Mice deficient in CRF were created using targeted gene inactivation in embryonic stem cells (Muglia et al., 1995). This model has added important information to our present understanding of CRF roles and in some cases has provided evidence that contradicts current hypotheses. CRF KO mice exhibit severe glucocorticoid deficiency and impaired HPA axis activation - features predicted to occur in the absence of CRF.
Effects of CRF absence on organ development and longevity
Histological examination revealed that pituitary structure and ACTH immunoreactivity are normal in CRF-KO mice, despite previous in vitro studies implicating CRF as a mitogenic stimulus for corti-cotroph development (Gertz et al., 1987). However, the adrenal gland of these mice shows marked atrophy, exclusively at the zona fasciculata, the region responsible for glucocorticoid production. Thus, corticosterone levels are extremely low in CRF KO mice. This defect has been attributed to altered ACTH input. While basal levels of ACTH are normal, CRF KO mice do not exhibit the normal circadian rise in ACTH (Muglia et al., 1997). In addition, neonatal alterations in ACTH may influence adrenal gland maturation, as revealed in CRF, receptor KO mice discussed below. It should be noted that although pituitary and circulating ACTH are similar to WT mice, these levels are lower than expected given the presumed lack of negative feedback from glucocorticoids.
Glucocorticoid deficiency in this model has dire consequences for neonates. Progeny of homozygous mating die within the first 12-24 h postnatally, due to lung dysplasia. Prenatal administration of glucocorticoids to homozygous mothers is required for fetal lung maturation and postnatal survival of homozygous offspring (Muglia et al., 1995). Thus, this model has allowed for extensive examination of glucocorticoid influence on fetal lung differentiation and maturation (Muglia et al., 1999). Interestingly, glucocorticoid treatment is not necessary beyond the fetal period. CRF KO mice exhibit normal longevity and fertility despite low circulating glucocorticoids (Muglia et al., 1995). In contrast to previous thought, this finding suggests that the many changes associated with adrenal insufficiency such as low body weight, fatigue, and decreased fertility may not be related directly to the lack of glucocorticoids.
CRF KO mice exhibit impaired HPA axis responses to a number of different stressors (ether inhalation, restraint stress, hypoglycemia, and hypovolemia) with little to no elevation in plasma ACTH and corticosterone (Muglia et al., 1995; Jacobson et al., 2000; Jeong et al., 2000). In addition, CRF KO mice have significantly lower plasma epinephrine basally and show a blunted and delayed epinephrine response to stress. It appears that conversion of noradrenaline to epinephrine is impaired in CRF KO mice. While basal noradrenaline levels are high in these mice, mRNA expression and activity levels of phenylethanolamine N-methyl-transferase (PNMT, the enzyme that catalyzes conversion of noradrenaline to epinephrine) in the adrenal medulla is severely diminished. It is suggested that glucocorticoid deficiency is responsible since blockade of corticosterone synthesis in WT mice with metyrapone similarly diminishes PNMT expression (Jeong et al., 2000). Thus, central CRF may mediate catecholamine responses to stress via autonomic input to the adrenal medulla as well as via paracrine actions of glucocorticoids produced in the adrenal cortex.
In contrast to psychological and homeostatic stress, immune activation induced by endotoxin produces a robust rise in plasma corticosterone in CRF KO mice (Karalis et al., 1997; Bethin et al., 2000). Furthermore, T cell activation with an anti-CD3 antibody increases corticosterone secretion in CRF KO mice to the same extent as in WT mice (Bethin et al., 2000). These results indicate that immune stimulation can activate HPA axis pathways independent of CRF and that the abnormally developed zona fasciculata is capable of corticosterone responses under conditions of sufficient stimulation. Evidence suggests that cytokines such as IL-6 induced during the inflammatory response act directly at the level of the pituitary or adrenal gland to stimulate glucocorticoid release in the absence of CRF (Bethin et al., 2000). A similar pattern of HPA axis activation was found in mice lacking the CRF) receptor as described below. These findings suggest that immune activation of the HPA axis is selectively preserved in the absence of CRF-CRF| receptor signaling and may underscore an adaptive role for immune-mediated pituitary-adrenal axis activation. Glucocorticoids are necessary to suppress pro-inflammatory cytokines, thereby limiting their deleterious effects. Such a pathway that permits activation of the pituitary adrenal axis independent of CRF would ensure the ability of this axis to improve survival during immune challenge.
When administered directly into the brain, CRF produces behaviors similar to those observed following exposure to stress (Dunn and Berridge, 1990). Furthermore, many stress-induced behaviors can be inhibited with CRF antagonism (Koob and Heinrichs, 1999). This has led to the widely held belief that CRF participates in behavioral components of the stress response. Surprisingly, behavioral responses following stress or exogenous CRF treatment are normal in mice lacking CRF.
