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Radiation may affect essential functions and behaviors such as locomotion, feeding, learning and memory. Although whole-body irradiation has been shown to reduce motility in the nematode Caenorhabditis elegans, the detailed mechanism responsible for this effect remains unknown. Targeted irradiation of the nerve ring responsible for sensory integration and information processing would allow us to determine whether the reduction of motility following whole-body irradiation reflects effects on the central nervous system or on the muscle cells themselves. We therefore addressed this issue using a collimating microbeam system. However, radiation targeting requires the animal to be immobilized, and previous studies have anesthetized animals to prevent their movement, thus making it impossible to assess their locomotion immediately after irradiation. We developed a method in which the animal was enclosed in a straight, microfluidic channel in a polydimethylsiloxane chip to inhibit free motion during irradiation, thus allowing locomotion to be observed immediately after irradiation. The head region (including the central nervous system), mid region around the intestine and uterus, and tail region were targeted independently. Each region was irradiated with 12 000 carbon ions (12C; 18.3 MeV/u; linear energy transfer = 106.4 keV/μm), corresponding to 500 Gy at a φ20 μm region. Motility was significantly decreased by whole-body irradiation, but not by irradiation of any of the individual regions, including the central nervous system. This suggests that radiation inhibits locomotion by a whole-body mechanism, potentially involving motoneurons and/or body-wall muscle cells, rather than affecting motor control via the central nervous system and the stimulation response., (© The Author 2017. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)

To clarify the relative biological effectiveness (RBE) values of carbon ion (C) beams in normal brain tissues, a rat organotypic slice culture system was used. The cerebellum was dissected from 10-day-old Wistar rats, cut parasagittally into approximately 600-µm-thick slices and cultivated using a membrane-based culture system with a liquid-air interface. Slices were irradiated with 140 kV X-rays and 18.3 MeV/amu C-beams (linear energy transfer = 108 keV/µm). After irradiation, the slices were evaluated histopathologically using hematoxylin and eosin staining, and apoptosis was quantified using the TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay. Disorganization of the external granule cell layer (EGL) and apoptosis of the external granule cells (EGCs) were induced within 24 h after exposure to doses of more than 5 Gy from C-beams and X-rays. In the early postnatal cerebellum, morphological changes following exposure to C-beams were similar to those following exposure to X-rays. The RBEs values of C-beams using the EGL disorganization and the EGC TUNEL index endpoints ranged from 1.4 to 1.5. This system represents a useful model for assaying the biological effects of radiation on the brain, especially physiological and time-dependent phenomena.

The study of radiation effect in Caenorhabditis (C.) elegans has been carried out over three decades and now allow for understanding at the molecular, cellular and individual levels. This review describes the current knowledge of the biological effects of ionizing irradiation with a scope of the germ line, aging and behavior. In germ cells, ionizing radiation induces apoptosis, cell cycle arrest and DNA repair. Lots of molecules involved in these responses and functions have been identified in C. elegans, which are highly conserved throughout eukaryotes. Radiosensitivity and the effect of heavy-ion microbeam irradiation on germ cells with relationship between initiation of meiotic recombination and DNA lesions are discussed. In addition to DNA damage, ionizing radiation produces free radicals, and the free radical theory is the most popular aging theory. A first signal transduction pathway of aging has been discovered in C. elegans, and radiation-induced metabolic oxidative stress is recently noted for an inducible factor of hormetic response and genetic instability. The hormetic response in C. elegans exposed to oxidative stress is discussed with genetic pathways of aging. Moreover, C. elegans is well known as a model organism for behavior. The recent work reported the radiation effects via specific neurons on learning behavior, and radiation and hydrogen peroxide affect the locomotory rate similarly. These findings are discussed in relation to the evidence obtained with other organisms. Altogether, C. elegans may be a good "in vivo" model system in the field of radiation biology.

Using heavy-ion microbeam, we report target irradiation of selected compartments within the diapause-terminated egg and its mutational consequences in the silkworm, Bombyx mori. On one hand, carbon-ion exposure of embryo to 0.5-6 Gy increased the somatic mutation frequency, suggesting targeted radiation effects. On the other, such increases were not observed when yolk was targeted, suggesting a lack of nontargeted bystander effect.

