Brain Corticotropin-releasing Factor Receptors Research Articles

Stimulation of brain corticotropin-releasing factor receptor type1 facilitates the rat micturition via brain glutamatergic receptors

Corticotropin-releasing factor (CRF), a representative stress-related neuropeptide, in the central nervous system reportedly both facilitates and suppresses the micturition, therefore, roles of central CRF in regulation of the micturition are still controversial. In this study, we investigated (1) effects of intracerebroventricularly (icv)-administered CRF on the micturition, and (2) brain CRF receptor subtypes (CRFR1/CRFR2) and glutamatergic receptors (NMDA/AMPA subtypes) involved in the CRF-induced effects in male Wistar rats under urethane anesthesia. Intercontraction intervals (ICI), and maximal voiding pressure (MVP), were evaluated by continuous cystometry 45 min before CRF administration or intracerebroventricular pretreatment with other drugs as follows and 3 h after CRF administration. Single-voided volume (Vv), post-voiding residual volume (Rv), bladder capacity (BC), and voiding efficiency (VE) were evaluated by single cystometry 60 min before CRF administration and 60–120 min after the administration. Icv-administered CRF reduced ICI, Vv, and BC without changing MVP, Rv, or VE. The CRF-induced ICI reduction was attenuated by icv-pretreated CP154526 (CRFR1 antagonist), MK-801 (NMDA receptor antagonist), and DNQX (AMPA receptor antagonist), but not by K41498 (CRFR2 antagonist). These results indicate that stimulation of brain CRFR1 can be involved in facilitation of the rat micturition via brain NMDA/AMPA receptors.

Molecular Basis for Hormone Recognition and Activation of Corticotropin-Releasing Factor Receptors.

Corticotropin-releasing factor (CRF) and the three related peptides urocortins 1-3 (UCN1-UCN3) are endocrine hormones that control the stress responses by activating CRF1R and CRF2R, two members of class B G-protein-coupled receptors (GPCRs). Here, we present two cryoelectron microscopy (cryo-EM) structures of UCN1-bound CRF1R and CRF2R with the stimulatory G protein. In both structures, UCN1 adopts a single straight helix with its N terminus dipped into the receptor transmembrane bundle. Although the peptide-binding residues in CRF1R and CRF2R are different from other members of class B GPCRs, the residues involved in receptor activation and G protein coupling are conserved. In addition, both structures reveal bound cholesterol molecules to the receptor transmembrane helices. Our structures define the basis of ligand-binding specificity in the CRF receptor-hormone system, establish a common mechanism of class B GPCR activation and G protein coupling, and provide a paradigm for studying membrane protein-lipid interactions for class B GPCRs.

Brain and Gut CRF Signaling: Biological Actions and Role in the Gastrointestinal Tract.

Corticotropin-releasing factor (CRF) pathways coordinate behavioral, endocrine, autonomic and visceral responses to stress. Convergent anatomical, molecular, pharmacological and functional experimental evidence supports a key role of brain CRF receptor (CRF-R) signaling in stress-related alterations of gastrointestinal functions. These include the inhibition of gastric acid secretion and gastric-small intestinal transit, stimulation of colonic enteric nervous system and secretorymotor function, increase intestinal permeability, and visceral hypersensitivity. Brain sites of CRF actions to alter gut motility encompass the paraventricular nucleus of the hypothalamus, locus coeruleus complex and the dorsal motor nucleus while those modulating visceral pain are localized in the hippocampus and central amygdala. Brain CRF actions are mediated through the autonomic nervous system (decreased gastric vagal and increased sacral parasympathetic and sympathetic activities). The activation of brain CRF-R2 subtype inhibits gastric motor function while CRF-R1 stimulates colonic secretomotor function and induces visceral hypersensitivity. CRF signaling is also located within the gut where CRF-R1 activates colonic myenteric neurons, mucosal cells secreting serotonin, mucus, prostaglandin E2, induces mast cell degranulation, enhances mucosal permeability and propulsive motor functions and induces visceral hyperalgesia in animals and humans. CRF-R1 antagonists prevent CRF- and stressrelated gut alterations in rodents while not influencing basal state. These preclinical studies contrast with the limited clinical positive outcome of CRF-R1 antagonists to alleviate stress-sensitive functional bowel diseases such as irritable bowel syndrome. The translational potential of CRF-R1 antagonists in gut diseases will require additional studies directed to novel anti-CRF therapies and the neurobiology of brain-gut interactions under chronic stress.

