CRF, receptor KO mice were generated by two independent groups using targeted gene inactivation in embryonic stem cells (Smith et al., 1998; Timpl et al., 1998). These two lines of mice generally show similar phenotypes despite differences in the genetic background of the embryonic stem cell lineage. Most notably, these mice display severe alterations in HPA axis regulation and marked glucocorticoid deficiency, along with impaired initiation of behavioral responses to stress.
Basal HP A axis regulation in CRF t receptor KO mice
The CRF, receptor is the predominant CRF receptor subtype in the anterior pituitary and mediates hypothalamic CRF-stimulated release of ACTH from pituitary corticotropes (Chalmers et al., 1995). CRF-induced ACTH release is impaired in cultured pituitary cells collected from CRF] receptor KO mice, although basal ACTH levels in vivo are normal in mice lacking CRF! receptor (Smith et al., 1998; Timpl et al., 1998). This suggests that other hypothalamic ACTH stimulating hormones may compensate for the lack of CRF input to maintain basal ACTH release. It is well known that hypothalamic vasopressin acts synergistically with CRF to stimulate ACTH secretion and thus is a likely candidate to amend perturbations in steady-state levels of ACTH. In initial reports, Turnbull and colleagues (Turnbull et al., 1999) showed that systemic administration of vasopressin antiserum significantly reduced basal levels of ACTH in mutant mice while having negligible effect on WT mice. These findings have been substantiated recently by studies showing that plasma vasopressin as well as vasopressin mRNA expression in the PVN and vasopressin immunoreactivity in the median eminence are increased in CRF, receptor KO mice (Muller et al., 2000). Interestingly, replacement of corticosterone in CRFj receptor KO mice returned plasma vaspressin to normal levels, which suggests that glucocorticoid deficiency is responsible for initiating this compensatory pathway. Taken together, vasopressin supplies important stimulatory influences on pituitary corticotropes to preserve ACTH levels in the absence of CRF stimulation. These data confirm that the CRF! receptor mediates CRF-induced release of pituitary ACTH but also demonstrate that circulating ACTH is actively conserved by other hormones.
Despite normal circulating ACTH levels, CRF] receptor KO mice exhibit pronounced glucocorticoid deficiency (Smith et al., 1998; Timpl et al., 1998). Mutant animals have low circulating corticosterone levels with no diurnal rhythm. Similar to CRF KO mice (Muglia et al., 1995), offspring of homozygous matings die within 48 h of birth, attributed to inadequate lung maturation due to low levels of glucocorticoids (Smith et al., 1998). Atrophy of the adrenal gland appears to be responsible for this deficiency. Smith et al. (1998) found a marked decrease in the size of the zona fasciculata region of the adrenal gland while the zona glomerulosa, zona reticularis, and medulla appeared normal. Postnatal treatment (days 10-21) with ACTH was found to prevent this atrophy (Smith et al., 1998). Thus, adrenal insufficiency appears to be due to lower levels of ACTH during neonatal adrenal maturation in CRF| receptor KO mice. In the other line of CRF] receptor KO mice, Timpl et al. (1998) also reported profound glucocorticoid deficiency, but the cause is less clear in this case. The zona fasciculata appeared normal in these mice; however, the size of the adrenal medulla was significantly reduced. It is not clear whether low sympatho-medullary drive could lead to reduced corticosterone levels. Alternatively, atrophy of the adrenal medulla could be a secondary effect of low corticosterone. Nonetheless, CRFi receptor KO mice have demonstrated the importance of CRF stimulatory influences on the development of the mature adrenal gland.