In order to study the radiobiological effects of low dose radiation, microbeam irradiation facilities have been developed in the world. This type of facilities now becomes an essential tool for studying bystander effects and relating signaling phenomena in cells or tissues. This review introduces you available microbeam facilities in Japan and in China, to promote radiobiology using microbeam probe and to encourage collaborative research between radiobiologists interested in using microbeam in Japan and in China.

Cells exposed to genotoxic stress, such as ionizing radiation and DNA damaging reagents, either arrest the cell cycle to repair the genome, or undergo apoptosis, depending on the extent of the DNA damage. DNA damage also has been implicated in various differentiation processes. It has been reported that gamma-ray exposure or treatment with DNA-damaging agents could induce myogenic differentiation in Drosophila Schneider cells. However, the mechanism underlying this process has been poorly understood. In this study, exposure of Schneider cells to X-rays or energetic carbon ion beams caused increase of TUNEL-positive cells and conversion of round-shaped cells to elongated cells. Both upregulation of genes related to myogenesis and increase of myosin indicate that the radiation-induced morphological changes of Schneider cells were accompanied with myogenic differentiation. Because the intracellular ceramide was increased in Schneider cells after exposure to X-ray, we examined whether exogenous ceramide could mimic radiation-induced myogenic differentiation. Addition of membrane-permeable C(2)-ceramide to Schneider cells increased apoptosis and expression of myogenic genes. These results suggest that ceramide plays important roles in both apoptosis and the radiation-induced myogenic differentiation process.

Locomotory behavior (motility) and mechanosensation are of vital importance in animals. We examined the effects of ionizing radiation (IR) on locomotory behavior and mechanosensation using a model organism, the nematode Caenorhabditis elegans. Bacterial mechanosensation in C. elegans induces the dopamine-mediated slowing of locomotion in the presence of bacteria (food), known as the basal slowing response. We previously reported an IR-induced reduction of locomotory rate in the absence of food. In the present study, we observed a similar IR-induced reduction of locomotory rate in the cat-2 mutant, which is defective in bacterial mechanosensation. The dose response pattern of the locomotory rate in the presence of food was relatively flat in wild-type animals, but not in cat-2 mutants. This suggests that the dopamine system, which is related to bacterial mechanosensation in C. elegans, might have a dominant effect on locomotory rate in the presence of food, which masks the effects of other stimuli. Moreover, we found that the behavioral responses of hydrogen peroxide-exposed wild-type animals are similar to those of IR-exposed animals. Our findings suggest that the IR-induced reduction of locomotory rate in the absence of food is mediated by a different pathway from that for bacterial mechanosensation, at least partially through IR-produced hydrogen peroxide.

Classical studies on root hydrotropism have hypothesized the importance of columella cells as well as the de novo gene expression, such as auxin-inducible gene, at the elongation zone in hydrotropism; however, there has been no confirmation that columella cells or auxin-mediated signaling in the elongation zone are necessary for hydrotropism. We examined the role of root cap and elongation zone cells in root hydrotropism using heavy-ion and laser microbeam. Heavy-ion microbeam irradiation of the elongation zone, but not that of the columella cells, significantly and temporarily suppressed the development of hydrotropic curvature. However, laser ablation confirmed that columella cells are indispensable for hydrotropism. Systemic heavy-ion broad-beam irradiation suppressed de novo expression of INDOLE ACETIC ACID 5 gene, but not MIZU-KUSSEI1 gene. Our results indicate that both the root cap and elongation zone have indispensable and functionally distinct roles in root hydrotropism, and that de novo gene expression might be required for hydrotropism in the elongation zone, but not in columella cells.

Learning impairment following ionizing radiation (IR) exposure is an important potential risk in manned space missions. We previously reported the modulatory effects of IR on salt chemotaxis learning in Caenorhabditis elegans. However, little is known about the effects of IR on the functional relationship in the nervous system. In the present study, we investigated the effects of gamma-ray exposure on the relationship between locomotion and salt chemotaxis learning behavior. We found that effects of pre-learning irradiation on locomotion were significantly correlated with the salt chemotaxis learning performance, whereas locomotion was not directly related to chemotaxis to NaCl. On the other hand, locomotion was positively correlated with salt chemotaxis of animals which were irradiated during learning, and the correlation disappeared with increasing doses. These results suggest an indirect relationship between locomotion and salt chemotaxis learning in C. elegans, and that IR inhibits the innate relationship between locomotion and chemotaxis, which is related to salt chemotaxis learning conditioning of C. elegans.

