Cellular and molecular mechanisms of hippocampal activation by acute stress are age-dependent.

The effects of stress, including their putative contribution to pathological psychiatric conditions, are crucially governed by the age at which the stress takes place. However, the cellular and molecular foundations for the impact of stress on neuronal function, and their change with age, are unknown. For example, it is not known whether 'psychological' stress signals are perceived by similar neuronal populations at different ages, and whether they activate similar or age-specific signaling pathways that might then mediate the spectrum of stress-evoked neuronal changes. We employed restraint and restraint/noise stress to address these issues in juvenile (postnatal day 18, [P18]) and adult rats, and used phosphorylation of the transcription factor CREB (pCREB) and induction of c-fos as markers of hippocampal neuronal responses. Stress-activated neuronal populations were identified both anatomically and biochemically, and selective blockers of the stress-activated hippocampal peptide, corticotropin-releasing hormone (CRH) were used to probe the role of this molecule in stress-induced hippocampal cell activation. Stress evoked strikingly different neuronal response patterns in immature vs adult hippocampus. Expression of pCREB appeared within minutes in hippocampal CA3 pyramidal cells of P18 rats, followed by delayed induction of Fos protein in the same cell population. In contrast, basal pCREB levels were high in adult hippocampus and were not altered at 10-120 min by stress. Whereas Fos induction was elicited by stress in the adult, it was essentially confined to area CA1, with little induction in CA3. At both age groups, central pretreatment with either a nonselective blocker of CRH receptors (alpha-helical CRH [9-41]) or the CRF1-selective antagonist, NBI 30775, abolished stress-evoked neuronal activation. In conclusion, hippocampal neuronal responses to psychological stress are generally more rapid and robust in juvenile rats, compared to fully mature adults, and at both ages, CRH plays a key role in this process. Enhanced hippocampal response to stress during development, and particularly the activation of the transcription factor CREB, may contribute to the enduring effects of stress during this period on hippocampal function.

1-Alkyl-3-amino-5-aryl-1H-[1,2,4]triazoles: novel synthesis via cyclization of N-acyl-S-methylisothioureas with alkylhydrazines and their potent corticotropin-releasing factor-1 (CRF(1)) receptor antagonist activities.

Cyclizations of alkylhydrazines with N-acyl-S-methylisothioureas, readily synthesized from acyl chlorides, sodium thioisocyanate, dialkylamines then methyl iodide in a one-pot reaction, gave 1-alkyl-3-dialkylamino-5-phenyltriazoles 7 as major products. The regioisomers were assigned through the use of NOE NMR experiments. While bearing a N-bis(cyclopropyl)methyl-N-propylamino group, this series of compounds shows very good binding affinity on the human CRF(1) receptor. Among them, 1-methyl-3-[N-bis(cyclopropyl)methyl-N-propylamino]-5-(2,4-dichlorophenyl)-1H-[1,2,4]triazole 7a had the best binding affinity for the CRF(1) receptor (K(i)=9 nM).

Neuroprotective effects of the CRF1 antagonist R121920 after permanent focal ischemia in the rat.

The neuroprotective effects of a systemically active, highly selective, corticotropin-releasing factor-1 (CRF1) receptor antagonist, R121920 ((7-(dipropylamino)-2,5-dimethyl-3- [2-(dimethylamino)-5-pyridyl] pyrazolo [1,5-a] pyrimidine), was assessed in two rat models of permanent focal cerebral ischemia, where the middle cerebral artery (MCA) was occluded either through the subtemporal approach or using the intraluminal suture technique. R121920 rapidly crossed the blood-brain barrier after intravenous (IV) bolus administration (10 mg/kg), with peak brain concentrations at 5 minutes (2.26 +/- 0.40 microg/mL), which were approximately 2-fold greater than those in plasma (0.98 +/- 0.24 microg/mL). Treatment with R121920 (10 mg/kg IV followed by 5 mg/kg subcutaneously at hourly intervals for 4 hours) significantly (P < 0.001) reduced total (by 40%) and cortical (by 37%) infarct volume at 24 hours after subtemporal MCA occlusion (MCAO). In the intraluminal suture MCAO model, IV administration of R121920 (10 mg/kg) at the time of ischemia onset (and at multiple times thereafter) reduced both hemispheric infarct volume (by 34%, P < 0.001) and brain swelling (by 50%, P < 0.001) when assessed at 24 hours. In this model of focal ischemia, significant reduction (P < 0.05) in both outcome measures was obtained when R121920 administration was delayed up to 1 hour after MCAO. These results further define the antiischemic properties of selective CRF 1 antagonists in two experimental models of permanent focal cerebral ischemia.

