Differential effects of x-irradiation on immature and mature hippocampal neurons in vitro.

X-irradiation to neuronal progenitor cells causes brain dysfunctions, such as a mental retardation, in adulthood. However, little has been known about the degree of radiosensitivity of neurons in the developmental stages at which they are most vulnerable. In this study we compared the effect of irradiation on mature neurons with that on immature neurons. Primary dissociated neuronal cultures were prepared from fetal rat hippocampi of embryonic day 18. X-irradiations were performed on the cultured cells at 7 or 21 days in vitro (DIV), and the cells were fixed at 12 or 24 h after irradiation. Then the cells were stained with 4',6-diamidino-2-phenylindole (DAPI) or terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The apoptotic changes were measured quantitatively by nuclear pyknosis and DNA fragmentation-both characteristic morphological changes of apoptosis. Light microscopy with differential interference contrast showed that 30 Gy of irradiation increased cellular shrinkage in 7-DIV neurons but not in 21-DIV neurons. Quantitative analysis using DAPI imaging showed that 30 Gy of irradiation significantly enhanced pyknotic changes in 7-DIV neurons after 24 h. In contrast, this irradiation did not enhance any pyknotic changes in 21-DIV neurons after 24 h. Further TUNEL staining also showed that the irradiation did not enhance any DNA fragmentation in nuclei of 21-DIV neurons after 24h. Hence, we showed that the radiosensitivity of 21-DIV postmitotic neurons was significantly lower than that of 7-DIV neurons, indicating that the susceptibility of such neurons depends on their developmental stage.