of the transcription factor cyclic AMP responsive element binding protein. This neuronal activation was abolished by selectively blocking the CRF1 receptor, suggesting that stress-evoked endogenous CRH release was involved in the activation of hippocampal principal cells.

The dysregulation of the activity of the hypothalamic-pituitary-adrenocortical (HPA) axis system is one of the major neuroendocrine abnormalities in major depression (MD). Many pieces of evidence supported that corticotropin-releasing hormone (CRH) play a role in the pathophysiology of major depression. In this article, whether genetic variations in the corticotropin-releasing hormone receptor1 (CRHR1) gene might be associated with increased susceptibility to major depression was studied by using a gene-based association analysis of single-nucleotide polymorphisms (SNPs). Three SNPs were identified in CRHR1 gene and genotyped in the samples of patients diagnosed with major depression and matched controls. We observed significant allele (P=0.0008) and genotype (P=0.0002) association with rs242939, and the haplotype defined by alleles G-G-T for the represent rs1876828, rs242939 and rs242941 was significantly over-represented in major depression patients compared to controls. These results support the idea that the CRHR1 gene is likely to be involved in the genetic vulnerability for major depression.

In recent studies to clone and characterize genes coding for the corticotropin-releasing factor-binding protein (CRF-BP), analysis of the tissue distribution of the CRF-BP gene indicated a high level of expression in the rat brain. We have now characterized by immunohistochemical and hybridization histochemical means the cellular localization of CRF-BP protein and mRNA expression, respectively. Results from both approaches converged to indicate that CRF-BP is expressed predominantly in the cerebral cortex, including all major archi-, paleo-, and neocortical fields. Other prominent sites of mRNA and protein expression include subcortical limbic system structures (amygdala, bed nucleus of the stria terminalis), sensory relays associated with the auditory, olfactory, vestibular, and trigeminal systems, severe raphe nuclei, and a number of cell groups in the brainstem reticular core. Expression in the hypothalamus appears largely limited to the ventral premammillary and dorsomedial nuclei; only isolated CRF-BP-stained cells are apparent in neurosecretory cell groups. Dual immunostaining for CRF and CRF-BP revealed a partial colocalization in some of these regions. In addition, prominent CRF-BP-stained terminal fields have been identified in association with CRF-expressing cell groups in circumscribed hypothalamic and limbic structures. In the anterior pituitary, CRF-BP mRNA and immunoreactivity were colocalized with corticotropin-immunoreactivity in a majority of corticotropes. Thus, CRF-BP could serve to modify the actions of CRF by intra- and intercellular mechanisms, in CRF-related pathways in the central nervous system and pituitary.

Hepatic gluconeogenesis is a main source of blood glucose during prolonged fasting and is orchestrated by endocrine and neural pathways. Here we show that the hepatocyte-secreted hormone fibroblast growth factor 21 (FGF21) induces fasting gluconeogenesis via the brain-liver axis. Prolonged fasting induces activation of the transcription factor peroxisome proliferator-activated receptor α (PPARα) in the liver and subsequent hepatic production of FGF21, which enters into the brain to activate the hypothalamic-pituitary-adrenal (HPA) axis for release of corticosterone, thereby stimulating hepatic gluconeogenesis. Fasted FGF21 knockout (KO) mice exhibit severe hypoglycemia and defective hepatic gluconeogenesis due to impaired activation of the HPA axis and blunted release of corticosterone, a phenotype similar to that observed in PPARα KO mice. By contrast, intracerebroventricular injection of FGF21 reverses fasting hypoglycemia and impairment in hepatic gluconeogenesis by restoring corticosterone production in both FGF21 KO and PPARα KO mice, whereas all these central effects of FGF21 were abrogated by blockage of hypothalamic FGF receptor-1. FGF21 acts directly on the hypothalamic neurons to activate the mitogen-activated protein kinase extracellular signal-related kinase 1/2 (ERK1/2), thereby stimulating the expression of corticotropin-releasing hormone by activation of the transcription factor cAMP response element binding protein. Therefore, FGF21 maintains glucose homeostasis during prolonged fasting by fine tuning the interorgan cross talk between liver and brain.

