The CRF(1) receptor antagonist DMP696 produces anxiolytic effects and inhibits the stress-induced hypothalamic-pituitary-adrenal axis activation without sedation or ataxia in rats

Abstract Rationale. CRF₁ antagonists may be effective in the treatment of anxiety disorders while having fewer side effects compared with classical benzodiazepines. Objectives. The effects of a small molecule selective CRF₁ antagonist DMP696 on anxiety-like behaviors and stress-induced increases in corticosterone in rats exposed to a novel environment and on locomotor activity and motor coordination were determined in rats. These effects of DMP696 were compared with those produced by the classical benzodiazepine chlordiazepoxide (CDP). Methods. DMP696 or CDP were administered PO, 60 minutes before behavioral testing in rats. Their effects on latency to exit a dark chamber and stress-induced increase in corticosterone in the Defensive Withdrawal test (an animal model of anxiety), locomotor activity, and rotorod performance (measure of ataxia) were determined. Results. DMP696 significantly reduced exit latency and reversed the stress-induced increase in corticosterone in the Defensive Withdrawal test at doses of 3.0–10 mg/kg and higher. In contrast, CDP significantly decreased exit latency at 10 and 30 mg/kg, but not at 100 mg/kg, due to concurrent non-specific side effects. Unlike DMP696, CDP had no effect on the stress-induced increase in corticosterone at lower doses, but resulted in a significant increase at higher doses. DMP696 did not reduce locomotor activity or impair motor coordination at doses up to 30-fold higher than doses effective in the Defensive Withdrawal model. In contrast, CDP produced significant sedation and ataxia at the same doses that were effective in reducing exit latency. Conclusions. These data suggest that the CRF₁ antagonist DMP696 might retain the therapeutic benefits of classical benzodiazepines but have fewer motoric side effects. Electronic Publication     

The anxiolytic CRF<Subscript>1</Subscript> antagonist DMP696 fails to function as a discriminative stimulus and does not substitute for chlordiazepoxide in rats

Rationale. Compounds with a mechanism of action different from benzodiazepines may retain the anxiolytic effects of benzodiazepines with fewer side effects. CRF₁ antagonists have anxiolytic-like effects but may have different discriminative stimulus (DS) effects compared with benzodiazepines. Objective. The present study evaluated the similarity of DS effects of a CRF₁ antagonist DMP696 to the benzodiazepine chlordiazepoxide and the ability of DMP696 to produce DS effects on its own using drug discrimination procedures, as well as its anxiolytic-like effects after acute or chronic administration. Methods. Rats were trained to discriminate chlordiazepoxide (5.0 mg/kg, IP, 30 min prior to session) from vehicle under a fixed-ratio 10 schedule of food reinforcement and drug- or vehicle-lever selection following administration of DMP696 was determined. The effects of DMP696 on latency to exit a dark chamber (defensive withdrawal model of anxiety) were used as an index of anxiolytic-like activity. Results. In chlordiazepoxide-trained rats, DMP696 (1.0–100 mg/kg, PO) resulted in most of the animals selecting the vehicle lever, as did another anxiolytic, the 5-HT_1A partial agonist buspirone (0.3–10 mg/kg, IP). DMP696 reduced exit latency in defensive withdrawal at 10 mg/kg administered either acutely or chronically for 14 days. Thus, the doses of DMP696 studied in drug discrimination were up to 10-fold higher than those active in the anxiety model. In addition, DMP696 (10–60 mg/kg, PO) could not be established as a DS under the conditions used in this study. In a subsequent study, chlordiazepoxide was established as a DS in these same animals. Conclusions. Lack of substitution of DMP696 for the chlordiazepoxide DS in rats and its inability to acquire DS properties suggest that the DS effects of DMP696 differ from those of benzodiazepines. Electronic Publication     

Anxiogenic and aversive effects of corticotropin-releasing factor (CRF) in the bed nucleus of the stria terminalis in the rat: role of CRF receptor subtypes

