The role of CRF2 receptor pathways in stress-related pathways remained elusive until the recent generation of CRF2 receptor KO mice and the development of a specific CRF2 receptor antagonist. In addition, the recent discovery of CRF2 receptor-selective ligands, urocortin 2 and urocortin 3, aid greatly in our understanding of CRF2 receptor pathways. CRF2 receptor KO mice were generated by us (Coste et al, 2000) and two other independent groups (Bale et al, 2000; Kishimoto et al, 2000) using targeted gene disruption in embryonic stem cells. This model has provided surprising insights into the role of CRF2 receptor in mediating central and peripheral responses of CRF-related peptides. Intriguingly, studies using these mice suggest a role for the CRF2 receptor in coordinating responses initiated through the CRFi receptor.
HP A axis responses to stress in CRF2 receptor KO mice
Mice that lack CRF or CRFi receptor revealed that both CRF and CRF] receptor are critical for maintaining normal HPA tone as well as initiating the HPA stress hormone cascade (Smith et al, 1998; Timpl et al., 1998). The presence of the CRF2 receptor in regions known to modulate HPA activity such as the PVN and the amygdala (Chalmers et al, 1995) suggests that CRF2 receptor pathways may also modify neuroendocrine activity. In the basal state, CRF2 receptor KO mice show normal levels of circulating ACTH and corticosterone. However, following a brief restraint stress, ACTH levels are more robust initially and decline more rapidly compared to WT mice (Bale et al, 2000; Coste et al, 2000). More rapid termination of ACTH in CRF2 receptor KO mice suggests that CRF2 receptors may sustain the early ACTH response possibly through CRF actions on CRF2 receptors in the PVN. That such a feedforward mechanism of regulation exists has been suggested by previous studies that show stress-induced pituitary-adrenal activation is further enhanced with exogenous CRF (Ono et al, 1985) and that CRF stimulates its own expression in the PVN (Parkes et al, 1993). Interestingly, CRF2 receptor KO mice also exhibit abnormal recovery from HPA axis activation. Corticosterone levels remained significantly elevated post-stress in CRF2 receptor KO mice compared to WT mice (Coste et al, 2000). Thus, CRF2 receptors may regulate the recovery phase of the stress response, perhaps by influencing negative feedback of the HPA axis - an effect likely to be independent of its feedforward actions in the hypothalamus. Collectively, the data suggest that the CRF2 receptor plays an integral part in shaping the HPA axis response to stress.
Behavioral stress responses in CRF2 receptor KO mice
While there is compelling evidence that the expression of anxiety-like behavior depends highly on CRFi receptor activation, the existence of CRF2 receptors in regions of the amygdala, BNST, and lateral septum suggests that a role for this receptor subtype should not be excluded. Analyses of anxiety-like behavior in the three lines of CRF2 receptor KO mice have yielded disparities, which render somewhat tenuous conclusions regarding this receptor subtype and anxiety. We have found no evidence for altered anxiety responses in CRF2 receptor KO mice using the elevated plus maze, open field activity (Coste et al, 2000), and the light/dark emergence test (unpublished data). Our results differ from those obtained with independently generated CRF2 receptor KO mice wherein increased anxiety-like behavior was observed. Such differences between mouse models highlight the potential caveat of genetic background, which may play a critical role, particularly when behavioral alterations are subtle (Nadeau, 2001). In addition, it should be noted that increased anxiety was found in some but not all anxiety paradigms (Bale et al, 2000; Kishimoto et al, 2000) and was apparent only in female mice in one of the lines, thus underscoring the difficulties in measuring anxiety. Given the mixed outcomes, it appears that the CRF2 receptor may not be a strong determinant of anxiety states per se. Rather, these data suggest that an effect of the CRF2 receptor activation may be to relieve anxiety, leading to speculation that CRF2 receptors may restrain CRF] receptor anxiogenic pathways during stress. Interestingly, recent pharmacological studies using a CRF2 receptor-selective ligand or antagonist (urocortin 2 and anti-sauvagine-30, respectively) show that CRF2 receptor activation stimulates anxiety-related behavior in distinct nuclei, particularly the lateral septum. Taken together, the data suggest that CRF2 receptor pathways may contribute to anxiety, perhaps acting in an ancillary role to that of CRF! receptor.
