Research reportEffects of antalarmin, a CRF type 1 receptor antagonist, on anxiety-like behavior and motor activation in the rat
Introduction
Corticotropin-releasing factor (CRF), a 41-amino-acid neuropeptide, is hypothesized to mediate behavioral, autonomic, endocrine and immunological responses to stress [22], [52]. Intracerebroventricular (i.c.v.) administration of CRF in rats produces behavioral effects similar to those induced by stress, including motor activation, anxiety-like behavior, anorexia, decreased sexual behavior and altered cognitive performance [6], [24], [53], [80]. Since the isolation of CRF in 1981 [93], several additional CRF-related molecules have been identified in the mammalian central nervous system (CNS), including three ligands, urocortin [98], urocortin II [76], and urocortin III [56]; a membrane-associated binding protein [8]; and two genes encoding separate families of G-protein-coupled receptors [75]. This complexity has led to uncertainty about not only the physiologic role of each CRF-related ligand in behavioral responses previously ascribed to CRF [25], [68], but also about which CRF receptor subtype mediates each behavioral effect [44], [72].
Recently developed nonpeptide, selective type 1 CRF (CRF1) receptor antagonists are promising tools for delineating the role of the CRF1 receptor in behaviors induced by CRF-like agonists and stress. All reported CRF1 receptor antagonists share a common structure of a proton-accepting cyclic core unit separately linked to a substituted aromatic ring and a hydrophobic portion [66]. The best behaviorally characterized of these antagonists is the heterocyclic pyrrolopyrimidine CP-154,526 [15]. In the rat, acute CP-154,526 treatment reduced CRF- and fear-potentiated startle responding [77], separation-induced ultrasonic vocalizations [49], shock-induced escape deficits [60], the acquisition and expression of conditioned fear [41], [51], and stress-induced reinstatement of operant drug-seeking behavior [78]. CP-154,526 also showed anxiolytic-like activity in the rat social interaction test [64]. In the mouse, acute CP-154,526 had anxiolytic-like effects on ethological measures of exploratory and defensive behavior as well as in the light/dark box test [32]. Broadly, these observations resonate with those from antisense and gene knockout studies indicating a role for the CRF1 receptor in mediating stress-like behavioral responses [4]. Such findings have spurred interest in the potential utility of CRF1 receptor antagonists in the treatment of stress-related psychopathology [43], [70], [97].
Despite these promising observations, several findings with CP-154,526 have raised lingering questions. First, acute treatment with CP-154,526 failed to show consistent anxiolytic-like activity in classic behavioral screens for anxiolytics, including the elevated-plus maze [32], [59], [64], defensive withdrawal (sometimes referred to as ‘situational anxiety’; [3]) [30], [36], and conflict tests ( [32], but see [64]), tests in which nonselective CRF receptor antagonists show anxiolytic-like activity [53], [63], [84], [88]. Second, CP-154,526 failed to attenuate significantly the expression of conditioned defeat [46], a stress-related behavior that nonselective CRF receptor antagonists can reverse [46], [63]. Third, in most studies with CP-154,526, systemic doses of 20–80 mg/kg were required to show efficacy [64], and these relatively high doses were associated with signs of malaise [3]. Whether these observations reflect problems with the solubility/bioavailability of CP-154,526 [16] as opposed to the lack of a role of the CRF1 receptor in the behaviors studied is unknown. Data obtained in similar paradigms with a different CRF1 receptor antagonist could help answer these questions.
Several other high affinity, selective CRF1 receptor antagonists have been recently developed and studied for their ability to reverse stress-related behaviors, including antalarmin [34], NBI 27914 [5], DMP695 [64], DMP696 [36], DMP904 [29], R121919 [48], CRA1000 and CRA1001 [66], and SSR125543A [33]. Of these, only antalarmin and R121919, like CP-154,526, are pyrrolopyrimidine derivatives. R121919 is reportedly water-soluble [97], but its structure has not been published. Moreover, antalarmin, N-butyl-N-ethyl-[2,5,6-trimethyl-7-(2,4,6)trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl] amine, is the antagonist most closely related structurally to CP-154,526 [66]. Antalarmin has slightly greater in vitro affinity for the CRF1 receptor compared to CP-154,526 (Ki=0.8 vs. 2.7 nM, respectively) [16], [77]. Therefore, antalarmin was used to determine whether negative findings obtained with CP-154,526 were compound-, as opposed to class-specific, a concern for drug development since improved pyrrolopyrimidines continue to be sought [45].
