Corticotropin

The Role of Corticotropin-Releasing Factor Receptors in Stress and Anxiety1

SYNOPSIS. Corticotropin releasing factor (CRF) is a critical integrator of the hypothalamic-pituitary-adrenal (HPA) axis in response to stress. CRF and its related molecule urocortin (UCN) bind CRF receptor 1 (CRFR1) and CRFR2 with distinct affinities. Mice deficient for CRFR1 or CRFR2 were generated in order to determine the physiological role of these receptors. While CRFR1-mutant mice show a depleted stress response and display anxiolytic-like behavior, CRFR2-mutant mice are hypersensitive to stress and display anxiogenic-like behavior. Both CRFR1- and CRFR2-mutant mice show normal basal feeding and weight gain, but CRFR2-mutant mice exhibit decreased food intake following a stress of food deprivation. While CRFR2-mutant mice display increased levels of CRF mRNA in the central nucleus of the amygdala (cAmyg) but not in the paraventricular nucleus of the hypothalamus (PVN), the CRFR1-mutant mice express high levels of CRF in the PVN but normal levels in the cAmyg. CRFR2-mutant mice also display increased levels of Ucn mRNA and protein in the edinger westphal nucleus (EW) as well as an increased number of cells expressing Ucn. The levels of these CRF-receptor ligands reflect the state of the receptor-deficient mice. These results demonstrate a possible modulatory function of CRFR2 in response to CRFR1 stimulation of the HPA axis or anxiety.

Corticotropin-releasing factor (CRF) is a critical coordinator of the hypothalamic-pituitary-adrenal (HPA) axis and is an essential component in mediation of endocrine and behavioral responses to stress (Vale et al., 1981; Vaughan et al., 1995). Following stress stimulation, the paraventricular nucleus of the hypothalamus (PVN) releases CRF which acts at receptors found on corticotrophs in the anterior pituitary resulting in a release of adrenocorticotropic hormone (ACTH) into the bloodstream. ACTH then acts on receptors in the cortex of the adrenal gland to increase the synthesize and release of glucocorticoids. These glucocorticoids perform a vast number of actions including a negative feedback to decrease further production and release of CRF Centrally, the CRF system also has numerous actions including mediation of anxiety, feeding, and stimulation of the sympathetic nervous system. These actions of CRF and related peptide family members including urocortin (Ucn) in regulation of homeostasis are mediated via activation of their two known receptors, CRFR1 (Chen et al., 1993) and CRFR2 (Kishimoto et al., 1995; Lovenberg et al., 1995; Perrin et al., 1995; Stenzel et al., 1995). These receptors share approximately 71% amino acid sequence similarity (Grigoriadis et al., 1996) and are distinct in both their localization in the CNS and periphery (Chalmers et al., 1995; Potter et al., 1994) and in their binding affinities for CRF and Ucn. Ucn binds with almost 40-fold higher affinity for CRFR2 than does CRF, suggesting it may be one of the endogenous ligands for CRFR2. In the CNS, CRFR1 is abundantly expressed in the cerebral cortex, cerebellum, medial septum, and anterior pituitary. CRFR2 is predominantly found in the lateral septum, ventromedial hypothalamus, and choroid plexus. In the periphery, CRFR2 is the predominant receptor and is widely distributed in the heart, skeletal muscle, vasculature, and gastrointestinal tract (Kishimoto et al., 1995; Lovenberg et al., 1995; Perrin et al., 1995; Stenzel et al., 1995).

The CRF receptors are also distinct in the phenotypes produced by null deletion in mice. Mice deficient for CRFR1 display decreased anxiety-like behavior (Fig. 1) and have an impaired stress response (Fig. 2) (Smith et al., 1998; Timpl et al., 1998). These mice fail to display the characteristic HPA-axis response to restraint stress due to agenesis of the zona fasciculata region of their adrenal gland resulting from a deficiency of ACTH during development. While CRFR1-mutant mice do show a response to stress with a slight increase in ACTH and concurrently elevated corticosterone levels in response to restraint, these levels never rise near their control littermates' levels. Behaviorally, the CRFR1-mutant mice display decreased anxiety-like behavior when examined on both an elevated plus maze and in the dark-light emergence task (Smith et al., 1998). CRF protein levels are increased in the paraventricular nucleus (PVN) in the CRFR1-deficient mice resulting from a diminished negative feedback due to decreased corticosterone levels (Fig. 3). Although the decreased basal level of corticosterone present in the mutant mice may account for the relative abundance of CRF protein found in the PVN, it does not appear to be responsible for the reduction in anxiety-like behaviors, as corticosterone-replaced mutant mice show no significant difference in their responses during anxiety testing (Smith et al., 1998).

In direct opposition to the CRFR1-mutant mice, the CRFR2-mutant mice display increased anxiety-like behaviors (Fig. 1) and are hypersensitive to stress (Fig. 2) (Bale et al., 2000; Coste et al., 2000). These mice respond not only more rapidly to stress but also with a greater amplitude of corticosterone than control animals (Bale et al., 2000). No histological differences for either adrenal or pituitary glands were detected between CRFR2-mutant and control mice. Behaviorally, the CRFR2-mutant mice display increased anxiety-like behavior when examined on the elevated plus maze and the open-field test (Bale et al., 2000; Kishimoto et al., 2000). Examination of CRF-family ligands reveals increased CRF mRNA levels in the null-mutant mice in the cAmyg. The amygdala is a nucleus important in transduction of stress and anxiety signals Allen and Allen, 1975; Beaulieu et al., 1986; King and Meyer, 1958; Liang et al., 1992). Interestingly, CRFR2-mutant mouse CRF mRNA levels are normal in the PVN, despite the heightened stress response detected in these mice (Fig. 3). These results were obtained under basal conditions and may be altered under an imposed stress. Ucn mRNA in the Edinger-Westphal nucleus (EW) was also increased in the CRFR2mutant mice. The increased levels of CRF and Ucn in the CRFR2-deficient mice may explain the stressed and anxious phenotype observed in these mice, as both ligands are able to activate the CRFR1 to produce these effects. No difference in CRFR1 mRNA levels were detected in the CRFR2-mutant mice when compared to controls, so it is unlikely that the phenotype reported for these mice is due to elevated CRFR1 levels (Bale et al., 2000).