Rationale Corticotropin-releasing factor (CRF) produces anxiety-like and aversive effects when infused directly into the various regions of the brain, including the bed nucleus of the stria terminalis (BNST). However, the CRF receptor subtypes within the BNST mediating these phenomena have not been established.Objectives We used selective CRF receptor antagonists to determine the receptor subtypes involved in the anxiogenic-like and aversive effects CRF in the BNST.Materials and methods Male Long–Evans rats were bilaterally infused with CRF (0.2 or 1.0 nmol) either alone or in combination with the CRF₁ receptor antagonist CP154,526 or the CRF₂ receptor antagonist anti-sauvagine 30 (AS30) before behavioral testing in the elevated plus maze or place conditioning paradigms.Results Intra-BNST administration of CRF produced a dose-dependent reduction in open arm entries and open arm time in the elevated plus maze, indicating an anxiogenic-like effect. These effects were inhibited by co-infusion of CP154,526 but not of AS30, indicating that the anxiogenic-like effects of CRF in the BNST are mediated by CRF₁ receptors. Place conditioning with intra-BNST administration of CRF produced a dose-dependent aversion to the CRF-paired environment that was prevented by co-infusion of either CP154,526 or AS30, indicating that both CRF receptor subtypes mediate the aversive effects of this peptide. Intra-BNST infusions of the CRF receptor antagonists alone produced no effects in either behavioral paradigm.Conclusions CRF₁ receptors in the BNST mediate the anxiogenic-like effects of CRF in this region, whereas both CRF₁ and CRF₂ receptor subtypes mediate the conditioned aversive effects of this peptide within the BNST.     

Dissociating anxiolytic and sedative effects of GABA<Subscript>A</Subscript>ergic drugs using temperature and locomotor responses to acute stress

Rationale  The stress-induced hyperthermia (SIH) model is an anxiety model that uses the transient rise in body temperature in response to acute stress. Benzodiazepines produce anxiolytic as well as sedative side effects through nonselective binding to GABA_A receptor subunits. The GABA_A receptor α₁ subunit is associated with sedation, whereas the GABA_A receptor α₂ and α₃ subunits are involved in anxiolytic effects. Objectives  We therefore examined the effects of (non)subunit-selective GABA_A receptor agonists on temperature and locomotor responses to novel cage stress. Results  Using telemetric monitoring of temperature and locomotor activity, we found that nonsubunit-selective GABA_A receptor agonist diazepam as well as the α₃ subunit-selective receptor agonist TP003 dose-dependently attenuated SIH and locomotor responses. Administration of GABA_A receptor α₁-selective agonist zolpidem resulted in profound hypothermia and locomotor sedation. The GABA_A receptor α₁-selective antagonist βCCt antagonized the hypothermia, but did not reverse the SIH response attenuation caused by diazepam and zolpidem. These results suggest an important regulating role for the α₁ subunit in thermoregulation and sedation. Ligands of extrasynaptic GABA_A receptors such as alcohol and nonbenzodiazepine THIP attenuated the SIH response only at high doses. Conclusions  The present study confirms a putative role for the GABA_A receptor α₁ subunit in hypothermia and sedation and supports a role for α_2/3 subunit GABA_A receptor agonists in anxiety processes. In conclusion, we show that home cage temperature and locomotor responses to novel home cage stress provide an excellent tool to assess both anxiolytic and sedative effects of various (subunit-selective) GABA_Aergic compounds.     

CRF1 receptor-deficiency induces anxiety-like vulnerability to cocaine

Rationale

The intake of psychostimulant drugs may induce cognitive dysfunction and negative affective-like states, and is associated with increased activity of stress-responsive systems. The corticotropin-releasing factor (CRF) system mediates neuroendocrine, behavioural and autonomic responses to stressors, and might be implicated in substance-related disorders. CRF signalling is mediated by two receptor types, named CRF₁ and CRF₂.

Objectives

The present study aims to elucidate the role for the CRF₁ receptor in cognitive dysfunction and anxiety-like states induced by cocaine.

Results

The genetic inactivation of the CRF₁ receptor (CRF₁+/? and CRF₁?/?) does not influence recognition memory in drug-naïve mice, as assessed by the novel object recognition (NOR) test. Moreover, the chronic administration of escalating doses of cocaine (5–20 mg/kg, i.p.) induces NOR deficits, which are unaffected by CRF₁ receptor-deficiency. However, the same drug regimen reveals an anxiety-like vulnerability to cocaine in CRF₁?/? but not in wild-type or CRF₁+/? mice, as assessed by the elevated plus maze test.