Targeted mutagenesis of the glucocorticoid receptor has revealed an essential function for survival and the regulation of multiple physiological processes. To investigate the effects of an increased gene dosage of the receptor, we have generated transgenic mice carrying two additional copies of the glucocorticoid receptor gene by using a yeast artificial chromosome. Interestingly, overexpression of the glucocorticoid receptor alters the basal regulation of the hypothalamo-pituitary-adrenal axis, resulting in reduced expression of corticotropin-releasing hormone and adrenocorticotrope hormone and a fourfold reduction in the level of circulating glucocorticoids. In addition, primary thymocytes obtained from transgenic mice show an enhanced sensitivity to glucocorticoid-induced apoptosis. Finally, analysis of these mice under challenge conditions revealed that expression of the glucocorticoid receptor above wild-type levels leads to a weaker response to restraint stress and a strongly increased resistance to lipopolysaccharide-induced endotoxic shock. These results underscore the importance of tight regulation of glucocorticoid receptor expression for the control of physiological and pathological processes. Furthermore, they may explain differences in the susceptibility of humans to inflammatory diseases and stress, depending on individual prenatal and postnatal experiences known to influence the expression of the glucocorticoid receptor.

A well established body of work indicates a crucial role for corticotropin-releasing factor (CRF) in neurobiological responses associated with excessive dependence-like ethanol drinking in ethanol-vapor-exposed rodents. Recent evidence demonstrates a role for CRF in the modulation of binge-like ethanol consumption by nondependent mice, a behavior that can precede ethanol dependence. The CRF circuitry that is engaged by binge-like ethanol exposure, however, is unknown. Using converging approaches, we provide evidence that, similar to ethanol-vapor-induced increases in ethanol intake, CRF signaling in the central nucleus of the amygdala (CeA) is engaged during binge-like ethanol consumption by C57BL/6J mice. Specifically, we found that binge-like consumption of an ethanol solution (20% ethanol v/v) was attenuated by pretreatment with the CRF1R antagonists antalarmin, 4-ethyl-[2,5,6-trimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]amino-1-butanol, and NBI-27914 at doses (30 mg/kg, i.p.) that did not alter nonbinge-like ethanol consumption. Binge-like ethanol consumption resulted in significant increases of CRF immunoreactivity in the CeA immediately following ethanol drinking and 18-24 h following ethanol removal and also blocked the ability of CRF to enhance GABAergic transmission in the CeA 18-24 h following ethanol removal. Pretreatment with bilateral injections of antalarmin (1 μg/0.5 μl per side) into the CeA, but not the adjacent basolateral amygdala, significantly attenuated binge-like ethanol consumption. These findings suggest that CRF signaling in the CeA is recruited during excessive ethanol intake, before the development of dependence. We hypothesize that plastic changes in CRF signaling develop with repeated binge-like drinking episodes, contributing to the transition to dependence.

Irritable bowel syndrome (IBS) is a common and potentially disabling functional gastrointestinal disorder characterized by abdominal pain and altered bowel patterns. A significant amount of clinical and research data suggest the importance of the brain-gut interaction in IBS. This review examines the observed high prevalence of psychiatric disorders in patients with IBS. The published literature indicates that fewer than half of individuals with IBS seek treatment for it. Of those who do, 50% to 90% have psychiatric disorders, including panic disorder, generalized anxiety disorder, social phobia, posttraumatic stress disorder, and major depression, while those who do not seek treatment tend to be psychologically normal. Both physiologic and psychosocial variables appear to play important roles in the development and maintenance of IBS. Recent information suggests that the association of IBS and psychiatric disorders may be more fundamental than was previously believed. A brain-gut model for IBS is presented, and the role of traumatic stress and corticotropin-releasing factor as modulators of the brain-gut loop is discussed. Finally, the rationale for the use of psychotropic agents in the treatment of IBS with or without psychiatric symptoms is presented.

The hypothalamo-pituitary-adrenal (HPA) axis represents a complex neuroendocrine feedback loop controlling the secretion of adrenal glucocorticoid hormones. Central to its function is the paraventricular nucleus of the hypothalamus (PVN) where neurons expressing corticotropin releasing factor reside. These HPA motor neurons are a primary site of integration leading to graded endocrine responses to physical and psychological stressors. An important regulatory factor that must be considered, prior to generating an appropriate response is the animal's reproductive status. Thus, PVN neurons express androgen and estrogen receptors and receive input from sites that also express these receptors. Consequently, changes in reproduction and gonadal steroid levels modulate the stress response and this underlies sex differences in HPA axis function. This review examines the make up of the HPA axis and hypothalamo-pituitary-gonadal (HPG) axis and the interactions between the two that should be considered when exploring normal and pathological responses to environmental stressors.

