Activating and 'anxiogenic' effects of corticotropin releasing factor are not inhibited by blockade of the pituitary-adrenal system with dexamethasone

Central administration of corticotropin releasing factor (CRF) in rats produces pituitary-adrenal activation and a variety of "anxiogenic-like" effects. The present study was designed to explore the contribution of the peripheral pituitary-adrenocortical axis in mediating these CRF responses. Intraventricularly administered CRF produced suppression of responding in the conflict test and a marked locomotor activation. Neither behavioral effect was altered by the prior administration of dexamethasone in a dose that blocked pituitary-adrenal activation to CRF. These results support the hypothesis that behavioral effects of CRF are mediated by its action at central sites and not via an action on the pituitary-adrenocortical system.     

Associated endocrine,physiological and behavioral changes in rhesus monkeys after intravenous corticotropin-releasing factor administration

The intravenous (IV) administration of synthetic ovine corticotropin-releasing factor (CRF) (10 and 125 μg/kg) to chair restrained rhesus monkeys stimulated the pituitary-adrenal axis. At these doses, increases in plasma concentrations of adrenocorticotropic hormone (ACTH) and cortisol were associated with blood pressure decreases and behavioral effects. These data demonstrate that synthetic ovine CRF (10 and 125 μg/kg) administered IV to the rhesus monkey results in associated endocrine, physiological, and behavioral changes.     

Corticotropin-releasing factor administered intraventricularly to rhesus monkeys

Synthetic ovine corticotropin-releasing factor (CRF) administered intraventricularly (ICV) to rhesus monkeys resulted in endocrine and behavioral changes. At doses of 20 and 180 micrograms, CRF stimulated the pituitary-adrenal axis in four chair-restrained monkeys. These monkeys showed concomitant increases in arousal. To study these animals in a less restrictive setting, three of the monkeys later received CRF ICV (20 and 180 micrograms) in their home cages. At the 180-micrograms dose the monkeys exhibited a combination of huddling and lying down behavior. These behavioral effects did not seem to be due to alterations in blood pressure.     

Cortagine infused into the medial prefrontal cortex attenuates predator-induced defensive behaviors and Fos protein production in selective nuclei of the amygdala in male CD1 mice

Corticotropin-releasing factor (CRF) plays an essential role in coordinating the autonomic, endocrine and behavioral responses to stressors. In this study, we investigated the role of CRF within the medial prefrontal cortex (mPFC) in modulating unconditioned defensive behaviors, by examining the effects of microinfusing cortagine a selective type-1 CRF receptor (CRF1) agonist, or acidic-astressin a preferential CRF1 antagonist, into the mPFC in male CD-1 mice exposed to a live predator (rat exposure test—RET). Cortagine microinfusions significantly reduced several indices of defense, including avoidance and freezing, suggesting a specific role for CRF1 within the infralimbic and prelimbic regions of the mPFC in modulating unconditioned behavioral responsivity to a predator. In contrast, microinfusions of acidic-astressin failed to alter defensive behaviors during predator exposure in the RET. Cortagine microinfusions also reduced Fos protein production in the medial, central and basomedial, but not basolateral subnuclei of the amygdala in mice exposed to the rat predatory threat stimulus. These results suggest that CRF1 activation within the mPFC attenuates predator-induced unconditioned anxiety-like defensive behaviors, likely via inhibition of specific amygdalar nuclei. Furthermore, the present findings suggest that the mPFC represents a unique neural region whereby activation of CRF1 produces behavioral effects that contrast with those elicited following systemic administration of CRF1 agonists.     