Conclusions

The present findings indicate dissociation of cognitive dysfunction and anxiety-like states induced by cocaine. Moreover, they unravel a novel mechanism of vulnerability to psychostimulant drugs.     

Behavioral,biological, and chemical perspectives on targeting CRF1 receptor antagonists to treat alcoholism

Background

Alcohol use disorders are chronic disabling conditions for which existing pharmacotherapies have only modest efficacy. In the present review, derived from the 2012 Behavior, Biology and Chemistry “Translational Research in Addiction” symposium, we summarize the anti-relapse potential of corticotropin-releasing factor type 1 (CRF₁) receptor antagonists to reduce negative emotional symptoms of acute and protracted alcohol withdrawal and stress-induced relapse to alcohol seeking.

Methods

We review the biology of CRF₁ systems, the activity of CRF₁ receptor antagonists in animal models of anxiolytic and antidepressant activity, and experimental findings in alcohol addiction models. We also update the clinical trial status of CRF₁ receptor antagonists, including pexacerfont (BMS-562086), emicerfont (GW876008), verucerfont (GSK561679), CP316311, SSR125543A, R121919/NBI30775, R317573/19567470/CRA5626, and ONO-2333Ms. Finally, we discuss the potential heterogeneity and pharmacogenomics of CRF₁ receptor pharmacotherapy for alcohol dependence.

Results

The evidence suggests that brain penetrant-CRF₁ receptor antagonists have therapeutic potential for alcohol dependence. Lead compounds with clinically desirable pharmacokinetic properties now exist, and longer receptor residence rates (i.e., slow dissociation) may predict greater CRF₁ receptor antagonist efficacy. Functional variants in genes that encode CRF system molecules, including polymorphisms in Crhr1 (rs110402, rs1876831, rs242938) and Crhbp genes (rs10055255, rs3811939) may promote alcohol seeking and consumption by altering basal or stress-induced CRF system activation.

Conclusions

Ongoing clinical trials with pexacerfont and verucerfont in moderately to highly severe dependent anxious alcoholics may yield insight as to the role of CRF₁ receptor antagonists in a personalized medicine approach to treat drug or alcohol dependence.     

Persistent anxiolytic affects after chronic administration of the CRF₁ receptor antagonist R121919 in rats

Corticotropin-releasing factor (CRF) functions as one of the major mediators of the mammalian stress response and appears to play a key role in the pathophysiology of mood and anxiety disorders. Small molecule CRF₁ receptor antagonists may represent a novel form of pharmacotherapy for these disorders. The therapeutic success of CRF₁ receptor antagonists will depend, in part, upon whether tolerance develops to the actions of these compounds and whether appropriate patterns of HPA axis function is maintained. This study evaluated the effects of long term (～4 week) treatment with the CRF₁ receptor antagonist R121919, on CRF receptor function, HPA axis activity, behavioral measures, adrenal gland size, and body weight gain.Animals treated with 20 mg/kg/day of R121919 spent significantly more time in the open field in a defensive withdrawal test (138 ± 36 s for R121919 vs 52 ± 12 s for vehicle, p = 0.01). No significant effect of chronic CRF₁ receptor blockade on basal ACTH or corticosterone concentrations were detected, nor were significant changes detected in an elevated plus maze test. Both vehicle- and R121919- treated rats showed increases in AUC and peak ACTH and corticosterone concentrations following air puff startle stress, without any overall group differences, although a clear but non-significant attenuation in HPA axis response was observable in R121919 treated animals. Chronic CRF₁ receptor blockade increased CRF peptide mRNA expression in the PVN and decreased CRF peptide mRNA expression in the central nucleus of the amygdala. Overall our results suggest that anxiolytic effects of chronic CRF₁ receptor antagonism persist following chronic administration without significant attenuation of the HPA axis’s ability to mount a stress response.This article is part of a Special Issue entitled ‘Trends in Neuropharmacology: In Memory of Erminio Costa’.     