Stress-induced changes in freezing, learning, and anxiety-like behaviors are similar to WT controls (Weninger et al., 1999). In addition, basal feeding as well as feeding responses to hypophagic stimuli are normal (Swiergiel and Dunn, 1999; Weninger et al., 1999). Interestingly, a CRF] receptor-specific antagonist blocks stress-induced freezing in these mice, which suggests a primary role for this receptor subtype in behavioral responses to stress. These data provide evidence that CRF is not required to initiate these behaviors and leave open intriguing questions regarding the identity of the CRF-related peptide that is responsible for CRF, receptor-mediated stress behaviors or substitutes for CRF in its absence. Urocortin 1 is upregulated basally in CRF KO and in WT animals upon stress; however, in both cases expression is restricted to the EW nucleus (Weninger et al., 2000). The function of the EW is still unresolved; however, it seems unlikely that urocortin 1 produced exclusively in the EW could mediate the full complement of complex behaviors involved in stress and anxiety. Moreover, recent results in urocortin 1 KO mice suggest that urocortin 1 is not anxiogenic (see below), although other behaviors have not yet been tested. Urocortins 2 and 3 are unlikely candidates as they are CRF2 receptor-specific. It is possible that CRF and urocortin 1 can substitute for one another - a hypothesis that can be tested readily with CRF/urocortin double KO mice. Alternatively, an as yet undiscovered CRF family member may mediate stress behaviors via the CRFi receptor.
Urocortin 1 knock-out mice (urocortin 1 KO)
Currently, little is known regarding the role of endogenous urocortin 1 in the CNS. Our understanding has been hampered by the fact that exogenous adminstration of urocortin 1 produces a profile similar to CRF, due to the ability of urocortin 1, like CRF to bind to both receptor subtypes. Recently, urocortin 1 KO mice were generated by two independent groups using standard targeted gene inactivation in embryonic stem cells (Vetter et al., 2002; Wang et al., 2002). The lines were founded and tested on different genetic backgrounds (129S7/ C57BL/6-7>rciW and 129Sv/C57BL/6J), which may account in part for the phenotypic differences observed.
Urocortin 1 immunoreactivity has been found in the pituitary (Bittencourt et al., 1999), raising the possibility that urocortin 1 may modulate HPA activation. However, normal HPA axis activity was observed in urocortin 1 KO mice. ACTH and corticosterone levels are comparable to WT mice basally and significantly increase with normal kinetics following acute restraint stress (Vetter et al., 2002; Wang et al., 2002). These findings demonstrate that endogenous urocortin 1 stimulation is not critical for HPA activation, consistent with a previous report showing that antiserum to urocortin 1 failed to inhibit stress-induced increases in ACTH (Turnbull et al., 1999). Currently, it is not known whether maintenance and recovery stages of the pituitary-adrenal response are normal in urocortin 1 KO mice. Such studies would be interesting given that HPA recovery is impaired in CRF2 receptor KO mice.
Behavioral and physiologic analyses in urocortin 1 KO mice
The finding that CRF KO mice exhibit normal stress-induced behaviors led to the suggestion that urocortin 1 may mediate these behaviors, acting either alone or in concert with other CRF congeners (Weninger et al., 2000). Behavioral responses tested thus far in urocortin 1 KO mice do not provide compelling support that urocortin alone is a pivotal mediator. Studies in both lines of mice suggest that endogenous urocortin 1 does not induce anxietylike behavior. Wang et al. (2002) report normal anxiety-related behaviors in urocortin 1 KO mice when exposed to three different anxiety paradigms. In contrast, Vetter et al. (2002) found that urocortin 1 KO mice display enhanced anxietylike behavior compared to WT mice, showing less time spent in the open arms of the elevated plus maze and the center of an open field. Overexpression of CRF or CRFj receptor does not appear to play a role in this enhanced anxiety as mRNA expression of these molecules are normal in urocortin 1 KO mice. However, a significant reduction of CRF2 receptors was found in the lateral septum, a region implicated in anxiety (Vetter et al., 2002). These data are consistent with heightened anxiety found in two of the three lines of CRF2 receptor KO mice. Collectively, the data suggest a subtle, anxiolytic role for endogenous urocortin 1/CRF2 receptor pathways that may be sensitive to genetic background. Other responses of urocortin 1 KO mice, including basal and post-deprivation feeding as well as stress-induced increases in heart rate are comparable to WT mice (Vetter et al., 2002; Wang et al., 2002).
A novel role for urocortin 1 pathways in the auditory system was revealed in urocortin 1 KO mice. These mice present with a hearing deficit at low sound frequencies as measured by auditory brainstem response (ABR) (Vetter et al., 2002). It is not clear whether such a defect exists in the other line of urocortin 1 KO mice. Wang et al. (2002) report a reduced acoustic startle reflex in male urocortin 1 KO, but suggests that hearing is normal in these mice. Differences in auditory examination prevent direct comparisons between the two lines of mice, thus further testing may be needed to resolve discrepancies in the auditory phenotype. Nevertheless, Vetter et al. (2002) show expression of urocortin 1 and both known CRF receptor subtypes in the cochlea, which suggests that paracrine signaling of a urocortin 1 system may influence peripheral auditory processing.
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