CRF and urocortin peptides as modulators of energy balance and feeding behavior during stress.

Early on, corticotropin-releasing factor (CRF), a hallmark brain peptide mediating many components of the stress response, was shown to affect food intake inducing a robust anorexigenic response when injected into the rodent brain. Subsequently, other members of the CRF signaling family have been identified, namely urocortin (Ucn) 1, Ucn 2, and Ucn 3 which were also shown to decrease food intake upon central or peripheral injection. However, the kinetics of feeding suppression was different with an early decrease following intracerebroventricular injection of CRF and a delayed action of Ucns contrasting with the early onset after systemic injection. CRF and Ucns bind to two distinct G-protein coupled membrane receptors, the CRF1 and CRF2. New pharmacological tools such as highly selective peptide CRF1 or CRF2 agonists or antagonists along with genetic knock-in or knock-out models have allowed delineating the primary role of CRF2 involved in the anorexic response to exogenous administration of CRF and Ucns. Several stressors trigger behavioral changes including suppression of feeding behavior which are mediated by brain CRF receptor activation. The present review will highlight the state-of-knowledge on the effects and mechanisms of action of CRF/Ucns-CRF1/2 signaling under basal conditions and the role in the alterations of food intake in response to stress.

Corticotropin-releasing factor receptor type-2 is involved in the cocaine-primed reinstatement of cocaine conditioned place preference in rats

Here we explored the in vivo role of brain corticotropin-releasing factor receptor type-2 (CRFR2) in cocaine-primed reinstatement of drug seeking. Conditioned place preference (CPP) procedure was used to assess the acquisition, extinction and reinstatement of cocaine-seeking behavior in rats. First, expressions of CRFR2 were shown to be affected in a brain region-specific manner within cocaine-induced CPP and cocaine-extinct CPP models. Bilateral blockade of CRFR2 in the dorsal portion of the medial prefrontal cortex (mPFC), or hippocampus (HP) was partially inhibited, but in the dorsal striatum (DS) did not affect, the cocaine-primed reinstatement of cocaine CPP.

CRF Facilitates Calcium Release from Intracellular Stores in Midbrain Dopamine Neurons

Changes in cytosolic calcium are crucial for numerous processes including neuronal plasticity. This study investigates the regulation of cytosolic calcium by corticotropin-releasing factor (CRF) in midbrain dopamine neurons. The results demonstrate that CRF stimulates the release of intracellular calcium from stores through activation of adenylyl cyclase and PKA. Imaging and photolysis experiments showed that the calcium originated from dendrites and required both functional IP3 and ryanodine receptor channels. The elevation in cytosolic calcium potentiated calcium-sensitive potassium channels (sK) activated by action potentials and metabotropic Gq-coupled receptors for glutamate and acetylcholine. This increase in cytosolic calcium activated by postsynaptic Gs-coupled CRF receptors may represent a fundamental mechanism by which stress peptides and hormones can shape Gq-coupled receptor-mediated regulation of neuronal excitability and synaptic plasticity in dopamine neurons.

Characterization and regulation of corticotropin-releasing factor receptors in the central nervous, endocrine and immune systems.

Corticotropin-releasing factor (CRF) plays a major role in coordinating the endocrine, autonomic, behavioural and immune responses to stress through actions in the brain and in 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 brain (58,000 Da) and pituitary and spleen (75,000 Da), which appeared to be due to differential glycosylation of the receptor proteins. In autoradiographic studies, CRF receptors were localized in highest densities in anterior and intermediate lobes of the pituitary, olfactory bulb, cerebral cortex, amygdala, cerebellum and the macrophage-rich marginal zones and red pulp regions of the spleen. CRF can modulate the number of CRF receptors in both the brain and pituitary in a reciprocal manner. The demonstration of functional CRF receptors in brain, pituitary and spleen suggests the importance of this neuropeptide in integrating the responses of the CNS, endocrine and immune systems to physiological, psychological and immunological stimuli.