Role of corticotropin releasing factor (CRF) receptors 1 and 2 in CRF-potentiated acoustic startle in mice

Rationale Hypersecretion of corticotropin releasing factor (CRF) has been implicated in both severe anxiety disorders and major depression. Although the role of the CRF₁ receptor in the anxiogenic effects of CRF is well supported, the role of CRF₂ receptors in anxiety-like behaviors is less clear. In rats, CRF increases the acoustic startle reflex (ASR) via its action in the extended amygdala, providing a putative measure of CRF-mediated anxiogenic activity.Objective To characterize the effect of CRF on ASR in mice and determine the respective roles of CRF₁ and CRF₂ receptors in CRF-potentiated ASR.Methods The present study examined: (1) the time course and dose response functions for the effects of human/rat (h/r)-CRF (0.02–0.6 nmol, ICV (intracerebroventricular)) on ASR in two inbred strains of mice; (2) the effects of the CRF₁ receptor antagonist NBI-30775 (20 mg/kg, intraperitoneal) and the CRF₂ receptor antagonist Antisauvagine-30 (1–10 nmol, ICV) on CRF-potentiated ASR and (3) the effects of the CRF₂ receptor agonist urocortin 2 (0.2–6 nmol, ICV) on ASR in mice.Results h/r-CRF significantly increased ASR in mice in a time-dependent manner with maximal efficacy at the 0.2 and 0.6 nmol doses. 129S6/SvEvTac mice exhibited a slightly increased duration of action and lower minimal effective dose threshold for CRF effects on ASR compared to C57BL/6J mice. Both selective CRF₁ and CRF₂ antagonists attenuated h/r-CRF-potentiated ASR without affecting acoustic startle when given alone. The selective CRF₂ receptor agonist urocortin 2 increased ASR (1 and 2 nmol), albeit with less efficacy than the non-selective CRF receptor agonist h/r-CRF.Conclusions Both CRF₁ and CRF₂ receptors appear to contribute to the h/r-CRF-induced increases in ASR in mice. These data support the hypothesis that both receptors contribute to the anxiogenic effects of CRF.     

Buspirone, chlordiazepoxide and diazepam effects in a zebrafish model of anxiety

Zebrafish are becoming more widely used to study neurobehavioral pharmacology. We have developed a method to assess novel environment diving behavior of zebrafish as a model of stress response and anxiolytic drug effects. In a novel tank, zebrafish dwell in the bottom of the tank initially and then increase their swimming exploration to higher levels over time. We previously found that nicotine, which has anxiolytic effects in rodents and humans, significantly lessens the novel tank diving response in zebrafish. The specificity of the diving effect was validated with a novel vs. non-novel test tank. The novel tank diving response of zebrafish was tested when given three anxiolytic drugs from two different chemical and pharmacological classes: buspirone, chlordiazepoxide and diazepam. When the test tank was novel the diving response was clearly seen whereas it was significantly reduced when the test tank was not novel. Buspirone, a serotonergic (5HT_1A receptor agonist) anxiolytic drug with some D₂ dopaminergic effect, had a pronounced anxiolytic-like effect in the zebrafish diving model at doses that did not have sedative effects. In contrast, chlordiazepoxide, a benzodiazepine anxiolytic drug, which is an effective agonist at GABA-A receptors, did not produce signs of anxiolysis in zebrafish over a broad dose range up to those that caused sedation. Diazepam another benzodiazepine anxiolytic drug did produce an anxiolytic effect at doses that did not cause sedation. The zebrafish novel tank diving task can be useful in discriminating anxiolytic drugs of several classes (serotonergic, benzodiazepines and nicotinic).     

Attaining in vivo selectivity of positive modulation of α3βγ2 GABAA receptors in rats: A hard task!

