To characterize further the establishment of the opioid system during prenatal mouse development, we have examined the spatial and temporal expression patterns of , , and ␦ opioid receptor mRNAs and find that the expression patterns of these mRNAs are distinct at all ages. Within the embryo, is the first opioid receptor expressed, with transcripts detected in the gut epithelium as early as embryonic day 9.5 (E9.5). By E10.5, receptor expression is first detected in the facial-vestibulocochlear preganglion complex, whereas ␦ receptor mRNA is first detected at E12.5 in several peripheral tissues, including the olfactory epithelium, heart, limb bud, and tooth. In the brain, both and mRNAs are first detected at E11.5 in the basal ganglia and midbrain, respectively. During mid-gestation and late gestation, the expression of both and receptors extends to other brain regions that exhibit high expression in the adult, including the medial habenula, hypothalamus, pons, and medulla for and the basal ganglia, thalamus, hypothalamus, raphe, and ventral tegmental area for . Thus by E17.5, many aspects of the adult expression patterns of and receptors already have been established. Compared with and , ␦ receptor mRNA expression in the brain begins relatively late, and the expression levels remain very low even at E19.5. In contrast to its late appearance in the brain, however, ␦ is the first opioid receptor expressed in the dorsal root ganglion, at E12.5, before its expression in the spinal cord begins at E15.5. receptor is the first opioid receptor expressed in the spinal cord, at E11.5. These results extend previous ligand-binding data to significantly earlier ages and suggest that early developmental events in both neural and non-neural tissues may be modulated by opioid receptors. Several examples of possible autocrine and paracrine loops of opioid peptide and receptor expression have been identified, suggesting a role for these local circuits in developmental processes.

IGFs (IGF-I and IGF-II) are essential for development, and their bioactivities are tightly regulated by six related IGFbinding proteins (IGFBPs). IGFBP-5 is the most highly conserved binding protein and is expressed in several key developmental lineages as well as in multiple adult tissues including the mammary gland. To explore IGFBP-5 actions in vivo, we produced IGFBP-5 knockout (KO) mice. Whole-body growth, selected organ weights, and body composition were essentially normal in IGFBP-5 KO mice, presumably because of substantial compensation by remaining IGFBP family members. The IGFBP-5 KO mice also exhibited normal mammary gland development and were capable of nursing their pups. We then directly evaluated the proposed role of IGFBP-5 in apoptosis and remodeling of mammary gland during involution. We found that the process of involution after forced weaning was delayed in IGFBP-5 KO mice, with both the appearance of apoptotic cells and the reappearance of adipocytes retarded in mutant mice, compared with controls. We also determined the effects of IGFBP-5 deletion on mammary gland development in pubertal females after ovariectomy and stimulation with estradiol/progesterone. In this paradigm, IGFBP-5 KO mammary glands exhibited enhanced alveolar bud formation consistent with enhanced IGF-I action. These results demonstrate that IGFBP-5, although not essential for normal growth, is required for normal mammary gland involution and can regulate mammary gland morphogenesis in response to hormone stimulation. (Endocrinology 148: 2138 -2147, 2007)

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The endogenous opioid system is involved in various physiological processes including neurogenesis in the dentate gyrus (DG) of the hippocampus. In the current study, we investigated the role of the mu opioid receptor (MOR-1) on DG neurogenesis and measured glucocorticoid levels following several injection paradigms to supplement the neurogenesis experiments. MOR-1 knockout (KO) mice on C57BL/6 and 129S6 backgrounds were injected with BrdU using either a single injection or two different repeated injection protocols and then sacrificed at different time points. The total number of BrdU and PCNA positive cells in the DG is significantly increased in MOR-1 KO mice compared to WT on both strains after repeated injection, but not after a single injection. Plasma corticosterone (CORT) levels increased similarly in MOR-1 KO and WT mice following both single and repeated injection, indicating that the stress response is activated following any injection protocol, but that the mechanism responsible for the increase in BrdU labeling in MOR-1 KO mice is CORT-level independent. Finally, WT 129S6 mice, independent of genotype, showed higher levels of plasma CORT compared to WT C57BL/6 mice in both non-injected controls and following injection at two separate time points; these levels were inversely correlated with low numbers of BrdU cells in the DG in 129S6 mice compared to C57BL/6 mice. In summary, these data demonstrate that loss of MOR-1 increases BrdU labeling in the DG independent of CORT levels, but only following a repeated injection, illustrating the capability of injection paradigms to influence cell proliferative responses in a genotype-dependent manner.

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