Effects of irradiation on cholinergic neurons and nerve growth factor mRNA in mouse foetal brain aggregation cultures

Neurochemical and morphological markers were used to study the dose-response relationship and effects of different doses (0.5, 1.0 and 2.0 Gy) of X-irradiation on foetal brain cells in reaggregating cell cultures. Cells, prepared from mouse forebrain were irradiated on culture day 2, corresponding to embryonic day 16 in vivo. The cell reaggregates were monitored continually up to day 40 in vitro. The level of a neuronal marker for cholinergic neurons, namely acetylcholine esterase (AChE), increased linearly with the dose of irradiation after 10 days in vitro. Furthermore, by day 30 the activity of AChE had decreased to a level below that found in the control aggregates. In the 2-Gy treatment the level of choline acetyltransferase (ChAT), another cholinergic-neuron marker, increased during the first 20-30 days in culture but had declined to control levels by day 40. Using Northern blot analysis of total RNA prepared from these cell reaggregates, we determined relative changes in the level of expression of mouse nerve growth factor (NGF) mRNA subsequent to irradiation after 10 and 30 days in culture. The ratio between the level of expression of NGF and that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was calculated and it was found that by day 10 in vitro the level of NGF had increased compared with control levels, in a dose-related manner. After 30 days in culture, the level of NGF mRNA was still high after the low-dose (0.5 Gy) treatment, whereas it had decreased below control levels after the higher-dose (1.0 and 2.0 Gy) treatment. The aggregates showed a tendency to deform and fuse together after irradiation: furthermore, the number of aggregates occurring as two or more grown together, increased with the X-ray dose. Aggregate size decreased with time in vitro and with irradiation dose. Thus, we showed that cholinergic and morphological markers are affected by irradiation and the alteration in AChE activity induced by irradiation correlated well with the changes in NGF mRNA.