Comparison of the radiosensitivities of neurons and glial cells derived from the same rat brain.

Non-proliferating cells, such as mature neurons, are generally believed to be more resistant to X-rays than proliferating cells, such as glial and vascular endothelial cells. Therefore, the late adverse effects of radiotherapy on the brain have been attributed to the radiation-induced damage of glial and vascular endothelial cells. However, little is known about the radiosensitivities of neurons and glial cells due to difficulties in culturing these cells, particularly neurons, independently. In the present study, primary dissociated neurons and glial cultures were prepared separately from the hippocampi and cerebrum, respectively, which had been obtained from the same fetal rat on embryonic day 18. X-irradiations of 50 Gy were performed on the cultured neurons and glial cells at 7 and 21 days in vitro (DIV). The cells were fixed at 24 h after irradiation. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was then performed to measure the apoptotic indices (AIs). The AIs of non-irradiated and irradiated neurons at 7 DIV were 23.7±6.7 and 64.9±4.8%, and those at 21 DIV were 52.1±17.4 and 44.6±12.5%, respectively. The AIs of non-irradiated and irradiated glial cells at 7 DIV were 5.8±1.5 and 78.4±3.3% and those at 21 DIV were 9.6±2.6 and 86.3±4.9%, respectively. Glial cells and neurons were radiosensitive at 7 DIV. However, while glial cells were radiosensitive at 21 DIV, neurons were not.

Myosin II ATPase activity mediates the long-term potentiation-induced exodus of stable F-actin bound by drebrin A from dendritic spines.

The neuronal actin-binding protein drebrin A forms a stable structure with F-actin in dendritic spines. NMDA receptor activation causes an exodus of F-actin bound by drebrin A (DA-actin) from dendritic spines, suggesting a pivotal role for DA-actin exodus in synaptic plasticity. We quantitatively assessed the extent of DA-actin localization to spines using the spine-dendrite ratio of drebrin A in cultured hippocampal neurons, and found that (1) chemical long-term potentiation (LTP) stimulation induces rapid DA-actin exodus and subsequent DA-actin re-entry in dendritic spines, (2) Ca(2+) influx through NMDA receptors regulates the exodus and the basal accumulation of DA-actin, and (3) the DA-actin exodus is blocked by myosin II ATPase inhibitor, but is not blocked by myosin light chain kinase (MLCK) or Rho-associated kinase (ROCK) inhibitors. These results indicate that myosin II mediates the interaction between NMDA receptor activation and DA-actin exodus in LTP induction. Furthermore, myosin II seems to be activated by a rapid actin-linked mechanism rather than slow MLC phosphorylation. Thus the myosin-II mediated DA-actin exodus might be an initial event in LTP induction, triggering actin polymerization and spine enlargement.

Effectiveness of carbon-ion beams for apoptosis induction in rat primary immature hippocampal neurons.

The direct biological effects of radiation, particularly accelerated heavy particle ions, on neurons are not fully known. Hence, the direct effect of carbon-ion beams on immature neurons was investigated by comparing to the effect of X-rays in vitro using primary hippocampal neurons. Primary neurons were prepared from hippocampi of fetal rats at embryonic day 18 from timed pregnant Wistar rats and cultured with Banker's methods. At 7 Days In Vitro (DIV), the cells were irradiated with 140 kV X-ray and 18.3 MeV/amu carbon-ion beams (LET = 108 keV/µm). The cells were fixed with 4% paraformaldehyde at 12 hours after irradiation. Then, the cells were treated with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and DAPI staining for measuring the percentage of apoptosis (apoptotic index: AI). AI in sham-irradiated hippocampal neurons was 18%. The value of AI (AIs) of the cells irradiated with X-rays at 10 or 30 Gy were 15% or 23%, respectively. AI in cells irradiated with carbon-ion beams at 1 Gy, 3 Gy, 5 Gy and 10 Gy were 22%, 23%, 24% and 33%, respectively. AI was significantly increased by carbon-ion beams at 10 Gy (p < 0.001). The apoptosis of hippocampal neurons increased in a dose-dependent manner following both X-ray and carbon-ion beams irradiation. Carbon-ion beams were about 10-fold more effective than X-rays for apoptosis induction in immature hippocampal neurons.

Effect of radiation on the development of immature hippocampal neurons in vitro.

Little is known about the direct biological effects of radiation on immature neurons, despite its relevance to the mental retardation caused by irradiation of the brains of fetuses and children. In this study, we investigated the effects of radiation using primary cultured hippocampal neuronal cells with exclusion of glial cells, focusing on cell survival and structural development. Primary neurons were prepared from the hippocampi of fetal rats at embryonic day 18 and cultured according to Banker's methods. After incubation for 7 days, cells were irradiated with X rays and incubated continuously for 7 or 14 days. The number of neurons, their rate of apoptosis, and the patterns of expression of synaptic proteins on the neural dendrites were investigated by immunohistochemical methods. The total numbers of neurons were the same regardless of whether they were irradiated. The number of TUNEL-positive neurons, which can be considered as undergoing apoptosis, increased significantly in a dose-dependent fashion at both 7 and 14 days after irradiation. The mean numbers of clusters of synaptic proteins on neural dendrites, which are considered to represent their developmental level, decreased dose-dependently at both 7 and 14 days after irradiation. These results suggest that radiation not only induces apoptosis but also produces structural defects in the surviving neurons that may directly suppress neural development.

Drebrin E is involved in the regulation of axonal growth through actin-myosin interactions.

Drebrin is a well-known side-binding protein of F-actin in the brain. Immunohistochemical data suggest that the peripheral parts of growing axons are enriched in the drebrin E isoform and mature axons are not. It has also been observed that drebrin E is concentrated in the growth cones of PC12 cells. These data strongly suggest that drebrin E plays a role in axonal growth during development. In this study, we used primary hippocampal neuronal cultures to analyze the role of drebrin E. Immunocytochemistry showed that within axonal growth cones drebrin E specifically localized to the transitional zone, an area in which dense networks of F-actins and microtubules overlapped. Over-expression of drebrin E caused drebrin E and F-actin to accumulate throughout the growth cone and facilitated axonal growth. In contrast, knockdown of drebrin E reduced drebrin E and F-actin in the growth cone and prevented axonal growth. Furthermore, inhibition of myosin II ATPase masked the promoting effects of drebrin E over-expression on axonal growth. These results suggest that drebrin E plays a role in axonal growth through actin-myosin interactions in the transitional zone of axonal growth cones.