Effects of urocortin 2 and 3 on motor activity and food intake in rats

Urocortin 2 (Ucn 2) and Ucn 3 are new members of the corticotropin-releasing factor (CRF) family and bind selectively to the CRF type 2 receptor (CRF2). The effects of these peptides on behavioral changes induced by CRF were examined in rats. In a familiar environment, intracerebroventricular injection of Ucn 2 attenuated the stimulatory effect of CRF on motor activity, although it alone produced no effect. Ucn 3 suppressed motor activity and attenuated the stimulatory effect of CRF. In an open field, CRF decreased locomotion and rearing but increased grooming behavior. Ucn 2 attenuated the inhibition of locomotor activity induced by CRF without affecting other activities, such as rearing or grooming behavior. Ucn 3 had no effect on the behavioral changes induced by CRF, although it alone decreased locomotion and rearing in a manner similar to CRF. Ucn 2 was thus found to have an antagonistic effect on bi-directional motor activation induced by CRF, while Ucn 3 had a suppressive effect on motor activity. Both Ucn 2 and Ucn 3 suppressed food intake in freely-fed rats, but not immediately after injection. These results suggest that the CRF2 receptor is involved in motor suppressive effects as well as anxiolytic and anorectic effects of Ucn 2 and Ucn 3.     

Effect of corticotropin-releasing factor on adrenal DBH and PNMT activity

The continuous administration of CRF (corticotropin-releasing factor) by the intraventricular route, 100 ng/day, to rats over a period of 7 days, results in significant increases of DBH (dopamine beta-hydroxylase) and PNMT (phenethanol N-methyltransferase) activities in the adrenal glands. The pattern of increase in DBH response to various doses of CRF does not correspond to the effects observed on plasma corticosterone, a result that suggests that CRF is acting to increase the adrenal enzyme by means other than through the pituitary-adrenal axis. In contrast, PNMT responds to CRF in a manner indicating a correlation with glucocorticoid availability. Moreover, PNMT induction by CRF persists even after adrenal denervation; it also occurs when CRF is given subcutaneously for 3 days, in a dose of 60 ng/day. Injection of reserpine did not potentiate the effect of intraventricularly administered CRF.     

Behavioral stress decreases plasma oxytocin concentrations in primates

Using rhesus monkeys, we studied the effects of a behavioral stress on plasma concentrations of adrenocorticotropin, oxytocin, and vasopressin. The stress resulted in significant increases in adrenocorticotropin and significant decreases in oxytocin concentrations. No significant changes were seen in vasopressin concentrations. To further explore the relationship between plasma oxytocin and pituitary-adrenal function, dexamethasone was administered to rhesus monkeys. This resulted in significant increases in plasma oxytocin concentrations, while adrenocorticotropin decreased.     

The effects of corticotropin-releasing factor and the urocortins on hypothalamic gamma-amino butyric acid release--the impacts on the hypothalamic-pituitary-adrenal axis

Corticotropin-releasing factor (CRF) and the urocortins (UCNs) are structurally and pharmacologically related neuropeptides which regulate the endocrine, autonomic, emotional and behavioral responses to stress. CRF and UCN1 activate both CRF receptors (CRFR1 and CRFR2) with CRF binding preferentially to CRFR1 and UCN1 binding equipotently to both receptors. UCN2 and UCN3 activate selectively CRFR2. Previously an in vitro study demonstrated that superfusion of both CRF and UCN1 elevated the GABA release elicited by electrical stimulation from rat amygdala, through activation of CRF1 receptors. In the present experiments, the same in vitro settings were used to study the actions of CRF and the urocortins on hypothalamic GABA release. CRF and UCN1 administered in equimolar doses increased significantly the GABA release induced by electrical stimulation from rat hypothalamus. The increasing effects of CRF and UCN1 were inhibited considerably by the selective CRFR1 antagonist antalarmin, but were not influenced by the selective CRFR2 antagonist astressin 2B. UCN2 and UCN3 were ineffective. We conclude that CRF1 receptor agonists induce the release of GABA in the hypothalamus as well as previously the amygdala. We speculate that CRF-induced GABA release may act as a double-edged sword: amygdalar GABA may disinhibit the hypothalamic CRF release, leading to activation of the hypothalamic-pituitary-adrenal axis, whereas hypothalamic GABA may inhibit the hypothalamic CRF release, terminating this activation.     