In the rat, antalarmin reverses CRF-induced pituitary-adrenal activation [95] and hypertension [12] at doses that are well-tolerated chronically [11]. Like its analog, antalarmin blocks the acquisition and expression of conditioned fear [23]. In a recent study, antalarmin reversed social stress-induced anxiety-like behavior in rhesus monkeys, restoring normal exploratory and sexual behavior [34].
To specify further the effects of this compound, and more generally of CRF1 receptor antagonists, on anxiety-like behavior, the present study tested the hypothesis that antalarmin could acutely reverse CRF- or novelty-induced anxiogenic-like behavior in the elevated plus maze (EPM) and defensive withdrawal tests in the rat. These tests are thought to rely on the approach–avoidance conflict present in rats during exploration of novel, unprotected spaces, with anxiety-like behavior reflected as increased preference for sheltered portions of the apparatus [73], [88]. Each test is sensitive to putative anxiogenic and anxiolytic drugs, including nonselective CRF receptor agonists and antagonists, respectively [6], [63], [65], [84], [88], [96].
The second aim of this study was to test the hypothesis that antalarmin could reverse the motor-activating effects of CRF, which are dissociable from its anxiogenic-like effects [37]. In familiar environments, nonselective CRF receptor agonists stimulate motor activity in the rat [55], [83], an effect that is blocked by nonselective CRF receptor antagonists [13], [63], [84]. Similarly, CRF1 KO mice do not show CRF-induced locomotor activation [20]. To date, however, only negative findings have been published on the ability of selective CRF1 receptor antagonists to reverse the motor activating effects of CRF in intact rats [67], and other CRF1 KO mice unexpectedly exhibit increased diurnal behavioral activity [74]. These contradictory findings question the role of the CRF1 receptor in behavioral arousal and locomotor activation. The ability of antalarmin to reverse the motor activating effects of d-amphetamine, which is thought to elicit motor activation via a different neural substrate [86], was studied as a potential negative control.
Section snippets
Subjects
On arrival, adult (250–300 g) male Wistar rats (n=203; Charles River, Hollister, CA, USA) were group-housed in a 12h:12h reverse-lit (lights on 2200 h), humidity- and temperature-controlled vivarium (22 °C) with standard rodent chow and water available ad libitum. Subjects were acclimated to the vivarium for 1 week prior to the start of experiments. Rats participating in the EPM and locomotor activity studies were handled daily for 5 min during the week prior to the start of testing. Those
Elevated plus maze
Antalarmin blocked the effect of CRF on avoidance of the open arms of the EPM, as indicated by an interaction between CRF and antalarmin for percentage open arm time [F(2,45)=3.84, P<0.05] and a similar trend for percentage open arm entries [F(2,45)=3.00 P<0.06]. For both measures, whereas CRF significantly reduced percentage open arm time and percentage open arm entries in rats pretreated with vehicle (P<0.05), it did not decrease these measures in rats pretreated with either the 10 or 20
Discussion
The main findings of the present study are that acute pretreatment with antalarmin, a nonpeptide CRF1 receptor antagonist, blocked CRF- and novelty-induced anxiety-like behavior in animal models of anxiety. Antalarmin pretreatment blocked the anxiogenic-like effect of CRF on the proportion of time spent and entries into the exposed arms of the EPM. Antalarmin pretreatment also decreased the latency to emerge from and time spent in a sheltered enclosure in a novel open field. In addition, acute
Acknowledgements
Supported by a Novartis Research Grant (AM) and DK26741 from the National Institute of Diabetes and Digestive and Kidney Diseases (GFK). EPZ was supported by a Minority Research Supplement to DK26741. GRV was supported by AA05563, an Individual National Research Service Award from the National Institute on Alcohol Abuse and Alcoholism. The authors would also like to thank Dr. Albert Enz from the Nervous System Research of Novartis Pharma AG, Basel, Switzerland for the pharmacokinetic analysis.
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