Preterm birth: associated neuroendocrine, medical, and behavioral risk factors.

Increased CRH secretion by the placenta of pregnant women has been associated with preterm birth. Certain indices of risk, both medical and psychosocial in nature, have been linked to preterm delivery. Levels of total, bound, and free CRH, CRH-binding protein (CRH-BP), and cortisol were measured prospectively in a large sample of pregnant Danish women who delivered preterm and term infants. Measures of maternal serum hormones were taken at 7--23 and 27--37 weeks gestation and, for those who delivered at term, at 37--43 weeks gestation. At 7--23 weeks gestation, maternal levels of total CRH (P = 0.01), bound CRH (P = 0.03), and CRH-BP (P = 0.01) were higher in the preterm than in the term group. At 27--37 weeks gestation, levels of total CRH (P < 0.0001), bound CRH (P < 0.0001), free CRH (P < 0.0001), and cortisol (P < 0.0001) were all higher in the preterm than the term group, whereas levels of CRH-BP (P < 0.0001) were lower in the preterm than in the term group. The best medical and behavioral factors associated with preterm delivery were, respectively, previous preterm delivery (P < 0.0001) and engagement in certain risk-taking behaviors (P = 0.008). The positive relations between preterm delivery and various adverse medical and socioeconomic variables with increases in placental secretion of CRH suggest that the latter may participate in the pathophysiology of preterm delivery.

Role of corticotropin-releasing factor (CRF) receptors in the anorexic syndrome induced by CRF.

Genetic manipulations of corticotropin-releasing factor (CRF)(1) and CRF(2) receptors have resulted in data suggesting that the CRF(2) receptor could mediate the effects of CRF on appetite or satiety. We have attempted to obtain pharmacological evidence for this hypothesis by comparing the ability of a high-affinity peptide, mixed CRF antagonist [cyclo 30-33,f12,L18,21E30, A32,K33]sucker fish urotensin (12-41)NH(2) [cUTSN (12-41)] with a small-molecule CRF(1)-selective antagonist, NBI-27914, and a CRF(2)-selective peptide antagonist, antisauvagine-30, to attenuate the anorexic effects of CRF. We also monitored other behaviors that accompanied CRF-induced anorexia. CRF-induced anorexia was significantly correlated with a reduction in locomotor activity and an increase in freezing behavior and piloerection. cUTSN (12-41) and antisauvagine-30 significantly attenuated the effects of CRF (0.04 nmol) on food intake along with the behavioral syndrome that accompanied anorexia. In contrast, NBI-27914 did not attenuate either of the above-mentioned CRF-induced phenomena when given centrally at doses ranging from 0.13 to 10 nmol/2.5 microl or when given orally at 20 to 40 mg/kg. Although these data support the hypothesis that the CRF(2) receptor mediates the appetite suppression induced by CRF, they also suggest that the CRF(2) receptor could mediate the stress-like behaviors that accompany CRF-induced appetite suppression.

Selective impairment of corticotropin-releasing factor1 (CRF1) receptor-mediated function using CRF coupled to saporin.

CRF is the main component in the brain neuropeptide effector system responsible for the behavioral, endocrine, and physiological activation that accompanies stress activation. Reduced CRF system activation plays a role in the etiology of a variety of psychiatric and metabolic disease states. We have developed a novel protein conjugate that joins native rat/human CRF to a ribosome-inactivating protein, saporin (CRF-SAP), for the purpose of targeted inactivation of CRF receptor-expressing cells. Cytotoxicity measurements revealed that CRF-SAP (1-100 nM) produced concentration-dependent and progressive cell death over time in CRF1 receptor-transfected L cells, but at similar concentrations had no effect on CRF2alpha receptor-transfected cells. The CRF-SAP-induced toxicity in CRF1-transfected cells was prevented by coincubation with the competitive CRF1/CRF2 receptor peptide antagonist, [D-Phe12]CRF-(12-41), or the selective nonpeptide CRF1 receptor antagonist, NBI 27914. Finally, in cultured rat pituitary cells that express native CRF1 receptors, CRF-SAP suppressed CRF-induced (1 nM) ACTH release. GnRH (1-10 nM) stimulated LH release was also assessed in the same pituitary cultures. Although there was a slight decrease in LH release from these cultures, this decrease was observed with CRF-SAP or SAP alone, suggesting that the response was nonspecific. Taken together, these results suggest the utility of CRF-SAP as a specific and subtype-selective tool for long term impairment of CRF1 receptor-expressing cells.

