Abstract:
Opioid receptors and their peptide ligands modulate numerous physiological functions, including analgesia, respiration, and brain development. A method was developed for the quantitative analysis of fluorescence in situ hybridisation (FISH) staining using cultured SH-SY5Y cells as a model system. Relative quantification with respect to a housekeeping gene was trialled using double-labelled FISH. This approach highlighted problems associated with the use of housekeeping genes as internal standards. The densitometric FISH analysis was used to demonstrate that preproenkephalin knockout mice, which lack the endogenous opioid receptor ligands [Leu]- and [Met]-enkephalin, show increased mu opioid receptor (MOR) mRNA expression within specific brain regions. Thus, FISH provided high-resolution information regarding the regional specific expression of MOR mRNA and showed that FISH could be used to quantitate relative changes in mRNA expression in fixed tissue sections. Using this technique the expression of MOR mRNA was mapped in the developing rat brain with an emphasis on the developing brainstem. High levels of MOR mRNA were detected in prenatal and postnatal brainstem in regions known to modulate respiration, including the nucleus of the solitary tract, nucleus ambiguous and spinal trigeminal tract. In addition, this study provides the first evidence that MOR mRNA is abundantly expressed in all of the embryonic germinal zones, including those of the developing cerebellum. The presence of MOR within these regions supports a role of MOR in the regulation of neurogenesis and brain development.
