In Situ and Immunohistochemical Analysis of Endogenous Opioid Receptor mRNA and Protein Expression in the Developing Human Brain. • 1745

Abstract

In the extremely premature neonate, much CNS development is undertaken ex utero, while the infant is exposed to multiple environmental and pharmacological stressors. Although there is a wealth of information concerning long-term outcomes of premature survivors, little is known regarding the effects of environmental influences on their developing brain, particularly on their developing opioid receptor systems. Premature neonates are regularly exposed to exogenous opiates (e.g., morphine, fentanyl) while critically ill, and this may permanently alter their opioid receptor profile as adults. In order to better study this, it is important to first learn the expression of opioid receptors during normal human CNS development. Fresh post-mortem brain specimens were obtained at various gestational ages (16 to 36 wk). Brain specimens were blocked, immersed in Zamboni's fixative for 100-200 days, soaked in 10% sucrose, quick-frozen in isopentane, then cryostat cut into 15μm sections. For human mu immunohistochemistry, tissue was incubated in primary rabbit antiserum directed against the C-terminal sequence of the rat mu receptor protein, then it was visualized using nickel chloride and diaminobenzidine. Pre-absorption controls were performed using 2μM protein absorption of primary antiserum prior to tissue incubation. For in situ hybridization, (35)S-labeled cRNA probes were generated against the human mu and kappa receptors and enkephalin peptide. After in situ hybridization was completed, sections were then opposed to X-ray film for several weeks prior to developing. Labeling of mRNA in parietal cortex was robust for enkephalin peptide in tissue at all gestational ages, and at 20-36 weeks for kappa receptor. Mu receptor mRNA was not detected in this area until 25 weeks gestation. Striatal labeling was strong at 16 weeks using enkephalin probe, but no kappa or mu mRNA was seen in this area until after 22 weeks. Mu immunostaining was observed in the striatum, entorhinal and cerebral cortex, and several additional forebrain and brainstem nuclei These regions are similar to those described previously in rat. Comparisons to distribution of mu receptor in the developing rat brain is also discussed. Supported by NIDA Grant T32DA07268.