Recent advances with the CRF1 receptor: design of small molecule inhibitors, receptor subtypes and clinical indications.

Corticotropin-releasing factor (CRF) has been widely implicated as playing a major role in modulating the endocrine, autonomic, behavioral and immune responses to stress. The recent cloning of multiple receptors for CRF as well as the discovery of non-peptide receptor antagonists for CRF receptors have begun a new era of CRF study. Presently, there are five distinct targets for CRF with unique cDNA sequences, pharmacology and localization. These fall into three distinct classes, encoded by three different genes and have been termed the CRF1 and CRF2 receptors (belonging to the superfamily of G-protein coupled receptors) and the CRF-binding protein. The CRF2 receptor exists as three splice variants of the same gene and have been designated CRF2a CRF2b and CRF2g. The pharmacology and localization of all of these proteins in brain has been well established. The CRF1 receptor subtype is localized primarily to cortical and cerebellar regions while the CRF2a receptor is localized to subcortical regions including the lateral septum, and paraventricular and ventromedial nuclei of the hypothalamus. The CRF2b receptor is primarily localized to heart, skeletal muscle and in the brain, to cerebral arterioles and choroid plexus. The CRF2g receptor has most recently been identified in human amygdala. Expression of these receptors in mammalian cell lines has made possible the identification of non-peptide, high affinity, selective receptor antagonists. While the natural mammalian ligands oCRF and r/hCRF have high affinity for the CRF1 receptor subtype, they have lower affinity for the CRF2 receptor family making them ineffective labels for CRF2 receptors. [125I]Sauvagine has been characterized as a high affinity ligand for both the CRF1 and the CRF2 receptor subtypes and has been used in both radioligand binding and receptor autoradiographic studies as a tool to aid in the discovery of selective small molecule receptor antagonists. A number of non-peptide CRF1 receptor antagonists that can specifically and selectively block the CRF1 receptor subtype have recently been identified. Compounds such as CP 154,526 (12), NBI 27914 (129) and Antalarmin (154) inhibit CRF-stimulation of cAMP or CRF-stimulated ACTH release from cultured rat anterior pituitary cells. Furthermore, when administered peripherally, these compounds compete for ex vivo [125I]sauvagine binding to CRF1 receptors in brain sections demonstrating their ability to cross the blood-brain-barrier. In in vivo studies, peripheral administration of these compounds attenuate stress-induced elevations in plasma ACTH levels in rats demonstrating that CRF1 receptors can be blocked in the periphery. Furthermore, peripherally administered CRF1 receptor antagonists have also been demonstrated to inhibit CRF-induced seizure activity. These data clearly demonstrate that non-peptide CRF1 receptor antagonists, when administered systemically, can specifically block central CRF1 receptors and provide tools that can be used to determine the role of CRF1 receptors in various neuropsychiatric and neurodegenerative disorders. In addition, these molecules will prove useful in the discovery and development of potential orally active therapeutics for these disorders.

Localization of ligand-binding domains of human corticotropin-releasing factor receptor: a chimeric receptor approach.

Two CRF receptors, CRFR1 and CRFR2, have recently been cloned and characterized. CRFR1 shares 70% sequence identity with CRFR2, yet has much higher affinity for rat/human CRF (r/hCRF) than CRFR2. As a first step toward understanding the interactions between rat/human CRF and its receptor, the regions that are involved in receptor-ligand binding and/or receptor activation were determined by using chimeric receptor constructs of the two human CRFR subtypes, CRFR1 and CRFR2, followed by generating point mutations of the receptor. The EC50 values in stimulation of intracellular cAMP of the chimeric and mutant receptors for the peptide ligand were determined using a cAMP-dependent reporter system. Three regions of the receptor were found to be important for optimal binding of r/hCRF and/or receptor activation. The first region was mapped to the junction of the third extracellular domain and the fifth transmembrane domain; substitution of three amino acids of CRFR1 in this region (Val266, Tyr267, and Thr268) by the corresponding CRFR2 amino acids (Asp266, Leu267, and Val268) increased the EC50 value by approximately 10-fold. The other two regions were localized to the second extracellular domain of the CRFR1 involving amino acids 175-178 and His189 residue. Substitutions in these two regions each increased the EC50 value for r/hCRF by approximately 7- to 8-fold only in the presence of the amino acid 266-268 mutation involving the first region, suggesting that their roles in peptide ligand binding might be secondary.