It is unclear whether GABA_A receptors (GABA_ARs) that contain the α3-subunit are substantially involved in the anxiolytic effects of benzodiazepines (BDZs). In the present study, we tested YT-III-31, a newer BDZ ligand with functional preference for α3βγ2 GABA_ARs, in two paradigms of unconditioned anxiety, the open field and elevated plus maze in rats. The effective dose of YT-III-31 (2?mg/kg) displayed a clear anxiolytic-like profile, unhampered by sedative action, in both tests. At a higher dose (10?mg/kg), YT-III-31 induced ataxia in the rotarod and sedation in spontaneous locomotor activity test. The latter effect was preventable by flumazenil and βCCt, the non-selective and α1βγ2 GABA_AR affinity-selective antagonist, respectively, demonstrating that sedative properties of YT-III-31, when attained, are mediated by the α1γ2 site. To elucidate the receptor substrate of subtle behavioral differences between YT-III-31 and diazepam, we approximated in vivo receptor potentiation for both ligands, based on estimated unbound concentrations in rat brains. Far different from diazepam, YT-III-31 has significantly lower affinity for the α1γ2 over other BDZ-sensitive sites, and at lower doses (1–2?mg/kg) was devoid of potentiation at α1βγ2 GABA_ARs. The approximation approach revealed a modest selectivity of YT-III-31 for α3γ2- in comparison to α2γ2 and α5γ2 binding sites, suggesting that its anxiolytic-like activity may not necessarily or predominantly reflect potentiation at α3βγ2 GABA_ARs. Nonetheless, as the anxiolytic effects are achievable at a dose devoid of any sedative potential, and having favorable safety (cytotoxicity) and metabolic stability profile, YT-III-31 represents a valuable candidate for further translational research.     

The CRF<Subscript>1</Subscript> receptor antagonist antalarmin attenuates yohimbine-induced increases in operant alcohol self-administration and reinstatement of alcohol seeking in rats

Rationale and objectives Yohimbine is an alpha-2 adrenoreceptor antagonist that provokes stress- and anxiety-like responses in both humans and laboratory animals. In rats, yohimbine increases operant alcohol self-administration and reinstates alcohol seeking. In this study, we assess whether these effects of yohimbine are attenuated by systemic injections of the corticotrotropin-releasing factor 1 (CRF₁) receptor antagonist antalarmin. Materials and methods In Exp. 1, we trained rats to lever press for alcohol solutions (12% w/v, 1 h/day) over several weeks; during training, the response requirement was increased from a fixed-ratio-1 (FR-1) to a fixed-ratio-3 (FR-3) reinforcement schedule. We then tested the effect of antalarmin (10 or 20 mg/kg) on yohimbine (1.25 mg/kg)-induced increases in operant alcohol self-administration (FR-3 reinforcement schedule). Subsequently, we assessed the effect of antalarmin on yohimbine-induced increases in plasma corticosterone levels in the previously self-administering rats. In Exp. 2, we trained the rats to self-administer alcohol as in Exp. 1, and after extinction of the alcohol-reinforced lever responding over 13 days, we tested antalarmin’s effect on yohimbine-induced reinstatement of alcohol seeking. Results Yohimbine increased operant alcohol self-administration and reinstated alcohol seeking after extinction. These effects of yohimbine were attenuated by antalarmin. Antalarmin injections in the absence of yohimbine had no effect on either operant alcohol self-administration or extinction responding. Antalarmin had no effect on yohimbine-induced corticosterone release in alcohol-experienced rats. Conclusions These results suggest that extrahypothalamic CRF₁ receptors are involved in the effect of yohimbine on operant alcohol self-administration and on relapse to alcohol seeking and support the notion that CRF₁ receptor antagonists should be considered in alcohol addiction treatment.     

Section Review—Central & Peripheral Nervous Systems: 5-HT3 Receptor Antagonists in Development As-Anxiolytics

Different serotonin (5-HT) receptor subtypes are targets for drugs of actual or potential interest in psychiatry. Among them, 5-HT₃ receptors may be of considerable interest in the development of new psychotropic drugs. Many 5-HT₃ receptor antagonists show an anxiolytic-like activity at very low doses in different models of anxiety, particularly in the light-dark test in mice. Conflicting results have been reported in other animal models such as the elevated plus-maze in rats or in conflict procedures. In general, the dose-response curve for the anxiolytic effect of 5-HT₃ receptor antagonists is bell-shaped and these compounds lose efficacy at high doses. Experimental studies suggest that 5-HT₃ antagonists offer some advantages over benzodiazepines such as apparent lack of tolerance and dependence, minimal or null effects of psychomotor performance and not only do not induce amnesia but may also enhance memory consolidation. Although few clinical studies have been published so far with 5-HT₃ receptor antagonists, these studies appear to suggest efficacy and quick onset of action in the treatment of generalised anxiety disorders. Further clinical investigation is nc doubt necessary until some obvious advantage of this class of compounds over benzodiazepines in some subgroup of human anxiety disorders is recognised.     