These results of deletion studies in mice support previous findings using infusion and antisense oligonucleotide techniques. Rats infused with either an agonist or an antagonist specific to the CRFR1 show a similar increase or decrease in HPA response and anxiety, accordingly (Britton et al., 1986; Koob and Thatcher-Britton, 1985; Menzaghi et al., 1994). Antisense studies also demonstrate that decreased levels of CRFR1 result in a decreased stress response and anxiety levels (Heinrichs et al., 1997; Liebsch et al., 1999).

Results from deletion of either CRF receptor indicate an important interaction between CRFR2 and CRFR1. Our hypothesis suggests that CRFR2 functions to "dampen" the activity of CRFR1 but may also have independent anti-stress and -anxiety functions. The septum, which contains an abundance of CRFR2, has been shown to modulate the activity of the amygdala (King and Meyer, 1958; Lee and Davis, 1997a, b; Melia and Davis, 1991). Previous findings have shown that lesions of the amygdala result in decreased ACTH secretion following restraint stress (Allen and Allen, 1975; Beaulieu et al., 1986, 1987; Marcilhac and Siaud, 1996). Therefore, it is possible that CRFR2 in the lateral septum modulates the amygdala, and in the absence of CRFR2, unimpeded CRFR1 activation and amygdala projections may result in a rapid HPA response and increased anxiety levels. As the CRFR2-- mutant mice display increased levels of CRF mRNA in the cAmyg, whereas CRFR1-mutant mice have normal levels, this again suggests that the cAmyg is a critical center for transduction of stress and anxiety signals. Interestingly, increased anxiety-like behavior was also reported for CRF binding-protein deficient-- mice where CRF levels are also increased in the brain (Karolyi et al., 1999), as well for the transgenic CRF-- overexpressing mice (Stenzel-Poore et al., 1994). While a close correlation exists between activation of stress and anxiety, evidence supports a disassociation between the HPA axis and the limbic system (Adamec and McKay, 1993; Pich et al., 1993; Smith et al., 1998). An amplified or exaggerated HPA axis response to stress can be a strong indication of animal's "state" and may therefore be indicative of the presence of anxiety (Allen and Allen, 1975). However, this is not necessarily the case as an animal can certainly be stressed without being anxious. As CRF has strong links to both induction of the HPA axis following stress and increased anxiogenic behaviors, it is somewhat difficult to tease apart these responses (Bale et al., 2000; Butler et al., 1990; Coste et al., 2000; Jones et al., 1998; Koob and Thatcher-Britton, 1985). While studies have shown that exogenous administration of glucocorticoids does not significantly elevate anxiety-like behaviors in mice (Smith et al., 1998), other studies provide evidence that depending on the type and duration of the stress, glucocorticoids can in fact promote anxiogenic behaviors (Calvo and Volosin, 2001; Kabbaj et al., 2000). This then supports a distinct disassociation between HPA axis stimulation and anxiety behavioral responses while still suggesting that neural connections between the limbic system and the hypothalamus exist.

CRFR2 in the PVN may also dampen the CRFR1-- activated HPA axis in response to stress (hypothesis is illustrated in Fig. 4). Preliminary data from our double-receptor deficient mice suggests that CRFR2 may play a role in regulation of the HPA axis not previously identified (unpublished results). Although the CRFR1-mutant mice have increased CRF mRNA in the PVN, it is difficult to determine at what level CRFR2 may affect the HPA axis response because in the absence of CRFR1 in the pituitary the ACTH response is diminished. Therefore, a comparison between the double-mutant mice and the CRFR1-mutant mice will allow a further evaluation of the role CRFR2 plays in stress. Thus, evidence exists in favor of opposite functional roles for the two known CRF receptors, where activation of CRFR1 may be responsible for increased anxiety-like responses, stimulation of CRFR2 may produce anxiolytic-like effects. Regulation of the relative contribution of the two CRF receptor subtypes to brain CRF pathways may be essential in coordinating physiological responses to stress. Examination of mice deficient for both of the CRF receptors will help delineate the independent as well as modulatory actions of each receptor. In addition, as new CRF-family ligands are being discovered, determination of their interaction and regulation in the stress response and in anxiety modulation will aid in the delineation of the role for each CRF receptor.

ACKNOWLEDGMENTS

The authors would like to thank K. Anderson for his assistance with the experiments and S. Guerra for her help in preparation of the manuscript. This work was supported in part by grants from the NIH (DK26741), the Robert J. and Helen Kleberg Foundation, the Ludwick Family Foundation and The Foundation for Research. TLB is supported by NRSA fellowship DK09841.

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TRACY L. BALE,2 KUO-FEN LEE, AND WYLIE W. VALE

Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North, Torrey Pines Rd., La Jolla, California 92037

From the Symposium Stress-Is It More Than a Disease? A Comparative Look at Stress and Adaptation presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 3-- 7 January 2001, at Chicago, Illinois.

2 E-mail: bale@salk.edu

Copyright Society for Integrative and Comparative Biology Jun 2002
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