Brain corticotropin-releasing factor immunoreactivity and receptors in five inbred rat strains: relationship to forced swimming behaviour.

In the present work we studied the relationship between behaviour in the forced swimming test (FST), a test that presumably measures depressive-like behaviour in rodents, and central corticotropin-releasing factor (CRF) concentration and binding in five strains of rats. The strains were: Brown-Norway (BN), Fisher (FIS) 344, Lewis (LEW), spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). The FST data corresponding to the pretest showed significant inter-strain differences in both struggling and immobility: BN and WKY rats displayed lower levels of struggling and longer periods of immobility, LEW and SHR rats showed intermediate levels, and FIS rats were the most active. The results of the pretest were roughly similar to those observed in the test, the activity of WKY being extremely low. The CRF binding revealed significant inter-strain differences in prefrontal cortex and hippocampus, but not in cerebellum, pons-medulla or hypothalamus: in the prefrontal cortex, BN and FIS rats showed greater CRF binding than LEW, SHR and WKY rats; in the hippocampus BN rats showed higher levels of CRF binding than the other strains. The study of CRF content in various brain areas revealed inter-strain differences in prefrontal cortex and pons-medulla, but not in parietal-temporal cortex or in hypothalamus (CRF concentrations in the hippocampus were not detectable): CRF content in the prefrontal cortex was higher in BN than in the other strains, although the differences with FIS were not statistically significant; in the pons-medulla, FIS and LEW showed significantly higher CRF content than the other strains. From the present results it appears that BN and WKY rats were more prone to adopt passive strategies in the FST, but they did not show higher brain CRF immunoreactivity or down-regulation of CRF receptors. Hence, although there were inter-strains differences in all variables studied, no evidence for a relationship between the FST behaviour and central CRF activity was found.

Localization of agonist- and antagonist-binding domains of human corticotropin-releasing factor receptors.

The CRF receptors, CRFR1 and CRFR2, are members of the G protein-coupled receptor superfamily. Despite their considerable sequence similarity, CRFR1 and CRFR2 have quite different affinities for the peptide ligand rat/human CRF. Previous studies using chimeric receptors between human CRFR1 and CRFR2 have identified three potentially important regions in the second and third extracellular domains of CRF receptor for the binding of rat/human CRF. The present report further demonstrates that these same three regions also affect the binding of urocortin and sauvagine, two other members of the CRF peptide family, albeit to different extents. We also show that a fourth region in the third extracellular domain, Asp254, has been identified to be important for sauvagine but not CRF or urocortin binding. Thus, the three peptide ligands not only interact with a different set of regions on CRFR1 and CRFR2 but also differentially interact with some of the same regions. These data could, at least in part, account for the much higher affinity of CRFR2 for urocortin and sauvagine compared with rat/human CRF. We have also identified two amino acid residues, His199 in the third transmembrane domain and Met276 in the fifth transmembrane domain, that are important for binding the non-peptide high-affinity CRFR1 antagonist NBI 27914. Mutations of His199 and Met276 to the corresponding amino acids in CRFR2 each decreased the binding affinity of NBI 27914 for CRFR1 by 40- and 200-fold, respectively. This suggests that the transmembrane regions are critically important in forming the binding pocket for the nonpeptide antagonist.

125I-Tyro-sauvagine: a novel high affinity radioligand for the pharmacological and biochemical study of human corticotropin-releasing factor 2 alpha receptors.