Antihyperalgesic Effect of the GABAA Ligand Clobazam in a Neuropathic Pain Model in Mice: A Pharmacokinetic–Pharmacodynamic Study

Facilitation of spinal GABAergic inhibition with benzodiazepines (BZDs) reverses pain sensitization in animals; however, the use of BZDs in man is limited by their sedative effect. The antihyperalgesic effects of GABA_A agonists are mediated by GABA_A receptors containing α2 subunits, whereas sedation is linked to α1 subunit‐containing receptors. α2 and α3 selective GABA_A receptor modulators have been tested in animals but are not yet available for use in human beings. Clobazam is a 1,5‐BZD, which exhibits less cognitive side effects than other benzodiazepines. Here, we studied its antihyperalgesic effects in a mouse model of neuropathic pain. Clobazam showed a dose‐dependent antihyperalgesic effect in the chronic constriction injury (CCI) model of neuropathic pain, peaking at 1 hr after administration and lasting for 4 hr with no relevant sedation at a dose of 3 mg/kg. At higher doses, the antihyperalgesic effect was stronger, but sedation became significant. The blood and brain kinetics of clobazam were linear over the range of doses tested with a short half‐life of the parent compound and a ready penetration of the blood–brain barrier. Clobazam blood concentrations decreased rapidly, falling below the limit of detection at 120 min. after drug application. Its main metabolite, N‐desmethyl‐clobazam, showed more delayed and prolonged pharmacokinetics, partly explaining why antihyperalgesia persisted when clobazam was no longer detectable in the blood. Considering its therapeutic margin and its pharmacokinetic properties, clobazam would be a valuable compound to assess the role of the GABAergic pathway in pain transmission in human beings.     

Disruption of CB<Subscript>1</Subscript> receptor signaling impairs extinction of spatial memory in mice

Rationale A growing body of in vitro and in vivo evidence indicates that a central endocannabinoid system, consisting of CB₁ receptors and endogenous cannabinoids, modulates specific aspects of mnemonic processes. Previous research has demonstrated that either permanent or drug-induced disruption of CB₁ receptor signaling interferes with the extinction of a conditioned fear response.Objectives In the present study, we evaluated whether the endocannabinoid system also plays a role in extinguishing learned escape behavior in a Morris water maze task.Methods CB₁ (–/–) mice and mice repeatedly treated with 3 mg/kg of the CB₁ receptor antagonist SR 141716 (Rimonabant) were trained to locate a hidden platform in the Morris water maze. Following acquisition, the platform was removed and subjects were assigned to either a massed (i.e., five consecutive sessions consisting of four 2-min trials/session) or a spaced (a single, 1-min trial every 2–4 weeks) extinction protocol.Results Strikingly, both 3 mg/kg SR 141716-treated mice and CB₁ (–/–) mice continued to return to the target location across all five trials in the spaced extinction procedure, while the control mice underwent extinction by the third or fourth trial. In contrast, both the 3-mg/kg SR 141716-treated and CB₁ (–/–) mice exhibited extinction in the massed extinction trial procedure.Conclusions These findings indicate that disruption of CB₁ receptor signaling impairs extinction processes in the Morris water maze, thus lending further support to the hypothesis that the endocannabinoid system plays an integral role in the suppression of non-reinforced learned behaviors.     

The effect of the mGlu5 receptor antagonist MPEP in rodent tests of anxiety and cognition: a comparison