Region-specific effects of brain corticotropin-releasing factor receptor type 1 blockade on footshock-stress- or drug-priming-induced reinstatement of morphine conditioned place preference in rats

Systemic injections of the selective corticotropin-releasing factor 1 (CRF1) receptor antagonist CP-154,526 attenuate footshock-stress-induced reinstatement of heroin and cocaine seeking and morphine conditioned place preference (CPP). Intracranial injections of the nonselective CRF receptor antagonist D-Phe-CRF into the bed nucleus of the stria terminalis (BNST), but not the amygdala, attenuate footshock-induced reinstatement of cocaine seeking. However, the brain sites involved in the effect of CP-154,526 on footshock-induced reinstatement of opiate seeking are unknown. We used a CPP version of the reinstatement model to examine the role of CRF1 receptors in the BNST, amygdala, and nucleus accumbens (NAc) in footshock- or drug-priming-induced reinstatement of extinguished morphine CPP. Rats acquired morphine CPP over a period of 8 days during which they were given four morphine (10 mg/kg s.c.) and four saline injections and were subsequently confined to distinct chambers for 50 min. Subsequently, the morphine CPP was extinguished in 14 daily sessions during which rats were given saline injections and given access to both the saline- and morphine-paired chambers. The rats were then tested for reinstatement of morphine CPP induced by priming injections of morphine (0 or 3.0 mg/kg s.c.) or by exposure to intermittent footshock (15 min, 0.5 mA). Prior to the test sessions, the rats were given intracranial injections of CP-154,526 (1.0 microg) or vehicle into the BNST, amygdala, or NAc. CP-154,526 injections into the BNST, but not the amygdala or NAc, attenuated footshock-stress-induced reinstatement of morphine CPP. In contrast, CP-154,526 injections into the amygdala or NAc, but not the BNST, attenuated morphine-priming-induced reinstatement of morphine CPP. The present results demonstrate dissociable roles of CRF1 receptors in the BNST, amygdala, and NAc in footshock-stress- vs morphine-priming-induced reinstatement of drug CPP.

Persistent corticotropin-releasing factor 1 receptor desensitization and downregulation in the human neuroblastoma cell line IMR-32

Brain corticotropin-releasing factor (CRF) systems integrate various responses to stress. Pathological responses to stress may result from errors in CRF receptor regulation in response to changes in synaptic CRF levels. To establish an in vitro model to study brain CRF receptors, we characterized the CRF-induced modulation of CRF 1 receptors in the human neuroblastoma cell line, IMR-32. Treatment with CRF decreased CRF 1 receptor binding and desensitized CRF-induced increases in cAMP. The decrease in binding had an EC 50 of ∼10 nM, was maximal by 30 min, and was blocked by the CRF receptor antagonist [ d-Phe 12, Nle 21,38, C α–MeLeu 37]CRF 12–41. The desensitization was homologous as vasoactive intestinal polypeptide-induced increases in cAMP were unchanged, and elevation of cAMP did not alter CRF 1 receptor binding. Treatment with CRF for up to 24 h did not alter CRF 1 receptor mRNA levels, suggesting that a posttranscriptional mechanism maintains the decrease in receptor binding. Interestingly, recovery of CRF receptor binding and CRF-stimulated cAMP production was only partial following exposure to 100 nM CRF. In contrast, receptor binding recovered to control levels following exposure to 10 nM CRF. These data suggest that exposure to high doses of CRF result in permanent changes characterized by only partial recovery. Identifying the mechanisms underlying this partial recovery may provide insights into mechanisms underlying the acute and chronic effects of stress on CRF receptor regulation.

Dissociation of arousal-like from anxiogenic-like actions of brain corticotropin-releasing factor receptor ligands in rats

Behavioral actions of centrally administered corticotropin-releasing factor (CRF) are likely subserved by multiple brain targets and functional effector systems. The present studies compared effects of two CRF ligands, a full, post-synaptic CRF receptor agonist (rat/human CRF(1–41)) and a CRF binding protein ligand inhibitor (rat/human CRF(6–33)) in a behavioral testing battery sensitive to arousal, fear-like and aversive processes in Wistar rats. The profile of global efficacy for the centrally administered CRF receptor agonist was characterized by low dose (0.5–1.0 μg) arousal-like effects in locomotor and conditioned ambulation contexts and by high dose (5–25 μg) conditioned immobility, taste aversion and place aversion. In contrast, a profile of limited efficacy for the centrally administered CRF binding protein ligand inhibitor included only dose dependent motor activating and facilitation of fear conditioning effects without any of the anxiogenic-like or aversive properties of CRF agonist administration. These results suggest that arousal-like activation is a fundamental, physiologically relevant consequence of brain CRF system stimulation whereas aversive and anxiety-like effects reflect pharmacological actions of a CRF receptor agonist.

Stress and the gastrointestinal tract III. Stress-related alterations of gut motor function: role of brain corticotropin-releasing factor receptors.