Corticotropin-releasing factor (CRF) receptors encoded by two distinct genes have recently been identified and termed CRF1 and CRF2. CRF and the non-mammalian-related peptide sauvagine bind to and activate CRF1 receptors with high affinity and equal potency. Although CRF is significantly weaker at the CRF2 receptor, sauvagine retains its high affinity interactions with this receptor subtype. We expressed the human CRF1 and CRF2 receptor subtypes in stable cell lines and characterized 125I-Tyr0-sauvagine, a high affinity radiolabel suitable for the pharmacological and functional profiles of these proteins. 125I-Tyr0-sauvagine has high affinity (200-400 PM) for CRF1 receptors and demonstrates a pharmacological profile identical to that of 125I-Tyr0-ovine CRF-labeled CRF1 receptors. 125I-Tyr0-sauvagine binding to human CRF2 alpha receptors is saturable and of high affinity (KD = 100-300 PM) and demonstrates guanine nucleotide sensitivity typical of agonist binding to receptors. The pharmacological profile of 125I-Tyr0-sauvagine binding to CRF2 alpha receptors with respect to inhibition by CRF-related analogs is similar to the agonist profile of potencies obtained by measurements of cAMP production stimulated by these analogs in CRF2 alpha expressing cell lines and distinct from the profile of the CRF1 receptor subtype. Thus, the related nonmammalian peptides sauvagine and urotensin have high affinity and rat/ human CRF and ovine CRF have lower affinity for CRF2 receptors labeled with 125I-Tyr0-sauvagine. Because the distribution of CRF1 and CRF2 alpha receptors has been demonstrated to be distinct, suggesting selective functional roles for each receptor subtype, the ability to label CRF2 alpha receptors with 125I-Tyr0-sauvagine in vitro represents a unique opportunity for the discovery of subtype-selective nonpeptide ligands, which would presumably target different aspects of CRF-mediated disorders. We have thus identified and characterized a novel high affinity radioligand for the labeling of CRF2 receptors.

Neurobiology of corticotropin releasing factor (CRF) receptors and CRF-binding protein: implications for the treatment of CNS disorders.

The actions of CRF in the brain and in the periphery are mediated through multiple binding sites. There are three receptors, CRF1, CRF2 alpha and CRF2 beta, which encode 411, 415 and 431 amino acid proteins and transduce signals via the stimulation of intracellular cAMP production. The recent identification of high-affinity non-peptide CRF receptor antagonists should allow for rapid progress in drug development of CRF receptor antagonists. In addition to the receptors, the actions of CRF in brain and in the periphery can also be modulated by a binding protein of 322 amino acids. Ligands of CRF-BP, such as CRF (6-33) can elevate brain levels of 'free' CRF and improve learning and memory without stress-like side effects of CRF receptor agonists. Urocortin, a mammalian CRF-related peptide with close sequence homology to fish urotensin, interacts with CRF1, CRF2 receptors and with CRF-BP. These data indicate that CRF receptor antagonists may be useful for the treatment of the disease states where CRF is elevated such as anxiety and depression, anorexia nervosa and stroke and that ligand inhibitors of CRF-BP may be used to elevate brain levels of 'free' urocortin and other CRF-related peptides.

Corticotrophin-releasing factor receptors: from molecular biology to drug design.

Corticotrophin-releasing factor (CRF) acts within both the brain and the periphery to coordinate the overall response of the body to stress. The involvement of the CRF systems in a variety of both CNS and peripheral disease states has stimulated great interest in this peptide as a potential site of therapeutic intervention. The recent cloning of multiple CRF receptor subtypes has precipitated a new era in CRF research that has allowed precise molecular, pharmacological and anatomical examination of mammalian CRF receptors. In this article, Derek Chalmers and colleagues highlight the major differences between the two classes of CRF receptors, CRF1 and CRF2, and a functionally related CRF-binding protein, and discuss the relevance of these sites to the ongoing development of CRF-based therapeutics.

Homologous desensitization of human corticotropin-releasing factor1 receptor in stable transfected mouse fibroblast cells.

Previous radioligand binding and second messenger studies have shown that corticotropin-releasing factor (CRF) modulates its receptor following both in vivo and in vitro treatment. In the present study, we determined the sequence of events leading to CRF-induced downregulation and desensitization of cloned CRF receptors in murine fibroblast cells (Ltk-) stably transfected with CRF1 DNA (from human pituitary). Treatment of cells with rat/human CRF produced a dose- and time-dependent decrease in [125I]Tyr degrees-ovine CRF ([125I]oCRF) binding and a concomitant decrease in CRF-stimulated adenylate cyclase activity. Significant decreases in [125I]oCRF binding and agonist-stimulated cAMP production were evident minutes after CRF treatment with maximal (60-80%) reductions seen following 1 h of CRF treatment. Scatchard analysis revealed that the decrease in [125I]oCRF binding was due to the downregulation of the receptor with no significant alteration seen in the affinity of the ligand. Since the transfected cell line is engineered using an artificial promoter, we did not detect any significant changes in CRF1 receptor mRNA levels following CRF treatment for up to 24 h.

