Development of phenylethanolamine N-methyltransferase (PNMT) in cultures of dissociated embryonic rat medulla oblongata

Abstract

The adrenergic phenotypic marker, phenylethanolamine N-methyltransferase (PNMT) is expressed in a subgroup of catecholaminergic neurons in the brain, as well as in the chromaffin cells of the adrenal medulla. Although PNMT in the rat adrenal is regulated by glucocorticoids, PNMT in the rat brainstem appears not to be regulated by glucocorticoids. Furthermore, little is known about factors required for the differentiation of this specific class of central neuron. The identification of such factors has been hampered not only by the heterogeneity of cell types in the brainstem, of which only a smaller number express PNMT, but also by the lack of a well characterized in vitro system in which the development of these neurons can be studied under defined conditions. The present study addresses this issue by establishing and characterizing a culture system for the study of adrenergic neurons.Dissociated cultures were prepared from embryonic rat medulla oblongata and the expression and development of PNMT was studied using immunocytochemistry and radioisotopic assay of PNMT activity. The survival of PNMT-immunoreactive (IR) neurons in vitro was found to be critically dependent on embryonic age. Numerous PNMT-IR neurons were observed in cultures prepared only from embryos of 46–51 somites (embryonic day E13–13.5). In contrast, cultures containing numerous neurons immunoreactive for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, could be successfully established from medulla oblongata of any age between E13 and E16. In cultures of the E13 rat, PNMT was found to be catalytically active at 4 days in vitro and the levels of PNMT activity per neuron, as estimated from the number of PNMT-IR neurons in sister cultures, increased 2-fold in cultures grown for 8 days in the presence of 10% fetal bovine serum and 1.5-fold in defined, serum-free conditions. Treatment of cultures with either corticosterone or dexamethasone had no effect on PNMT activity, supporting previous studies suggesting that PNMT in central neurons is not regulated by glucocorticoids.These studies provide a convenient and defined in vitro system for studying factors that influence the development of central adrenergic neurons. The differential development of PNMT-IR and TH-IR neurons in these cultures suggests that the factors involved in adrenergic neuron survival and/or differentiation are not common to catecholaminergic neurons in general.