Rat embryonic septal neurons survive and express cholinergic properties in isolation and without nerve growth factor

We studied survival and expression of cholinergic properties in embryonic septal neurons grown in very low density microcultures (1–7 cells per Terasaki well). Even in cultures containing only a single neuron, at least 10% of plated neurons survived for 2 weeks or more in medium containing fetal calf serum or an acid-stable fraction (55,000 Da) of horse serum. Of these surviving neurons, 30–40% stained positively for acetylcholinesterase (AChE) or nerve growth factor (NGF) receptor, even though the culture medium lacked detectable levels of NGF, brain-derived neurotrophic factor, and fibroblast growth factor. Addition of NGF or an antibody against NGF had no effect on either neuronal survival or the percentage of neurons staining positively for AChE or NGF receptor after 18–20 days in vitro. There was no cell division in medium containing the serum fraction, but when 10% fetal calf serum was present cell division occured in some of the cultures, and in half of these cases at least one of the clonal progeny became AChE-positive. These results demonstrated that some embryonic septal cells can survive at least 2 weeks and develop cholinergic neuronal properties in the absence of other cells or NGF.