Cloning and characterization of the human corticotropin-releasing factor-2 receptor complementary deoxyribonucleic acid.

Two CRF receptor subtypes (CRF1 and CRF2 receptors) with distinct brain localizations and pharmacological profiles have recently been cloned and characterized. For the CRF2 receptor subtype, at least 2 splice forms with different 5'-coding sequences (CRF2 alpha and CRF2 beta) have been identified in rat. In this article, we report the genomic structure and the corresponding complementary DNA (cDNA) sequence of the human CRF2 receptor. The gene coding for human CRF2 receptor consists of at least 12 exons and spans approximately 30 kilobases. The cDNA sequence in the protein-coding region is 94% identical to that of the reported rat CRF2 alpha receptor. At present, there is no evidence for the existence of a CRF2 beta receptor homolog in humans. The encoded receptor is 411 amino acids in length and is 70% identical to the human CRF1 receptor, with least sequence homology in the N-terminal extracellular domain (47% identical). Cells transfected with the full-length human CRF2 receptor cDNA responded to rat/human CRF and sauvagine by increasing the intracellular cAMP level, with EC50 values of approximately 20 and 1 nM, respectively. The CRF- and sauvagine-induced accumulation of intracellular cAMP could be competitively inhibited by the CRF receptor antagonist D-Phe-CRF. This pharmacological profile was comparable to that of the rat CRF2 alpha receptor. The relative abundance of the CRF2 receptor messenger RNA appears to be lower in humans than in rats for the tissues studied thus far.

Relative affinities of dopaminergic drugs at dopamine D2 and D3 receptors.

Quantitative autoradiography was used to evaluate the pharmacological profile of dopamine D2-like receptors labeled by [125I]iodosulpiride. Caudate/putamen, a brain region associated primarily with dopamine D2 receptor mRNA, was used as a prototypical D2 tissue; cerebellar lobule X (D3 mRNA associated), as a D3 tissue. 7-OH-DPAT ((+/-)-2-dipropylamino-7-hydroxy-1,2,3,4- tetrahydronaphthalene) exhibited selectively for cerebellar receptors (24-fold), followed by quinpirole (6-fold). Haloperidol and domperidone were 4- and 18-fold more potent at striatal receptors, respectively. These data are in close agreement with that derived from dopamine D2 and D3 receptor-expressing cell lines.

Previous stress alters corticotropin-releasing factor neurotransmission in the locus coeruleus.

Spontaneous and stress-evoked discharge of locus coeruleus neurons were characterized in rats with a history of stress. Rats exposed to one or five daily 30-min sessions of footshock were anesthetized with halothane and surgically prepared for locus coeruleus single-unit recording immediately following the last session. Locus coeruleus spontaneous discharge rate and discharge evoked by sciatic nerve stimulation were comparable between acutely and repeatedly stressed rats and controls. In contrast, locus coeruleus activation produced by intracerebroventricular administration of corticotropin-releasing factor (3 micrograms) or by hypotensive challenge (which requires endogenous corticotropin-releasing factor release in the locus coeruleus) was greatly attenuated in acutely stressed rats. The corticotropin-releasing factor dose-response curve was shifted to the right in acutely stressed rats compared with controls. In repeatedly stressed rats, the effects of 3 micrograms corticotropin-releasing factor on locus coeruleus discharge were similarly diminished. Although the maximum effect produced by corticotropin-releasing factor was decreased in these rats, the dose-response curve was shifted to the left, indicative of sensitization. Hypotensive challenge, which was ineffective in acutely stressed rats, increased locus coeruleus discharge of repeatedly stressed rats by a similar magnitude as in matched controls. The return of locus coeruleus responsiveness to hypotension in repeatedly stressed rats may be related to the sensitization to corticotropin-releasing factor. Finally, the protocol of repeated stress did not alter the affinity or density of corticotropin-releasing factor receptors in either the frontal cortex or brainstem. Taken together, the results suggest that a history of stress alters corticotropin-releasing factor neurotransmission in the locus coeruleus at the postsynaptic level. However, these effects are not reflected by corticotropin-releasing factor binding kinetics in brainstem. Stress-induced changes in corticotropin-releasing factor neurotransmitter function in the locus coeruleus may play a role in certain symptoms of stress-related psychiatric disorders.