Pressor, tachycardic and behavioral excitatory responses in conscious rats following ICV administration of ACTH and CRF are blocked by naloxone pretreatment

Experiments were conducted to compare the blood pressure and heart rate responses of conscious rats given intracerebroventricular (ICV) injections of adrenocorticotropin (ACTH 1-24) and corticotropin releasing factor (CRF). Under sodium pentobarbital anaesthesia, rats were implanted with a stainless-steel cannula into the lateral cerebral ventricle and had their right femoral artery and vein cannulated. Upon recovery (24-48 hr later) conscious, unrestrained rats were given ICV injections (total volume 5 microliter by gravity flow) of sterile saline, ACTH (1-24) (0.85 and 1.7 nmoles) or CRF (0.55 and 1.1 nmoles) and blood pressure and heart rate were monitored over the next 2 hr (from the abdominal aorta via the femoral arterial catheter). Both ACTH and CRF caused mean arterial pressure (MAP) to increase, which was paralleled with increases in mean heart rate (MHR). Moreover, these elevations in MAP and MHR were temporally associated with excessive grooming (for ACTH) and locomotor activity (for CRF), which occurred before and lasted as long as MAP and MHR were enhanced. Intravenous (IV) pretreatment whereby naloxone was given 10 min before ICV administration of ACTH (1.7 nmoles) or CRF (1.1 nmoles), showed that naloxone blocked the behavioral, pressor and tachycardic effects of both ACTH and CRF. The results demonstrate that the pressor, tachycardic and locomotor effects evoked in conscious rats by ICV administration of ACTH or CRF are antagonized by naloxone and that their hemodynamic changes may, in part, be mediated by prior behavioral activation.     

Dopamine-dependent responses to cocaine depend on corticotropin-releasing factor receptor subtypes

The effects on locomotor response to cocaine challenge, acquisition of cocaine conditioned place preference and cocaine-induced dopamine (DA) release in nucleus accumbens and ventral tegmental area by the non-specific corticotropin-releasing factor (CRF) receptors antagonist alpha-helical CRF, the selective CRF receptor subtype 1 antagonist CP-154,526 and the selective CRF receptor subtype 2 antagonist anti-sauvagine-30 (AS-30) were investigated in rats. Both alpha-helical CRF (10 microg, i.c.v.) and CP-154,526 (3 microg, i.c.v.) decreased the cocaine-induced distance travelled, whereas AS-30 (3 microg, i.c.v.) did not show such an effect. The CRF receptor antagonists also have significant effects on stereotype counts induced by cocaine injection, in which the alpha-helical CRF or CP-154,526 but not AS-30 did significantly reduce the stereotype counts. alpha-Helical CRF (10 microg) prior to each injection of cocaine blocked cocaine conditioned place preference with no significant difference observed in the time spent in the drug-paired side between post- and pre-training and both 1 and 3 microg CP-154,526 also had significant inhibitory effects on cocaine-induced place preference. However, pre-treatment with an i.c.v. infusion of AS-30 (1 or 3 microg) prior to each injection of cocaine did not affect the acquisition of conditioned place preference. The alpha-helical CRF and CP-154,526 reduced extracellular DA levels of nucleus accumbens and ventral tegmental area in response to the injection of cocaine. However, both alpha-helical CRF and CP-154,526 did not modify extracellular DA levels under basal conditions. In contrast, the i.c.v. infusion of AS-30 had no effects on either the basal DA or the cocaine-induced increase in DA release in nucleus accumbens and ventral tegmental area. These findings demonstrate that activation of the CRF receptor is involved in behavioral and neurochemical effects of cocaine challenge and cocaine reward and that the role of CRF receptor subtypes 1 and 2 in cocaine-induced locomotion, reward and DA release is not identical. The CRF receptor subtype 1 is largely responsible for the action of the CRF system on cocaine locomotion and reward. These results suggest that the CRF receptor antagonist, particularly the CRF receptor subtype 1 antagonist, might be of some value in the treatment of cocaine addiction and cocaine-related behavioral disorders.     