Stress-induced HP A axis activation in CRFi receptor KO mice
Studies using CRF, receptor KO mice demonstrate that the CRF, receptor is critical for the initiation of the neuroendocrine stress response. CRFi receptor KO mice exhibit severely compromised HPA axis activation in response to behavioral stress (Smith et al., 1998; Timpl et al., 1998). Circulating ACTH and corticosterone levels were not significantly increased following acute restraint stress, forced-swim stress or acute alcohol injection in mutant mice (Smith et al., 1998; Timpl et al, 1998; Lee et al, 2001). Exposure to a stressor with a strong sympathetic nervous system component (social defeat stress) caused a modest increase in ACTH in mutant mice; however, a concomitant elevation in corticosterone was not observed. In contrast to behavioral stress, CRF] receptor KO mice were able to mount a robust HPA response to turpentine-induced local inflammation (Turnbull et al, 1999). Both circulating ACTH and corticosterone were increased significantly in mutant mice following inflammation. Similar to findings in CRF KO mice, it appears that cytokine production may be responsible for stimulating the pituitary-adrenal axis during immune challenge. While neither antiserum to CRF nor vasopressin prevented the rise in ACTH as it did in WT mice, mutant mice responded to inflammation with a marked increase in the cytokine, IL-6 (Turnbull et al„ 1999). It is known that IL-6 is capable of stimulating HPA activation at the level of the pituitary and adrenal gland (Bethin et al, 2000). Thus, pronounced increases in IL-6 in mutant mice may contribute to HPA axis activation, independent of CRF or vasopressin pathways. Robust increases in IL-6 in these mice could reflect low circulating corticosterone or lack of CRF input as both have been shown to restrain cytokine production.
Behavioral studies in CRF/ receptor KO mice
As discussed above, CRF KO mice revealed the unexpected finding that a number of stress-induced behaviors do not depend on the presence of CRF, but instead require the presence of the CRFi receptor. Current findings suggest that the CRF| receptor may contribute to the initiation of the classical behavioral response to stress. Thus, CRF] receptor pathways may be involved in stimulating arousal, anxiety, selective memory enhancement and locomotion while suppressing vegetative functions such as feeding and reproductive behavior. Most notably, it is clear that CRFi receptor activation is critical in the expression of anxiety. CRF i receptor KO mice show significantly reduced anxiety-related behaviors under basal conditions and during alcohol withdrawal (Smith et al, 1998; Timpl et al, 1998; Contarino et al, 1999). These results are consistent with reports using CRF| receptor-specific antagonists and are reciprocal to findings of increased anxiety in transgenic mice with CRF overproduction (discussed above). In addition, CRF) receptor KO mice exhibit altered spatial recognition memory during novelty exploration, an effect that has been attributed to inadequate levels of arousal necessary for successful memory performance. Furthermore, CRF| receptor activation may contribute to stress-induced changes in locomotor activity as CRF] receptor KO mice do not display characteristic enhancement of locomotor activity following exogenous administration of CRF (Contarino et al, 2000).
The CRF i receptor is located in several brain regions that participate in the regulation of feeding behavior (hypothalamic nuclei, amygdala) and has been implicated in mediating stress-related suppression of feeding (Hotta et al, 1999). CRF] receptor KO mice show normal body weight and 24-h food intake. However, the circadian pattern of food intake is altered wherein CRF| receptor KO mice consume more food during the light phase compared to WT mice (Muller et al, 2000). These alterations in feeding appear to be related to glucocorticoid deficiency as corticosterone treatment restores diurnal feeding. When exogenous urocortin 1 is given icv, CRF, receptor KO mice do not show reduced feeding initially as WTs do however, hypophagia occurs at later timepoints (3+ hours post injection) at comparable values of WT mice (Bradbury et al, 2000). These findings suggest that CRF, receptor activation is necessary for initial suppression of feeding but not for maintaining this response. As discussed below, late hypophagia is mediated by CRF2 receptor activation. Collectively, these behavioral studies suggest that the CRF, receptor may be involved in eliciting the behavioral response to stress. Thus, CRF | receptor and CRF2 receptor pathways appear to mediate distinct temporal components of the stress response - a theme that is emergent across many CRF-related responses.
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