The noradrenergic system in cultured aggregates of fetal rat brain cells: Morphology of the aggregates and pharmacological indices of noradrenergic neurons

Abstract

Spherical aggregates formed rapidly in culture by re-aggregation of trypsin-dissociated brain cells from the 17-day-old fetal rat. Over about 10 days in initially random distribution of cells evolved into a 3-layered arrangement; cells with characteristics of neurons were found largely in the intermediate layer. The survival of neuronal and glial cell types was evaluated histologically and verified by electron microscopy, which revealed synaptic and myelin structures that rapidly increased in number after 18 days in culture. Levels of norepinephrine (NE) and dopamine (DA) reached peaks of 9.5 and 4.4 ng/mg protein, respectively, at culture day 21. Uptake of [3H]NE paralleled these amine levels and was blocked by desipramine or pretreatment with either reserpine or 6-OH-DA. Autoradiographs of aggregates labeled with [3H]NE showed a high density of silver grains over cells, apparently neurons, with branching processes traced for 120 micrometer. Previously accumulated [3H]NE was released under depolarizing conditions (high [K+] or vertridine) only in the presence of Ca2+. Release was induced to a lesser extent by kainic greater than glutamic acid. Thus, such aggregates appear to contain catecholaminergic neurons capable of synthesis, uptake and release of NE. The time course of development of these functions supports suggestions that aggregate preparations might be useful in studying neurochemical or morphological aspects of brain development and function in vitro.