Other behavioral studies support the notion that the CRF2 receptor may countermand the effects of CRF! receptors by modulating late stages of the stress response. For example, CRF2 receptor KO mice and WT mice differ in their anorectic responses to urocortin 1. Both genotypes show similar inhibition of feeding immediately following urocortin 1 treatment, however, CRF2 receptor KO mice recover to normal intake levels more rapidly than WT animals. As mentioned above, urocortin 1-treated CRF) receptor KO mice are the mirror image -hypophagic only during the late but not early phase (Bradbury et al., 1999). Taken together, the early phase of urocortin 1-induced hypophagia occurs via CRF, receptor, but late-phase suppression critically depends on the CRF2 receptor. Such findings have led us to explore whether other, secondary behaviors that occur in later stages of the stress response are mediated by the CRF2 receptor. Such behaviors include stress-coping behaviors that are recruited during the stress response and serve to reduce the effect of the aversive stimulus, thereby aiding the return to steady state. Indeed, studies using antisense modulation of the CRF2 receptor implicated a role for this receptor subtype in stress-coping behaviors (Liebsch et al., 1999). We explored this possibility further using self-grooming, a behavior thought to reflect de-arousal and coping following stress (Spruijt et al., 1992). Compared to WT mice, CRF2 receptor KO mice exhibit significantly reduced grooming behavior in a novel, open field, or following restraint stress (unpublished data), which suggests that CRF2 receptors may be involved in recovery from stress.
CRF is well-recognized for its ability to modulate cardiovascular function. CRF delivered icv increases arterial blood pressure and heart rate similar to the effects of stress (Overton and Fisher, 1991). In contrast, urocortin 1 or CRF delivered systemically induces a marked decrease in blood pressure (Vaughan et al., 1995) due to vasodilation in specific vascular beds (Overton and Fisher, 1991). The predominance of CRF2 receptors in the heart and vasculature suggests that this receptor subtype mediates the peripheral actions of CRF or urocortin 1. We found that systemic urocortin 1 administration fails to decrease mean arterial pressure in CRF2 receptor KO mice, whereas WT mice show a marked reduction, which demonstrates that CRF2 receptor mediates the hypotensive effect of systemically administered urocortin 1 (Bale et al., 2000; Coste et al., 2000). CRF2 receptors localized on endothelial and/or smooth muscle cells of blood vessels likely mediate urocortin 1-induced hypotension; however, the central and/or peripheral source of CRF-related peptides that modulate peripheral cardiovascular changes has not been determined. In addition to effects on blood pressure, a recent report suggests that CRF2 receptors in the vasculature is necessary for tonic inhibition of new vascularization in the adult via effects on smooth muscle cell proliferation and endothelial growth factors (Bale et al., 2002). Careful analysis showed that CRF2 receptor KO mice exhibit hypervascularization postnatally with increases in both the number and the size of blood vessels in various tissues.
In addition, several studies indicate that CRF or urocortin 1 may have direct actions on the heart leading to increased contractile function (Grunt et al., 1992; Parkes et al., 1997). Both CRF and urocortin 1 have been shown to increase cardiac contractility in vitro (Grunt et al., 1992) and in vivo following systemic administration (Parkes et al., 1997). Cardiomyocytes express CRF2 receptors and respond to CRF and urocortin 1 with robust increases in cAMP production (Heldwein et al., 1996), which suggests that cardiac contractile responses to urocortin 1 are CRF2 receptor-dependent. Indeed, we found that CRF2 receptor KO mice do not exhibit detectable cardiac responses to urocortin 1 whereas WT mice show a pronounced increase in cardiac function (Coste et al., 2000). Increased cardiac function in WT mice is likely due to direct actions of urocortin 1 on cardiomyocytes since CRF2 receptor activation in these cells increases cAMP (Heldwein et al., 1996), which is known to stimulate cardiac contractility (Miyakoda et al., 1987). However, decreased blood pressure following urocortin 1 injection may also contribute to the increase in contractile function.
