Nerve growth factor and phorbol esters increase the number of choline acetyltransferase-positive cells in two morphologically distinct classes of basal forebrain neurons in primary cultures

Abstract

Nerve growth factor (NGF) has been shown to be active in the CNS as a neurotrophic agent. Cholinergic neurons of the basal forebrain are one cell type in the CNS which have been identified as a target for NGF. When dissociated cell cultures from the basal forebrain were treated for 7 days with NGF (20 ng/100 μl), the number of choline acetyltransferase (ChAT) -immunopositive cells was increased from 30 ± 6 to 58 ± 3. Cholinergic cells taken from the basal forebrain exhibit 3 different morphologies: stellate, pyramidal, and bipolar. The NGF treatment was found to increase the number of stellate cells from 7 ± 2 to 23 ± 2 and the number of pyramidal cells from 14 ± 2 to 26 ± 2, but had no effect on the number of bipolar cells. The activation of protein kinase C by phorbol 12-myristate, 13-acetate (TPA) also increased the number of ChAT-positive cells in a dose-dependent manner. A maximal increase was observed with 10 ng/ml of TPA which increased the number of positive cells from a basal level of 21 ± 4 to 42 ± 4. As was the case with NGF, only the stellate and pyramidal cells were affected by the phorbol ester treatment. In co-addition experiments, the cultures were treated with 10 ng/100 μl of NGF and 10 ng/ml of TPA, with the result that there was no further increase in the number of immunopositive cells over the NGF controls. These results suggest that the mechanisms by which NGF and TPA increase the number of ChAT-positive cells are interactive at some point. The effect of TPA at the higher doses of NGF was distinctly different. When cells were treated with 20 ng/100 μl of NGF and 0.05–50 ng/ml of TPA, the NGF response was down-regulated to the level of the vehicle-treated controls.