Alterations of gastrointestinal (GI) motor function are part of the visceral responses to stress. Inhibition of gastric emptying and stimulation of colonic motor function are the commonly encountered patterns induced by various stressors. Activation of brain corticotropin-releasing factor (CRF) receptors mediates stress-related inhibition of upper GI and stimulation of lower GI motor function through interaction with different CRF receptor subtypes. CRF subtype 1 receptors are involved in the colonic and anxiogenic responses to stress and may have clinical relevance in the comorbidity of anxiety/depression and irritable bowel syndrome.

Distribution of mRNAs encoding CRF receptors in brain and pituitary of rat and mouse.

Two G protein-coupled receptors have been identified that bind corticotropin-releasing factor (CRF) and urocortin (UCN) with high affinity. Hybridization histochemical methods were used to shed light on controversies concerning their localization in rat brain, and to provide normative distributional data in mouse, the standard model for genetic manipulation in mammals. The distribution of CRF-R1 mRNA in mouse was found to be fundamentally similar to that in rat, with expression predominating in the cerebral cortex, sensory relay nuclei, and in the cerebellum and its major afferents. Pronounced species differences in distribution were few, although more subtle variations in the relative strength of R1 expression were seen in several forebrain regions. CRF-R2 mRNA displayed comparable expression in rat and mouse brain, distinct from, and more restricted than that of CRF-R1. Major neuronal sites of CRF-R2 expression included aspects of the olfactory bulb, lateral septal nucleus, bed nucleus of the stria terminalis, ventromedial hypothalamic nucleus, medial and posterior cortical nuclei of the amygdala, ventral hippocampus, mesencephalic raphe nuclei, and novel localizations in the nucleus of the solitary tract and area postrema. Several sites of expression in the limbic forebrain were found to overlap partially with ones of androgen receptor expression. In pituitary, rat and mouse displayed CRF-R1 mRNA signal continuously over the intermediate lobe and over a subset of cells in the anterior lobe, whereas CRF-R2 transcripts were expressed mainly in the posterior lobe. The distinctive expression pattern of CRF-R2 mRNA identifies additional putative central sites of action for CRF and/or UCN. Constitutive expression of CRF-R2 mRNA in the nucleus of the solitary tract, and stress-inducible expression of CRF-R1 transcripts in the paraventricular nucleus may provide a basis for understanding documented effects of CRF-related peptides at a loci shown previously to lack a capacity for CRF-R expression or CRF binding. Other such "mismatches" remain to be reconciled.

Corticotropin-releasing factor and systemic capsaicin-sensitive afferents are involved in abdominal surgery-induced Fos expression in the paraventricular nucleus of the hypothalamus

We previously reported that abdominal surgery induces Fos expression in specific hypothalamic and medullary nuclei and also causes gastric stasis. The gastric ileus is reduced by systemic capsaicin and abolished by central injection of corticotropin-releasing factor (CRF) antagonist. We studied the influence of systemic capsaicin and intracerebroventricular (i.c.v.) injection of the CRF antagonist, α-helical CRF 9–41, on Fos expression in the brain 1 h after abdominal surgery in conscious rats using immunocytochemical detection. In control groups (vehicle s.c. or i.c.v.), abdominal surgery (laparotomy with cecal manipulation) performed under 7–8 min of enflurane anesthesia induced Fos staining in neurons of the spinal trigeminal, C1/A1 group, ventrolateral medulla, central amygdala, parabrachial nucleus, cuneate nucleus, nucleus tractus solitarii (NTS), paraventricular nucleus of the hypothalamus (PVN) and supraoptic nucleus (SON). Capsaicin (125 mg/kg s.c., 2 weeks before) or α-helical CRF 9–41 (50 μg i.c.v., before surgery) reduced the number of Fos-positive cells by 50% in the PVN while not modifying the number of Fos-labelled cells in the other nuclei. These results indicate that capsaicin-sensitive primary afferents and brain CRF receptors are part of the pathways and biochemical coding through which abdominal surgery activates PVN neurons 1 h post surgery.

