Fate of mu receptors during rat skeletogenesis

Studies have intimated a role for endogenous opioids in skeletal ontogeny. We hypothesized that this role may be confined to the perinatal period. We, therefore, examined both fetal and postnatal rat long bones to determine the pattern, if any, of mu receptor expression. Perfused long bones and brains were harvested from 3 to 4 each of near-term fetuses, 3- to 4-week-old neonates, and 6-week-old juveniles. Tissues were decalcified, embedded, sectioned, quenched of peroxidase, and then blocked. Sections were incubated overnight with either rabbit antirat mu receptor IgG or naïve rabbit IgG (control) at 4 degrees C. The next day, sections were washed, blocked again, and incubated with biotin-labeled secondary antibody, streptavidin-peroxidase conjugate, and a chromogen substrate with intervening wash steps. Slides were counterstained with hematoxylin and coverslipped. Digital photomicrographs were then imported into an image analysis program and the percent area of stained cortical and trabecular bone quantified. Mu receptor expression decreased significantly with age. Approximately 25% of the area of fetal long bones stained positively, including the endosteum, periosteum, and the growth plate. Little or no nonspecific staining occurred. Staining in neonatal tissue was diminished to <11% of the area and involved areas of apparent remodeling; chondrocytes in the growth plates failed to stain. Finally, juvenile bone evidenced staining approaching background levels produced by control slides (approximately 2%). Mu receptors are abundant in developing rat long bones during fetal development but become progressively less abundant postnatally. This infers a role for endogenous opioids during skeletal ontogeny.