A 35S-labeled 48-base synthetic oligonucleotide complementary to a portion of the rat corticotropin-releasing factor (CRF) mRNA was used for in situ hybridization histochemistry. CRF-synthesizing cells were located in the paraventricular nucleus of the hypothalamus. These cells were observed in the medial parvocellular subdivision where there was a 90% increase in the amount of CRF mRNA per unit volume after adrenalectomy.

Drug addiction is a chronically relapsing disorder that is characterized by a compulsion to take drugs and loss of control in limiting intake. Medications that are on the market for the treatment of drug addiction target either the direct reinforcing effects of abuse (eg, naltrexone) or the consequent protracted abstinence syndrome (eg, acamprosate). Both conceptual and neurobiological advances in research have suggested that brain stress systems contribute to the withdrawal/negative affect and preoccupation/anticipation stages of the addiction cycle that promote the compulsivity of drug-taking in addiction. Validated animal models of the stress component of addiction and improved understanding of the neurocircuitry and neuropharmacological mechanisms involved in perturbations of this component suggest that corticotropin-releasing factor systems are a viable target for the development of future medications for drug addiction.

The nonpeptide CRH antagonist antalarmin has been shown to block both behavioral and endocrine responses to CRH. However, it's potential activity in blunting behavioral and endocrine sequelae of stressor exposure has not been assessed. Because antagonism of central CRH by alpha-helical CRH attenuates conditioned fear responses, we sought to test antalarmin in this regard. In addition, it remains unclear as to whether this is a result of receptor blockade during conditioning or during testing. Thus, we explored whether CRH mediates the induction or expression of conditioned fear (freezing in a context previously associated with 2 footshocks; 1.0 mA, 5 sec each). Furthermore, because rats previously exposed to inescapable shock (IS; 100 shocks, 1.6 mA, 5 sec each), demonstrate enhanced fear conditioning, we investigated whether this effect would be blocked by antalarmin. Antalarmin (20 mg/kg x 2 ml i.p.) impaired both the induction and expression of conditioned fear. In addition, antalarmin blocked the enhancement of fear conditioning produced by prior exposure to IS. Despite the marked behavioral effects observed in antalarmin-treated rats, antalarmin had no effect on IS-induced rises in ACTH or corticosterone. However, antalarmin did block the ACTH response produced by exposure to 2 footshocks.

The central nucleus of the amygdala is a CRF-containing limbic brain site which mediates both fear-like and avoidance behaviors, and intra-amygdala administration of a CRF antagonist blocks the increase in anxiogenic-like behavior characteristic of ethanol withdrawal. In order to evaluate the role of brain CRF in negative motivational states associated with other classes of abused drugs, the present studies examined the effects of suppression of amygdala CRF systems on the characteristic aversive state of precipitated withdrawal in morphine-dependent subjects. In a place conditioning paradigm, administration of a CRF antagonist, alpha-belical CRF (9-41) [250ng], bilaterally into the central nucleus of amygdala, reversed the withdrawal-induced conditioned place aversion produced by injection of the opiate antagonist, methylnaloxonium [500ng], into the same site. In a conditioned operant suppression paradigm, impairment of CRF neurons by immuno-targeted toxins administered into the central nucleus of amygdala, one month prior to testing, attenuated the decrease in response rate produced by exposure to distinctive sensory cues associated previously with systemic administration of naloxone [25µg/kg s.c.] in morphine-dependent subjects. These results indicate that suppression of intra-amygdala CRF systems weakens the aversive stimulus properties of conditioned opiate withdrawal, and suggest a general role for CRF in coordinating behavioral responses to negative motivational effects of drug withdrawal.

OBJECTIVE

Although it has been shown that des-acyl ghrelin decreases food intake and gastric emptying, no previous studies have examined the effects of des-acyl ghrelin on physiologic fed and fasted motor activity in the gastrointestinal tract.