Affinity purification of 61- and 65-kDa rat brain corticotropin-releasing factor receptors and receptor-associated G proteins

Corticotropin-releasing factor (CRF) has been shown to have a central role in physiological adaptation to stress. It is recognized for stimulating the release of adrenocorticotropin from the anterior pituitary gland, and has more recently been implicated as a regulator of autonomic and immunological responses to stress. Much confusion has surrounded the characterization of CRF receptors, with proteins of varying molecular weights having been identified but never purified and characterized. Recently, two CRF receptors have been cloned from brain and pituitary gland, but evidence from in-situ hybridization studies suggests that further CRF receptor types exist. We therefore developed two techniques which enable the isolation of CRF receptors from whole rat brain. The use of a solid-phase CRF analogue affinity column and elution using a competing ligand resulted in the purification of a single protein of 61 kDa. A second technique was devised which allowed the co-isolation of associated signalling proteins and the identification of CRF bound species following purification. CRF was covalently cross-linked to receptors and the complex purified using antibodies specific for the ligand. This enabled the purification of a CRF receptor of approximately 65 kDa and associated α and βγ G protein subunits. This study demonstrates the successful isolation of CRF receptors which are of different molecular weights to those previously observed from affinity cross-linking studies or predicted from cloned genes. In addition, we confirm the involvement of G proteins in CRF stimulated cell signalling by demonstrating their association with purified CRF receptor.

Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor.

Corticotropin-releasing factor (CRF), a peptide first isolated from mammalian brain, is critical in the regulation of the pituitary-adrenal axis, and in complementary stress-related endocrine, autonomic and behavioural responses. Fish urotensin I and amphibian sauvagine were considered to be homologues of CRF until peptides even more closely related to CRF were identified in these same vertebrate classes. We have characterized another mammalian member of the CRF family and have localized its urotensin-like immunoreactivity to, and cloned related complementary DNAs from, a discrete rat midbrain region. The deduced protein encodes a peptide that we name urocortin, which is related to urotensin (63% sequence identity) and CRF (45% sequence identity). Synthetic urocortin evokes secretion of adrenocorticotropic hormone (ACTH) both in vitro and in vivo and binds and activates transfected type-1 CRF receptors, the subtype expressed by pituitary corticotropes. The coincidence of urotensin-like immunoreactivity with type-2 CRF receptors in brain, and our observation that urocortin is more potent than CRF at binding and activating type-2 CRF receptors, as well as at inducing c-Fos (an index of cellular activation) in regions enriched in type-2 CRF receptors, indicate that this new peptide could be an endogenous ligand for type-2 CRF receptors.

Corticotropin-releasing factor (CRF) receptors in infant rhesus monkey brain and pituitary gland: biochemical characterization and autoradiographic localization.

A large body of data suggests that the corticotropin-releasing factor (CRF) system serves to coordinate the autonomic, endocrine, immune and behavioral aspects of the stress response. In rats, the distribution of CRF receptors in brain and pituitary has been well characterized, however, little information is available in primates. In this study, CRF receptors were characterized by radioligand binding and localized using autoradiography with [125I]-oCRF in the pituitary gland and in discrete brain regions of 2-week-old, 12-week-old and adult rhesus monkeys. Autoradiographic localization studies in slide-mounted tissue sections in the 2- and 12-week-old monkeys demonstrated high CRF receptor densities in both anterior and intermediate lobes of the pituitary as well as in discrete regions of the brain. The distribution of CRF receptors in the anterior pituitary demonstrated a 'cluster-like' appearance reminiscent of corticotrope distribution. In contrast, receptors in the intermediate lobe were more uniformly distributed. No significant differences were evident in the pattern of localization or the number of CRF receptors in the pituitaries of 2- compared to 12-week-old animals. However, marked differences were observed in the dentate gyrus of the hippocampus. Receptors in this region were absent in 1- to 2-week old animals but quite dense by 11-12 weeks of age. Conversely, in the lateral and medial geniculate nuclei, high levels of CRF receptors were identified early in life that virtually disappeared by 11-12 weeks of age. Thus, there is considerable correspondence in the development of the CRF system between the rat and rhesus monkey and this presents further evidence for the functional role of this peptide in brain development.

Effect of corticotropin-releasing factor antagonist on behavioral and neuroendocrine responses during exposure to defensive burying paradigm in rats

Defensive burying behavior is a coping strategy in rodents in response to an aversive stimulus where fear will facilitate burying and treatment with anxiolytics will result in less burying. To test the hypothesis that endogenous corticotropin-releasing factor (CRF) is involved in the defensive burying response, the effects of an ICV CRF antagonist were tested on defensive burying behaviors as well as the plasma adrenocorticotropic hormone (ACTH) and plasma corticosterone response. Rats were allowed to self-administer one mild electric shock (1.0 mA) through a probe mounted on the wall of the home cage by briefly touching it. Following this shock, control rats typically display burying behavior, as defined as moving the bedding material toward and/or over the shock probe. A CRF antagonist, α-hel CRF 9–41, was administered ICV (1, 5, and 25 μg) 1 min after they received the shock. The 5 μg dose of the CRF antagonist significantly attenuated the time spent in defensive burying compared to vehicle-treated controls. The 5 and 25 μg doses of α-hel CRF 9–41 increased the latency to display defensive burying. However, the enhanced release of plasma ACTH and plasma corticosterone concentrations in the stressed animals was not significantly modified over the 15-min period by either of the three doses of CRF antagonist. The results suggest that endogenous CRF is involved in the expression of defensive burying behavior and that the brain CRF receptors modulating the behavioral response may be different from the CRF receptors modulating the hormonal response of defensive burying.

