ACTH (i.e., corticotropin) is the principal regulator of the hypothalamus-pituitary-adrenal axis and stimulates steroidogenesis in the adrenal gland via the specific cell-surface melanocortin 2 receptor (MC2R). Here, we generated mice with an inactivation mutation of the MC2R gene to elucidate the roles of MC2R in adrenal development, steroidogenesis, and carbohydrate metabolism. These mice, the last of the knockout (KO) mice to be generated for melanocortin family receptors, provide the opportunity to compare the phenotype of proopiomelanocortin KO mice with that of MC1R-MC5R KO mice. We found that the MC2R KO mutation led to neonatal lethality in three-quarters of the mice, possibly as a result of hypoglycemia. Those surviving to adulthood exhibited macroscopically detectable adrenal glands with markedly atrophied zona fasciculata, whereas the zona glomerulosa and the medulla remained fairly intact. Mutations of MC2R have been reported to be responsible for 25% of familial glucocorticoid deficiency (FGD) cases. Adult MC2R KO mice resembled FGD patients in several aspects, such as undetectable levels of corticosterone despite high levels of ACTH, unresponsiveness to ACTH, and hypoglycemia after prolonged (36 h) fasting. However, MC2R KO mice differ from patients with MC2R-null mutations in several aspects, such as low aldosterone levels and unaltered body length. These results indicate that MC2R is required for postnatal adrenal development and adrenal steroidogenesis and that MC2R KO mice provide a useful animal model by which to study FGD.adrenocorticotropic hormone (ACTH) ͉ familial glucocorticoid deficiency (FGD) ͉ hypothalamus-pituitary-adrenal ͉ zona fasciculata T he adrenal gland regulates a number of essential physiological functions in adult organisms through the production of steroids and catecholamines. Maintenance of adrenal structure and function is regulated through the integration of extra-and intracellular signals. The pituitary hormone ACTH (i.e., adrenocorticotropic hormone), which is derived from the proopiomelanocortin (POMC) polypeptide precursor, is the principal regulator that stimulates adrenal glucocorticoid (GC) biosynthesis and secretion via the membrane-bound specific receptor for ACTH, ACTH receptor/melanocortin 2 receptor (MC2R) (1).It was previously demonstrated that, although POMC knockout (KO) mice are born at the expected Mendelian frequency, three-quarters of POMC KO mice undergo neonatal death. Furthermore, those mice surviving to adulthood exhibit obesity, pigmentation defects, and adrenal insufficiency (2-4). POMC KO mice possess macroscopically detectable adrenal glands that lack normal architecture (2, 4, 5). These results demonstrate the importance of POMC-derived peptides in regulating the hypothalamus-pituitary-adrenal axis and adrenal development.Familial glucocorticoid deficiency (FGD), or hereditary unresponsiveness to ACTH [Online Mendelian Inheritance in Man (OMIM) no. 202200; www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?idϭ202200], is an autosomal recessive disorder ...

The expression of corticotropin releasing factor (CRF) and urocortin in hypothalamic magnocellular neurones increases in response to osmotic challenge. To gain a better understanding of the physiological roles of CRF and urocortin in fluid homeostasis, CRF, urocortin and CRF type 1 receptor (CRFR-1) gene expression was examined in the hypothalamic-hypophyseal system usingin situ and double-label in situ hybridization following chronic salt loading. CRFR-1 expression was further examined by immunohistochemistry and receptor binding. Ingestion of hypertonic saline by Sprague-Dawley rats for 7 days induced CRF mRNA exclusively in the oxytocin neurones of the magnocellular paraventricular nucleus (PVN) and the supraoptic nucleus (SON), but induced CRFR-1 mRNA in both oxytocin and vasopressin-containing magnocellular neurones. Hypertonic saline treatment also increased urocortin mRNA expression in the PVN and the SON. In the SON, urocortin was localized to vasopressin and oxytocin neurones but was rarely seen in CRF-positive cells. Changes in CRFR-1 mRNA expression in magnocellular neurones by hypertonic saline treatment were accompanied by changes in CRFR-1 protein levels and receptor binding. Hypertonic saline treatment increased CRFR-1-like immunoreactivity in the magnocellular PVN and SON, and decreased it in the parvocellular PVN. CRF receptor binding in the PVN and SON was also increased in response to osmotic stimulation. Finally, hypertonic saline treatment increased CRFR-1 mRNA, CRFR-1-like immunoreactivity and CRF receptor binding in the intermediate pituitary. These results demonstrate that the increase in the expression of CRF and urocortin message in magnocellular neurones induced by salt loading is accompanied by an increase in CRF receptor levels and binding in the hypothalamus and intermediate pituitary. Thus, CRF and urocortin may exert modulatory effects locally within magnocellular neurones as well as at the pituitary gland in response to osmotic stimulation.

We have previously proposed the existence of ultrashort loop-positive feedback regulation of corticotropin-releasing hormone (CRH) in the hypothalamus. To gain a better understanding of this effect, we performed double-label in situ hybridization to identify the neurons in the paraventricular nucleus (PVN) that express CRH type 1 receptor (CRH-R1) following stress. We also conducted immunohistochemistry to determine whether CRH-R1 mRNA was translated to CRH-R1 protein in the PVN. Thirty-minute restraint stress given to male Wistar rats increased c-fos mRNA expression primarily in the CRH-producing neurons of the parvocellular PVN. Small numbers of vasopressin and oxytoxin-producing cells were also labeled by c-fos probes. Approximately 70% of CRH-R1 positive neurons exhibited CRH mRNA 2 h after the beginning of stress, while only a small percentage of the vasopressin and oxytocin-producing cells coexpressed CRH-R1 mRNA. CRH-R1 immunoreactivity, which was detected in the perikarya and fibers of PVN neurons, appeared to increase in response to stress, though this was not statistically significant. Pretreatment with a selective CRH-R1 antagonist, CP-154,526, significantly attenuated stress-induced corticotropin (ACTH) secretion as well as c-fos mRNA expression in the PVN. These results demonstrate that acute stress increases neuronal activation and CRH-R1 mRNA expression primarily in CRH-producing neurons of the parvocellular PVN, that CRH-R1 message is translated to CRH-R1 protein, and that PVN neurons are activated at least in part through CRH-R1 under acute stress. The data further support the possibility of feedback regulation of CRH itself in CRH-producing neurons.

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Multiple sequential actions, performed during parental behaviors, are essential elements of reproduction in mammalian species. We showed that neurons expressing melanin concentrating hormone (MCH) in the lateral hypothalamic area (LHA) are more active in rodents of both sexes when exhibiting parental nursing behavior. Genetic ablation of the LHA-MCH neurons impaired maternal nursing. The post-birth survival rate was lower in pups born to female mice with congenitally ablated MCH neurons under control of tet-off system, exhibiting reduced crouching behavior. Virgin female and male mice with ablated MCH neurons were less interested in pups and maternal care. Chemogenetic and optogenetic stimulation of LHA-MCH neurons induced parental nursing in virgin female and male mice. LHA-MCH GABAergic neurons project fibres to the paraventricular hypothalamic nucleus (PVN) neurons. Optogenetic stimulation of PVN induces nursing crouching behavior along with increasing plasma oxytocin levels. The hypothalamic MCH neural relays play important functional roles in parental nursing behavior in female and male mice.