Ciliary neurotrophic factor supports p75NGFR-immunoreactive non-cholinergic, but not cholinergic, developing septal neurons in vitro.

Abstract

Ciliary neurotrophic factor is known to exert both survival and differentiative actions on a number of neuronal populations of the peripheral and central nervous systems. In this study we have compared the trophic effects of ciliary neurotrophic factor and nerve growth factor on developing septal neurons of the rat in vitro. Fetal septal neurons were grown in vitro under glass coverslips in sandwich culture. Septal cultures grown for 14 days in the continual presence of nerve growth factor contain a population of cholinergic neurons that stain intensely for the low-affinity nerve growth factor receptor (p75NGFR), choline acetyltransferase and acetylcholinesterase. Without added nerve growth factor, few neurons stain for these markers. Ciliary neurotrophic factor addition for 14 days from plating in the absence of exogenous nerve growth factor results in the appearance of a population of neurons that stains for p75NGFR. This population is similar in number to that seen in nerve growth factor-treated cultures but is not immunoreactive for choline acetyltransferase and is significantly smaller in mean cross-sectional area. Delayed addition of nerve growth factor to ciliary neurotrophic factor-supported cultures at 14 days for a further seven days fails to induce choline acetyltransferase immunoreactivity in these p75NGFR-positive septal neurons. In cultures grown in the continual presence of nerve growth factor from plating, removal of nerve growth factor and addition of nerve growth factor antibodies at 14 days results in the death of over 80% of the cholinergic neurons after a further four days. Addition of ciliary neurotrophic factor during the period of nerve growth factor withdrawal appears to preserve a p75NGFR-positive, choline acetyltransferase-negative neuronal population. However, seven day re-addition of nerve growth factor to ciliary neurotrophic factor-treated, nerve growth factor-withdrawn cultures fails to induce choline acetyltransferase immunoreactivity in the ciliary neurotrophic factor-supported p75NGFR-positive septal neurons. Simultaneous treatment of cultures with both ciliary neurotrophic factor and nerve growth factor for 14 days from plating approximately doubles the number of p75NGFR-positive neurons relative to cultures treated with either ciliary neurotrophic factor or nerve growth factor alone, but the number of choline acetyltransferase-positive neurons in these cultures is not significantly greater than that found in cultures treated solely with nerve growth factor. These results suggest that ciliary neurotrophic factor does not support the survival and differentiation of developing septal cholinergic neurons in vitro, but can support the development of a p75NGFR-immunoreactive population of non-cholinergic septal neurons.