Effects of synthetic ovine CRF on ACTH, cortisol and blood pressure in sheep

Intravenously administered synthetic ovine CRF at doses of 0.1, 1.0 and 10.0 micrograms/kg increased plasma ACTH and cortisol concentrations in a dose-dependent fashion in unanesthetized sheep. In two unanesthetized sheep, aortic blood pressure remained relatively unaffected after the intravenous administration of CRF at 5 and 20 micrograms/kg. These results suggest that peripherally administered ovine synthetic CRF specifically stimulates the sheep pituitary-adrenal axis. Unlike other species receiving intravenous synthetic ovine CRF, sheep did not show hypotensive effects.     

Demonstration that corticotropin-releasing factor binding to rat peripheral tissues is modulated by glucocorticoid treatment in vivo and in vitro

In a recent study we reported the presence of specific binding sites for corticotropin-releasing factor (CRF) in peripheral tissues of the rat (Endocrinology, 116, 2151, 1985). The objective of this study was to determine if CRF binding to peripheral tissues was modified following adrenalectomy and glucocorticoid replacement therapy. Adult male rats were adrenalectomized and CRF binding to liver, spleen and testicular membranes was determined at 5, 7 or 14 days following adrenalectomy. An additional group of adrenalectomized rats received subcutaneous injections of dexamethasone (75 micrograms/day) for 14 days. Adrenalectomy of rats for 14 days increased CRF binding to liver, kidney, testis, spleen and ventral prostate by approximately 65%-125% above sham-control values. CRF binding to membrane preparations obtained from the pancreas of sham-operated rats was undetectable; however, adrenalectomy produced detectable CRF binding in this tissue. Adrenalectomy produced a time-related increase in CRF binding to ventral prostate, spleen and liver tissue. Administration of dexamethasone to adrenalectomized animals prevented increased CRF binding to peripheral tissues observed following adrenalectomy alone. In vitro dexamethasone treatment of prostatic or hepatic homogenates from adrenalectomized rats resulted in a dose-related decrease in CRF binding activity. However, similar in vitro treatment of prostatic or hepatic homogenate with progesterone exhibited no significant effects on CRF binding. Our results suggest that glucocorticoids may be a regulator of peripheral CRF receptors.     

The Effects of Corticoptropin-Releasing Factor and the Urocortins on Striatal Dopamine Release Induced by Electrical Stimulation—An in vitro Superfusion Study

The members of the CRF peptide family, corticotropin-releasing factor (CRF), urocortin I (Ucn I), urocortin II (Ucn II) and urocortin III (Ucn III) coordinate endocrine and behavioral responses to stress. CRF has also been demonstrated to stimulate dopamine (DA) synthesis.In our study, a superfusion system was used to investigate the effects of this peptide family on striatal DA release following electrical stimulation. The involvement of the CRF receptors was studied by pretreatment of rat striatal slices with selective CRF antagonists. CRF and Ucn I increased the release of [³H]DA while Ucn II and Ucn III were ineffective. The CRFR1 antagonist antalarmin inhibited the [³H]DA release induced by electrical stimulation and enhanced by CRF and Ucn I. The CRFR2 antagonist astressin-2B was ineffective.These results suggest that CRF and Ucn I mediate DA release through the activation of CRFR1. Ucn II and Ucn III are not involved in this process.Special Issue Dedicated to Miklós Palkovits.     