Rationale Antagonists at the metabotropic glutamate 5 (mGlu5) receptor produce robust anxiolytic effects in a number of rat tests. However, there is evidence that mGlu5 receptor antagonists may also impair working memory and spatial learning following intracerebroventricular administration.Objectives The aim of this study is to compare the effect of the potent and selective mGlu5 receptor antagonist, 2-methyl-6-(phenylethynyl)-piperidine (MPEP), administered systemically on rodent tests of cognition and anxiety.Methods MPEP was assessed in the following rodent tests, 60 min following oral administration: Geller–Seifter conflict, conditioned emotional response (CER), Vogel conflict, delayed match to position (DMTP) and Morris water maze. Diazepam was also tested as a comparator.Results MPEP had a significant anxiolytic effect, comparable in magnitude to diazepam, at 10–30 mg/kg in the two conflict and CER tasks. There was no effect of MPEP up to 30 mg/kg on working memory in the DMTP task, but at 100 mg/kg, there was a significant reduction in choice accuracy at the longest delay interval (24 s). MPEP (3–30 mg/kg) did not significantly impair spatial learning in the Morris water maze, although during the last probe trial, 30-mg/kg-treated rats were significantly less accurate than controls. In contrast, diazepam significantly impaired performance in both the DMTP and Morris water maze tests. Assessment of plasma and brain concentration of MPEP 75 min following oral administration showed a dose linearity from 3 to 30 mg/kg and good brain penetration, i.e. a brain/plasma ratio of 3.1.Conclusions Oral administration of the selective mGlu5 receptor antagonist MPEP induces a robust anxiolytic-like effect in rat conflict tests comparable to that seen with diazepam, but in contrast to diazepam, MPEP does not impair working memory or spatial learning at anxiolytic doses.     

Importance of CRF Receptor-Mediated Mechanisms of the Bed Nucleus of the Stria Terminalis in the Processing of Anxiety and Pain

Corticotropin-releasing factor (CRF)-mediated mechanisms in the bed nucleus of the stria terminalis (BNST) have a pivotal role in stress-induced anxiety and hyperalgesia. Although CRF is known to activate two receptor subtypes, CRF₁ and CRF₂, attempts to delineate the specific role of each subtype in modulating anxiety and nociception have been inconsistent. Here we test the hypothesis that CRF₁ and CRF₂ receptor activation in the anteriolateral BNST (BNST_AL) facilitates divergent mechanisms modulating comorbid anxiety and hyperalgesia. Microinfusions of the specific antagonists CP376395 and Astressin₂B into the BNST_AL were used to investigate CRF₁ and CRF₂ receptor functions, respectively. We found that CRF₁ and CRF₂ receptors in the BNST_AL had opposing effects on exploratory behavior in the elevated plus-maze, somatic mechanical threshold, and the autonomic and endocrine response to stress. However, CRF₁ or CRF₂ receptor antagonism in the BNST_AL revealed complementary roles in facilitating the acoustic startle and visceromotor reflexes. Our results suggest that the net effect of CRF₁ and CRF₂ receptor activation in the BNST_AL is pathway-dependent and provides important insight into the CRF receptor-associated circuitry that likely underpins stress-induced pathologies.     

Both corticotropin-releasing factor receptor type 1 and type 2 are involved in stress-induced inhibition of food intake in rats

Rationale Stress-induced inhibition of food intake is reportedly blocked by a selective corticotropin-releasing factor (CRF) type 1 receptor (CRF₁) antagonist, suggesting the involvement of CRF₁ in the inhibitory mechanism. CRF₁ and CRF₂ are considered to function in the inhibition of food intake by CRF-related peptides with different time courses.Objectives This study was designed to clarify whether CRF₂ is also involved in stress-induced inhibition of food intake and to examine the relation of CRF₁ to CRF₂ in the inhibitory mechanism.Methods Antisauvagine-30 (AS-30), a selective CRF₂ antagonist, and/or CRA1000, a selective CRF₁ antagonist, were pre-administered intracerebroventricularly and intraperitoneally, respectively, to male Wistar rats deprived of food for 24 h before the animals were exposed to a 1-h period of stressors and food intake in 1 h after stress exposure was examined. The effect of both antagonists on locomotor activity was also examined.Results Pre-administration of 5–30 g of AS-30 attenuated inhibition of food intake induced by restraint, electric footshock or emotional stress using a communication box. CRA1000 also attenuated the restraint-induced inhibition of food intake at doses of 5 and 10 mg/kg body weight. The reversal of restraint-induced inhibition of food intake by co-administration of AS-30 and CRA1000 was not larger than that by AS-30 or CRA1000 alone. Both antagonists did not affect locomotor activity.Conclusions These results suggest that not only CRF₁, but also CRF₂, are involved in stress-induced inhibition of food intake, and that both subtypes of CRF receptor function probably in series in 1 h after stress exposure.     