METHODS

We examined the effects of intraperitoneal (IP) administration of des-acyl ghrelin on food intake and the effects of intracerebroventricular (ICV) or intravenous (IV) administration of des-acyl ghrelin on gastroduodenal motility using freely moving conscious rat models. The brain nuclei responding to these effects were examined by c- fos immunohistochemistry of the brain sections.

RESULTS

IP injection of des-acyl ghrelin decreased food intake, and this effect was not altered by capsaicin treatment. IP injection of des-acyl ghrelin enhanced c- fos expression in the arcuate and paraventricular nucleus but not in the nucleus of the solitary tract. Both ICV and IV injection of des-acyl ghrelin disrupted fasted motor activity in the antrum but not in the duodenum. Changes in gastric motility induced by IV injection of des-acyl ghrelin were completely antagonized by ICV injection of a selective corticotropin-releasing factor (CRF) 2 receptor antagonist; however, the CRF 1 receptor antagonist had no effects.

CONCLUSIONS

The results suggest that des-acyl ghrelin decreases food intake and disrupts the fasted motor activity of the antrum in freely moving conscious rats. Peripheral des-acyl ghrelin may induce this function by direct activation of brain receptor by crossing the blood-brain barrier but not by the activation of vagal afferent pathways. In the brain, CRF 2 receptor, but not CRF 1 receptor, is involved in this action.

In the present study, we established and characterized an animal model of vulnerability to repeated stress. We found that control Sprague-Dawley (SD) rats showed a gradual decrease in the HPA axis response following 14 days of repeated restraint stress, whereas Fischer 344 (F344) rats did not show such HPA axis habituation. Similar habituation was observed in the expression of c-fos mRNA, corticotropin-releasing hormone hnRNA, and phospho-CREB and phospho-ERK proteins in the hypothalamic paraventricular nucleus (PVN) of SD rats, but not in the F344 rats. In addition, repeatedly restrained F344 rats exhibited decreased cell proliferation in the dentate gyrus of the hippocampus and increased anxiety-related behaviours, while repeatedly restrained SD rats exhibited a selective enhancement of hippocampal cell proliferation in the ventral area. Moreover, we found a lower expression of glucocorticoid receptor (GR) protein, but not mRNA, in the PVN of F344 rats compared to SD rats. We also identified that microRNA (miR)-18a inhibited translation of GR mRNA in cultured neuronal cells and that increased expression of miR-18a in the PVN was observed in F344 rats compared with SD rats. These strain differences in GR protein levels were not found in the hippocampus and prefrontal cortex, and the expression of miR-18a was much lower in these brain regions than in the PVN. Our results suggest that F344 rats could be a useful animal model for studying vulnerability to repeated stress, and that miR-18a-mediated down-regulation of GR translation may be an important factor to be considered in susceptibility to stress-related disorders.

The serotonergic system arising from the dorsal raphe nucleus (DR) has long been implicated in psychiatric disorders, and is considered one site of action of classical anxiolytic and antidepressant agents. Recent studies implicate the DR as a site of action of novel anxiolytic and antidepressant agents that target neuropeptide systems, such as corticotropin-releasing factor (CRF) and neurokinin 1 (NK1) antagonists. The present study identified unique characteristics of the dorsomedial DR that implicate this particular subregion as a key component of a circuit, which may be targeted by these diverse psychotherapeutic agents. First, it was observed that a cluster of CRF-containing cell bodies was present in the dorsomedial DR of colchicine-treated rats. Dual-labeling immunohistochemistry revealed that almost all CRF-containing neurons were serotonergic, implicating CRF as a cotransmitter with serotonin in this subpopulation of DR neurons. Moreover, dendrites laden with immunoreactivity for NK1 had a striking topographic distribution surrounding and extending into the dorsomedial subregion of the DR, suggesting that NK1 receptor ligands may selectively impact the dorsomedial DR. Finally, anterograde tract tracing from the dorsomedial DR combined with CRF immunohistochemistry revealed that CRF-containing axons from this subregion project to CRF-containing neurons of the central nucleus of the amygdala. Taken together, the present results reveal a circuit whereby NK1 receptor activation in the dorsomedial DR can impact on limbic sources of CRF that have been implicated in emotional responses. This circuit may be relevant for understanding the mechanism of action of novel psychotherapeutic agents that act through NK1 or CRF receptors.