Abstract:
Few satisfactory protocols exist for primary culture of postnatal brainstem neurons, and commonly used procedures often give poor survival rates in older foetal (>E16) and early postnatal brainstem cultures. The present study describes the first reliable method for establishing stable in vitro cultures of foetal and postnatal brainstem neurons up to six days postnatal age in a defined, serum-free culture medium. This novel culture method was used to study opioid receptor expression and distribution in developing brainstem cells. Opioids play an important role in brainstem functions, being involved in respiratory and cardiovascular modulation and pain control (Olsen et al., 1995; Olson et al., 1997; Vaccarino et al., 1999; Vaccarino and Kastin, 2001). These brainstem functions are particularly important for survival at birth, and opioid receptor distribution patterns and sensitivities to opioid ligands change during development. Using cultured cells and frozen sections of brainstem tissue, mu (MOR) and delta (DOR) opioid receptor localisation in neuronal and glial cells at different stages of foetal and postnatal development in the rat were examined by immunocytochemistry and confocal microscopy. Bipolar and multipolar neurons showed similar immunoreactivities; whereas, glial cells were more lightly stained than neurons. Developmentally advanced stages were more intensely stained for MOR (P<0.006, Mann-Whitney test); whereas, DOR immunoreactivity did not change during development. These developmental expression patterns observed in culture for MOR were similar to those obtained from Western blots of electrophoreses brainstem lysates. DOR, however, decreased in expression in brainstem lysates with increased developmental age, even though there was no difference in DOR expression in cultured cells. MOR and DOR were colocalised in specific brainstem regions and in the cerebellum of foetal and postnatal animals, although the distribution of both opioid receptors in the foetal brain was more diffuse than in the older animals. The intracellular distributions of MOR and DOR were investigated by confocal microscopy. In addition to plasma membrane staining, a population of internalised cytoplasmic receptors was present in neurons. MOR was down-regulated after exposure of either cultured brainstem cells or transfected or non-transfected SH-SY5Y neuroblastoma cells to the MOR agonist DAMGO. From the above investigation, it was concluded that opioid receptors are developmentally regulated during maturation of the brainstem of the rat, and that primary cell culture, immunocytochemistry, and immunoblotting of cell lysates are suitable techniques for investigating opioid systems in the foetal, postnatal, and adult rat.