Corticotropin-releasing Factor in the Rat Dorsal Raphe Nucleus Promotes Different Forms of Behavioral Flexibility Depending on Social Stress History

The stress-related neuropeptide, corticotropin-releasing factor (CRF) regulates the dorsal raphe nucleus–serotonin (DRN–5-HT) system during stress and this may underlie affective and cognitive dysfunctions that characterize stress-related psychiatric disorders. CRF acts on both CRF₁ and CRF₂ receptor subtypes in the DRN that exert opposing inhibitory and excitatory effects on DRN-5-HT neuronal activity and 5-HT forebrain release, respectively. The current study first assessed the cognitive effects of intra-DRN microinfusion of CRF or the selective CRF₂ agonist, urocortin II in stress-naive rats on performance of an operant strategy set-shifting task that is mediated by the medial prefrontal cortex (mPFC). CRF (30 ng) facilitated strategy set-shifting performance, whereas higher doses of CRF and urocortin II that would interact with CRF₂ were without effect, consistent with a CRF₁-mediated action. This dose decreased 5-HT extracellular levels in the mPFC, further supporting a role for CRF₁. The effects of CRF were then assessed in rats exposed to repeated social stress using the resident–intruder model. Repeated social stress shifted the CRF effect from facilitation of strategy set shifting to facilitation of reversal learning and this was most prominent in a subpopulation of rats that resist defeat. Notably, in this subpopulation of rats 5-HT neuronal responses to CRF have been demonstrated to shift from CRF₁-mediated inhibition to CRF₂-mediated excitation. Because 5-HT facilitates reversal learning, the present results suggest that stress-induced changes in the cellular effects of CRF in the DRN translate to changes in cognitive effects of CRF. Together, the results underscore the potential for stress history to shift cognitive processing through changes in CRF neurotransmission in the DRN and the association of this effect with coping strategy.     

Diazepam impairs place learning in naive but not in maze-experienced rats in the Morris water maze

Anxiolytic benzodiazepines have been shown to impair place learning in the Morris water maze. However, a clear-cut demonstration of a direct and specific effect on mnemonic processes has not yet been offered. In the present study, the effects of diazepam on place navigation in the Morris water maze were studied in rats. Three conditions were examined: learning, reversal learning and learning after familiarisation of animals with the maze. In view of the anxiolytic and sedative properties of diazepam, appropriate doses of the drug, i.e. those that produced an anxiolytic effect but no major motor impairment, were initially selected in the water-lick conflict and rotarod tests, respectively. Doses of 2.5 and 5 mg/kg PO increased punished drinking in the water-lick conflict test without significantly decreasing rotarod performance. These doses were then used to assess the effects of diazepam on spatial behaviour. Diazepam, at both doses, impaired place learning in behaviourally naive rats. Such an effect appeared to be transient: diazepam-treated rats eventually reached control performance. Moreover, analysis of the probe trial at the end of training revealed adoption of a spatial strategy to locate the submerged platform. Neither reversal learning nor learning after familiarisation was affected. These results do not replicate previous findings in the Morris water maze and provide some evidence that the diazepam-induced place learning deficit may be primarily anxiolytic in nature.     

The therapeutic potential of CRF1 antagonists for anxiety

Preclinical studies suggest that the brain corticotropin-releasing factor (CRF) systems mediate anxiety-like behavioural and somatic responses through actions at the CRF₁ receptor. CRF₁ antagonists block the anxiogenic-like effects of CRF and stress in animal models. Cerebrospinal fluid levels of CRF are elevated in some anxiety disorders and normalise with effective treatment, further implicating CRF systems as a therapeutic target. Prototypical CRF₁ antagonists are highly lipophilic, non-competitive antagonists of peptide ligands. Modification of the chemotype and the identification of novel pharmacophores are yielding more drug-like structures with increased hydrophilicity at physiological pHs. Newer compounds exhibit improved solubility, pharmacokinetic properties, potency and efficacy. Several clinical candidates have entered Phase I/II trials. However, unmet challenges await resolution during further discovery, clinical development and therapeutic application of CRF₁ antagonists.