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Cultured cells from the rat brainstem were used to study opioid receptor (OpR) expression during late fetal and early postnatal development. Mu and delta opioid receptor (MOR and DOR) expression was investigated from embryonic day 16 (E16) to 6 days postnatal (P6). Postnatal neurons showed more intense MOR immunoreactivity (IR) than neurons cultured from fetal brainstem (P < 0.006). DOR IR showed a similar pattern, but the differences between fetal and neonatal animals were not statistically significant. Using confocal microscopy, MOR and DOR IR were shown to be present on both the cell membrane and within the cytoplasm, in a similar pattern to the IR seen in SH-SY5Y neuroblastoma cells that endogenously express both MOR and DOR. Double-labeling experiments demonstrated colocalization of MOR and DOR in the same brainstem neurons; however, not all MOR IR regions of a single neuron were also positively stained for DOR, and not all DOR IR regions were also positive for MOR. MOR was down-regulated after a 1- or 2-h treatment with 1 microM DAMGO, a potent mu opioid agonist, in both non-transfected and MOR-transfected SH-SY5Y cells and in primary cell cultures. It was concluded that many brainstem neurons express functional MOR or DOR or coexpress both receptors, although intracellular distributions of the receptors are unique for each receptor type.

Opioid receptors are expressed in the brain during fetal and postnatal development, and the expression patterns vary with developmental age. To investigate the role of opioids in brain development, immunoblotting and immunohistochemical techniques were used to determine mu (MOR) and delta (DOR) opioid receptor expression levels and regional distributions in fetal, early postnatal and adult rat brainstem. Two immunoreactive bands were seen on Western blots of brainstem lysates for both MOR (50 and 70 kDa) and DOR (30 and 60 kDa). The expression levels of the isoforms changed dramatically between 6 and 15 days after birth. Total MOR protein was expressed at low levels in fetal and early postnatal animals with the 50-kDa band predominating. MOR expression then increased in the older animals and the 70-kDa isoform became dominant. Total DOR protein showed the opposite pattern, being high in the fetal and neonatal brainstem and low in the juvenile and adult. A postnatal switch in isoform expression for DOR was not evident in our study. In general, regional brainstem distributions in developing and adult animals were comparable to those reported in the literature, and both receptors were localized in the same areas where opioid receptor expression was high. It was concluded that MOR and DOR are developmentally regulated in the brainstem of the rat, that the isoform ratio switches postnatally from a fetal-neonatal pattern to a juvenile-adult pattern and that both receptors are generally expressed in the same brainstem regions from E16 to adult.

Primary culture of postnatal brainstem neurons in defined medium has not been described in the literature. Successful primary culture of brainstem neurons is typically restricted to embryonic ages E14-E18. This study describes a method for culture of late fetal and early postnatal brainstem neurons using a serum-free culture medium. The culture system is based on Neurobasal medium supplemented with antioxidant-rich B27 (Life Technologies). Neuron survival was optimized by replacing glutamine with GlutaMaxI, by matching osmolality with neuronal age, and by using Hibernate medium to increase neuron survival during tissue dissociation. This paper describes the first reliable method for culturing brainstem neurons from late fetal and early postnatal stages of the rat for up to 6 days postpartum.

Serum-free medium is essential for cell culture studies in which complete control of the environment is required. Primary culture of post-natal brainstem neurons in defined medium has not been described in the literature, and successful culture of primary brainstem neurons is typically restricted to embryonic ages E14-E18. This study describes a method for culture of fetal and post-natal brainstem neurons using a serum-free culture medium. The culture system is based on Neurobasal medium supplemented with antioxidant-rich B27. Media and supplements are commercially available products from Life Technologies. Neuron survival was optimized by replacing glutamine with GlutaMaxI, by matching osmolality with neuronal age, and by using Hibernate medium to increase neuron survival during tissue dissociation. Fetal E14, E16, E20, and post-natal P3 and P6 cultures were examined after 4, 7, and 9 days in culture. Neuron and glial cells present in the cultures were identified using immunocytochemistry with antibodies raised against microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP), respectively. Fetal E14 cultures had more bipolar neurons than multipolar neurons compared with developmentally older P6 cultures. Early fetal cultures had a higher percentage of neurons than late fetal and early post-natal cultures. Neuron survival was similar between 4 and 9 days in culture for all age groups tested. This is the first reliable, defined culture medium that supports brainstem neurons from late fetal and early post-natal stages of the rat for up to 6 days post-partum.