Collectively, these findings show that changes in cardiac function and blood pressure depend critically on the CRF2 receptor. It is noteworthy that stress-induced effects and those induced by injection of CRF into the CNS lead to similar cardiovascular changes that are favorable during the "fight or flight" response; namely, elevation in arterial pressure and heart rate, and a marked change in regional blood flow resulting in shunting from mesentery to skeletal muscle (Overton and Fisher, 1991). It is possible that systemic or paracrine actions of urocortin 1 may oppose these CNS effects by redirecting local blood flow thereby restoring regional hemodynamics to a basal state while maintaining increased cardiac function. Thus, it is tempting to speculate that a common theme exists wherein neuroendocrine, behavioral and cardiovascular responses to stress are initiated through CRF] receptors and are then tailored further within various stress pathways through the actions of the CRF2 receptor.
CRF type 1 and type 2 receptor knock-out mice (CRF-R DKO)
Two independent laboratories recently generated mice deficient in both CRF receptor subtypes by mating CRF! receptor KO and CRF2 receptor KO mice. The double mutation exists currently on a heterogenous genetic background of either 129Svj:C57BL/6 (Bale et al., 2002) or 129Svj:C57BL/6:12901a:CDl (Preil et al., 2001).
In both lines, the endocrine profile of the double mutants follows closely the CRF! receptor KO mice, which demonstrates the dominance of CRFi receptors in mediating HPA axis activity. Thus, CRF-R DKO mice show atrophy of the zona fasciculata region of the adrenal gland, with resultant low circulating levels of corticosterone in the basal state and consequent lung dysplasia and neonatal mortality in progeny of homozygous matings. Plasma ACTH levels are normal basally, most likely due to compensatory upregulation of hypothalamic vasopressin as CRF-R DKO mice show elevations in vasopressin mRNA expression in the PVN and immunoreactivity in the median eminence, similar to findings in CRF, receptor KO mice. During behavioral stress, HPA axis activation is severely impaired with no substantial elevation in circulating ACTH or corticosterone in CRF-R DKO mice.
While these studies confirm the importance of the CRF! receptor in the neuroendocrine response, this genetic model has provided the unique opportunity to glimpse the subtle role of CRF2 receptors in modulating HPA axis activity, which would likely be unattainable using pharmacological methods. In these studies, the loss of functional CRF2 receptors in the double mutants exacerbates neuroendocrine deficiencies. Thus, CRF-R DKO mice show lower basal and stress-induced corticosterone levels compared to mice deficient in the CRF, receptor. This finding is most pronounced in the mutant line generated by Bale et al. (2002), while Preil et al. (2002) reports such findings in female mice only. These differences may be due to genetic modifiers of the background strain. Nonetheless, the data suggest that CRF2 receptor may be involved in fine-tuning HPA axis activity. There has been some investigation as to whether such influences may occur at the level of the adrenal gland where both CRF] receptor and CRF2 receptor are expressed (Muller et al., 2001). However, the corticosterone response to a CRF challenge (i.p. injection) remains significantly impaired in both CRFj receptor KO and double receptor mutant mice, which suggests that CRF2 receptor activation does not stimulate adrenocortical steriodogenesis (Preil et al., 2001).
Behavioral analyses have not been undertaken extensively in either line of CRF-R DKO mice. To date, it is known that viability, body weight, and total food intake are normal in double mutants, as described previously in single-receptor mutant mice (Preil et al., 2001). Bale et al. (2002) report that female CRF-R DKO mice displayed decreased anxiety-like behavior compared to WT mice, showing more frequent entries and increased total time spent in the open arms of the elevated plus maze, similar to female CRF, receptor KO mice. However, male double mutant mice showed a normal anxiety profile during testing, as did WT, CRFi receptor KO and CRF2 receptor KO mice derived from this line. Such findings of normal anxiety in male CRFi receptor KO mice are discrepant from initial reports of robust anxiolytic-like behavior in the original mouse lines. The authors provide preliminary data that suggests the manifestation of anxiety in male mice may be attributable to the mother's genotype (Bale et al., 2002). Thus, anxiety in male mice appeared to be associated with the loss of at least one allele of the CRF2 receptor. However, testing of a large breeding colony is necessary to confirm this claim.
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