Reduction in brain immunoreactive corticotropin-releasing factor (CRF) in spontaneously hypertensive rats

The brain CRF concentration of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) was examined by rat CRF radioimmunoassay. Anti-CRF serum was developed by immunizing rabbits with synthetic rat CRF. Synthetic rat CRF was also used as tracer and standard. The displacement of 125I-rat CRF by serially diluted extracts of male Wistar rats hypothalamus, thalamus, midbrain, pons, medulla oblongata, cerebral cortex, cerebellum and neurointermediate lobe was parallel to the displacement of synthetic rat CRF. In both WKY and SHR the highest levels of CRF immunoreactivity were shown by the hypothalamus and neuro-intermediate lobe, and considerable CRF immunoreactivity was also detected in other brain regions. The CRF immunoreactivity in the hypothalamus, neurointermediate lobe, midbrain, medulla oblongata and cerebral cortex was significantly reduced in SHR and it may suggest that CRF abnormality may be implicated in the reported abnormalities in the pituitary-adrenal axis, autonomic response and behavior of SHR.     

Effects of corticotropin releasing factor on locomotor activity in hypophysectomized rats

Corticotropin releasing factor (CRF) injected intracerebroventricularly to hypophysectomized and sham hypophysectomized rats produced a dose dependent increase in locomotor activity, but in untreated hypophysectomized rats 10× more CRF was needed to produce a significant increase in activity. Concomitant daily supplements of rat growth hormone, thyroxine, and corticosterone to the hypophysectomized rats eliminated locomotor activity differences between the two groups. There was no statistically significant difference in locomotor response to either saline, 0.1 μg CRF, 1.0 μg CRF or 10.0 μg CRF in the group of animals receiving hormonal supplements. These results demonstrate that CRF can produce behavioral activation in rats independently of its effects on releasing hormones from the pituitary gland.     

Tonic modulation of anxiety-like behavior by corticotropin-releasing factor (CRF) type 1 receptor (CRF1) within the medial prefrontal cortex (mPFC) in male mice: Role of protein kinase A (PKA)

The medial prefrontal cortex (mPFC) and the neuropeptide corticotropin-releasing factor (CRF) have recently been receiving more attention from those interested in the neurobiology of anxiety. Here, we investigated the CRF pathway in the modulation of anxiety-like behaviors in male mice exposed to the elevated plus-maze (EPM), through intra-mPFC injections of CRF, CP376395 [N-(1-ethylpropyl)-3,6-dimethyl-2-(2,4,6-trimethylphenoxy)-4-pyridinamine hydrochloride, a CRF type 1 receptor antagonist (CR F1)] or H-89 [N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide dihydrochloride, a protein kinase (PKA) inhibitor]. We also investigated the effects of intra-mPFC injections of H-89 on the behavioral effects induced by CRF. Mice received bilateral intra-mPFC injections of CRF (0, 37.5, 75 or 150 pmol), CP376395 (0, 0.75, 1.5 or 3 nmol) or H-89 (0, 1.25, 2.5 or 5 nmol) and were exposed to the EPM, to record conventional and complementary measures of anxiety for 5 min. Results showed that while CRF (75 and 150 pmol) produced an anxiogenic-like effect, CP376395 (all doses) and H-89 (5 nmol) attenuated anxiety-like behavior. When injected before CRF (150 pmol), intra-mPFC H-89 (2.5 nmol, a dose devoid of intrinsic effects on anxiety) completely blocked the anxiogenic-like effects of CRF. These results suggest that (i) CRF plays a tonic anxiogenic-like role at CRF1 receptors within the mPFC, since their blockade per se attenuated anxiety indices and (ii) the anxiogenic-like effects following CRF1 receptor activation depend on cAMP/PKA cascade activation in this limbic forebrain area.     