Research concerning cellular responses to low dose irradiation, radiation-induced bystander effects, and the biological track structure of charged particles has recently received particular attention in the field of radiation biology. Target irradiation employing a microbeam represents a useful means of advancing this research by obviating some of the disadvantages associated with the conventional irradiation strategies. The heavy-ion microbeam system at JAEA-Takasaki, which was planned in 1987 and started in the early 1990's, can provide target irradiation of heavy charged particles to biological material at atmospheric pressure using a minimum beam size 5 mum in diameter. A variety of biological material has been irradiated using this microbeam system including cultured mammalian and higher plant cells, isolated fibers of mouse skeletal muscle, silkworm (Bombyx mori) embryos and larvae, Arabidopsis thaliana roots, and the nematode Caenorhabditis elegans. The system can be applied to the investigation of mechanisms within biological organisms not only in the context of radiation biology, but also in the fields of general biology such as physiology, developmental biology and neurobiology, and should help to establish and contribute to the field of "microbeam biology".

To carry out the radio-microsurgery study using silkworm, Bombyx mori, we have already developed the specific irradiation systems for eggs and third to fifth instar larvae. In this study, a modified application consisting of the first instar silkworm larvae was further developed using heavy-ion microbeams. This system includes aluminum plates with holes specially designed to fix the first instar silkworm larvae during irradiation, and Mylar films were used to adjust energy deposited for planning radiation doses at certain depth. Using this system, the suppression of abnormal proliferation of epidermal cells in the knob mutant was examined. Following target irradiation of the knob-forming region at the first instar stage with 180-mum-diameter microbeam of 220 MeV carbon (12C) ions, larvae were reared to evaluate the effects of irradiation. The results indicated that the knob formation at the irradiated segment was specially suppressed in 5.9, 56.4, 66.7 and 73.6% of larvae irradiated with 120, 250, 400 and 600 Gy, respectively, but the other knob formations at the non-irradiated segments were not suppressed in either irradiation. Although some larva did not survive undesired non-targeted exposure, our present results indicate that this method would be useful to investigate the irradiation effect on a long developmental period of time. Moreover, our system could also be applied to other species by targeting tissues, or organs during development and metamorphosis in insect and animals.

We have examined in vitro radiosensitivities and radioresponses to (60)Co gamma-rays irradiation in feline T-lymphocyte cell lines, FeT-J and FL-4. There seemed to be no significant difference in clonogenic survival between the two lines. The mean lethal dose for both was both 1.9 Gy, and surviving fraction at 2 Gy was 0.30 and 0.48 for FeT-J and FL-4 cells, respectively. However, TUNEL assay indicated much higher degrees of apoptosis induction in FeT-J cells (>40%) than in FL-4 cells (<10%) at 4 days after 15 Gy irradiation. Microscopic examination revealed a larger population of multi-nucleate cells in FL-4 cells (60.3%) than in FeT-J cells (16.0%) at 4 days after 15 Gy irradiation, suggesting that a larger ratio of mitotic catastrophe occurred in FL-4 cells. These results suggest that FeT-J is more likely to be induced into apoptosis and FL-4 is more likely to fall into mitotic catastrophe, and eventually necrosis; both of them showed a similar surviving fraction against gamma-rays. The results also indicate that FL-4 cells follow a process other than apoptosis to cell death, suggesting the presence of a regulatory mechanism that may control the relationship between mitotic catastrophe and apoptosis in feline T-lymphocytes.

The protective effects of Trolox on the inhibition of negative gravitaxis in Euglena gracilis exposed to 200 Gy 60Co gamma-rays were examined using different concentrations (1, 10 and 100 microM). The orientation precision of the negative gravitaxis was quantified using the r-value. A significant decrease in the r-value was observed in gamma-irradiated samples (0.18+/-0.03) compared to those of non-irradiated samples (0.47+/-0.03). There were no significant changes in the r-value of cells exposed to 200 Gy gamma-rays by the addition of 1 or 10 microM of Trolox. A significant increase (0.19) in the r-value of cells exposed to 200 Gy with 100 microM Trolox was observed. The results indicates that Trolox at a concentration of 100 microM protects negative gravitaxis against 60Co gamma-ray irradiation at a dose of 200 Gy. It also suggests that the negative gravitaxis of Euglena gracilis is affected by free radicals.