Cloning and functional expression of a rat brain corticotropin releasing factor (CRF) receptor

Corticotropin releasing factor (CRF), a key neuroregulator of the hypothalamic-pituitary-adrenal cortical axis, also displays a broad range of effects on the endocrine, central nervous and immune systems. Having recently characterized the human pituitary CRF receptor by expression cloning of cDNA from a human Cushing's corticotropic adenoma, we report here the structure of the cDNA for a rat brain CRF receptor (rCRF-R) which was cloned by hybridization from a rat brain cDNA library. The sequence of the rCRF-R encodes a 415 amino acid protein comprising seven membrane spanning domains. The rCRF-R is 97% identical at the amino acid level to the human pituitary tumor CRF receptor, differing by only 12 amino acids. When expressed in COSM6 cells, the rCRF-R binds CRF with high affinity (Kd = 1.7 (0.8-3.8)nM). The receptor transduces a CRF stimulated accumulation of intracellular cAMP which is inhibited by the CRF antagonist, alpha helCRF(9-41). These results suggest that the brain expresses a CRF receptor similar to that in the pituitary.

Cloning and functional expression of a rat brain corticotropin releasing factor (CRF) receptor.

Corticotropin releasing factor (CRF), a key neuroregulator of the hypothalamic-pituitary-adrenal cortical axis, also displays a broad range of effects on the endocrine, central nervous and immune systems. Having recently characterized the human pituitary CRF receptor by expression cloning of cDNA from a human Cushing's corticotropic adenoma, we report here the structure of the cDNA for a rat brain CRF receptor (rCRF-R) which was cloned by hybridization from a rat brain cDNA library. The sequence of the rCRF-R encodes a 415 amino acid protein comprising seven membrane spanning domains. The rCRF-R is 97% identical at the amino acid level to the human pituitary tumor CRF receptor, differing by only 12 amino acids. When expressed in COSM6 cells, the rCRF-R binds CRF with high affinity (Kd = 1.7 (0.8-3.8)nM). The receptor transduces a CRF stimulated accumulation of intracellular cAMP which is inhibited by the CRF antagonist, alpha helCRF(9-41). These results suggest that the brain expresses a CRF receptor similar to that in the pituitary.

Effects of chronic stress on anterior pituitary and brain corticotropin-releasing factor receptors

[ 125I]Corticotropin-releasing factor binding was measured in membrane homogenates prepared from the anterior pituitary, frontal cortex, motor cortex, somatosensory cortex, mesolimbic area (olfactory tubercle and nucleus accumbens), caudate putamen, hypothalamus, midbrain, and cerebellum from control and chronically stressed rats. The stressor consisted of 3 or 14 days of around-the-clock intermittent foot-shock (approximately one trial per 5-min frequency) that could be avoided or escaped on 90% of the trials presented by pulling a ceiling chain. Plasma corticosterone levels were almost doubled in stressed rats following 3 days of chronic stress and remained significantly elevated in rats stressed for 14 days as compared to controls. Plasma corticotropin levels were similar in controls and stressed animals in both the 3- and 14-day experiments. [ 125I]Corticotropin-releasing factor binding was decreased in anterior pituitary and frontal cortex following 3 ways of chronic stress; binding affinity of anterior pituitary membranes was not different between control and stressed animals. [ 125I]Corticotropin-releasing factor binding was similar in control and 3-day-stressed animals in the other brain areas examined. After 14 days of chronic stress, hypothalamic [ 125I]corticotropin-releasing factor binding was decreased in stressed rats as compared to control animals but no other differences were seen. The decrease in the apparent number of anterior pituitary corticotropin-releasing factor receptors following 3 days of stress may be due, in part, to increased plasma corticosterone levels and/or increased corticotropin-releasing factor secretion during that time. The downregulation of frontal cortex and hypothalamic corticotropin-releasing factor receptors, however, is more likely to be primarily in response to sustained stress-induced corticotropin-releasing factor release at those sites.