Multireceptor-induced release of adrenocorticotropin from anterior pituitary tumor cells

Corticotropin releasing factor (CRF), (?) isoproterenol and vasoactive intestinal peptide (VIP) induced cyclic AMP synthesis and the release of immunoreactive adrenocorticotropin hormone (ACTH) from clonal mouse AtT-20 pituitary tumor cells. CRF and (?) isoproterenol together produced an additive increase in cyclic AMP formation but a less than additive effect on ACTH secretion. VIP with either CRF or (?) isoproterenol produced additive increases in both cyclic AMP and ACTH secretion. Forskolin, an activator of adenylate cyclase stimulated the release of ACTH suggesting that cyclic AMP mediates some of the effects of hormone-receptor activation on ACTH secretion. The action of all three receptor agonists and forskolin on ACTH release was blocked by dexamethasone treatment. The release process, but not the changes in cyclic AMP synthesis was calcium dependent with all these hormones. The calcium ionophore, A-23187, increased ACTH secretion without altering intracellular cyclic AMP content. Its effect on secretion was not additive with either CRF, (?) isoproterenol or VIP. These observations indicate that hormone-induced regulation of ACTH secretion converges at varying intracellular locations.     

Neuropeptide Y blocks anxiogenic-like behavioral action of corticotropin-releasing factor in an operant conflict test and elevated plus maze

Central administration of neuropeptide Y (NPY) produces anxiolytic-like behavioral effects in rat models of anxiety. Because previous evidence has suggested a relationship between NPY and corticotropin-releasing factor (CRF) in the brain, we have focused on the interaction of these neuropeptide systems in emotional responsiveness to stressful stimuli. Intracerebroventricular administration of CRF produced a marked response suppression in an operant incremental shock conflict paradigm. NPY [(1 microg, intracerebroventricularly (i.c.v.)] significantly antagonized the response-suppressing effects of CRF (0.75 microg, i.c.v.) on punished responding in the conflict test at doses that produced little or no behavioral effect when administered alone. Central administration of the CRF antagonist [D-Phe(12), Nle(21,38),C(alpha) MeLeu(37)]CRF (D-Phe CRF(12-41)) alone did not alter punished or unpunished responding in the conflict test. However, pretreatment with the CRF antagonist before a subthreshold dose of NPY (1 microg, i.c.v.) produced a significant potentiation of the release of punished responding relative to NPY alone and untreated controls. NPY also antagonized the "anxiogenic-like" behavioral effects of CRF in the elevated plus maze. These findings support the hypothesis that NPY and CRF may reciprocally modulate an animal's behavioral response to stressful stimuli.     

The effect of corticotropin-releasing factor (CRF) on autonomic and behavioral responses during shock-prod burying test in rats

When administered intracerebroventricularly (ICV) in rats, corticotropin-releasing factor (CRF) possesses arousing and anxiogenic properties, which may be found reflected in autonomic and behavioral activation. As these responses are dependent on dose and situation, ICV-injected CRF may affect behavioral responses to a defined stimulus in a different fashion than autonomic concomitants. Two experiments were conducted in order to test this hypothesis. In both experiments, rats were treated ICV with CRF or an artificial cerebrospinal fluid (aCSF) 5 min prior to a 15-min exposure to an electrified prod (shock-prod burying test, SPB test) in their home cages. In the first experiment, 0.3 ng CRF injected ICV in unhandled rats significantly reduced the prod-burying response to electric shock, in favor of immobility, whereas following 300 ng CRF ICV, the predominant behavioral response was grooming behavior. In contrast, habituated rats, implanted with telemetric devices to measure heart rate, core temperature, and gross activity in the second experiment, showed a significant increase of burying behavior after 0.3 ng CRF ICV, in comparison to vehicle-treated controls. However, simultaneous cardiac acceleration was of the same magnitude and duration in both groups. In addition, whereas similar rises in CT were observed in both groups during the SPB test, CRF-treated rats showed more marked rise in core temperature during the first 15 min of the posttest period. At the 24-h retention test, rats belonging to the CRF group showed burying behavior and HR responses, in onset, magnitude, and duration similar to day 1, whereas extinction of the burying response and tachycardia was found in controls. Changes in CT, although less marked, showed the same pattern as on day 1 in both groups. These results show a differential effect of central CRF on behavioral and autonomic activation induced by a well-defined stressful stimulus. The response to CRF seems to be not only situation related, but also dependent on the pretest experience of the animal.