Zebrafish possess two isoforms of vertebrate ancient long (VAL)-opsin, val-opsinA (valopa) and val-opsinB (valopb), which probably mediate non-visual responses to light. To understand the diurnal and light-sensitive regulation of the valop genes in different cell groups, the current study used real-time quantitative PCR to examine the diurnal changes of valopa and b mRNA levels in different brain areas of adult male zebrafish. Furthermore, effects of the extended exposure to light or dark condition, luminous levels and the treatment with a melatonin receptor agonist or antagonist on valop transcription were examined. In the thalamus, valop mRNA levels showed significant diurnal changes; valopa peaked in the evening, while valopb peaked in the morning. The diurnal change of valopa mRNA levels occurred independent of light conditions, whereas that of valopb mRNA levels were regulated by light. A melatonin receptor agonist or antagonist did not affect the changes of valop mRNA levels. In contrast, the midbrain and hindbrain showed arrhythmic valop mRNA levels under light and dark cycles. The differential diurnal regulation of the valopa and b genes in the thalamus and the arrhythmic expression in the midbrain and hindbrain suggest involvement of deep brain VAL-opsin in time- and light-dependent physiology. We show diurnal expression changes of vertebrate ancient long (VAL) opsin genes (valopa and valopb), depending on brain area, time of day and light condition, in the adult male zebrafish. Differential regulation of the valop genes in the thalamus and arrhythmic expression in the midbrain and hindbrain suggest their involvement in time- and light-dependent physiology to adjust to environmental changes.
Synchrotron microbeam radiation treatment (MRT) is a preclinical radiotherapy technique with considerable clinical promise, although some of the underlying radiobiology of MRT is still not well understood. In recently reported studies, it has been suggested that MRT elicits a different tumor immune profile compared to broad-beam treatment (BB). The aim of this study was to investigate the effects of synchrotron MRT and BB on eosinophil-associated gene pathways and eosinophil numbers within and around the tumor in the acute stage, 48 h postirradiation. Balb/C mice were inoculated with EMT6.5 mouse mammary tumors and irradiated with microbeam radiation (112 and 560 Gy) and broad-beam radiation (5 and 9 Gy) at equivalent doses determined from a previous in vitro study. After tumors were collected 24 and 48 h postirradiation, RNA was extracted and quantitative PCR performed to assess eosinophil-associated gene expression. Immunohistochemistry was performed to detect two known markers of eosinophils: eosinophil-associated ribonucleases (EARs) and eosinophil major basic protein (MBP). We identified five genes associated with eosinophil function and recruitment (Ear11, Ccl24, Ccl6, Ccl9 and Ccl11) and all of them, except Ccl11, were differentially regulated in synchrotron microbeam-irradiated tumors compared to broad-beam-irradiated tumors. However, immunohistochemical localization demonstrated no significant differences in the number of EAR- and MBP-positive eosinophils infiltrating the primary tumor after MRT compared to BB. In conclusion, our work demonstrates that the effects of MRT on eosinophil-related gene pathways are different from broad-beam radiation treatment at doses previously demonstrated to be equivalent in an in vitro study. However, a comparison of the microenvironments of tumors, which received MRT and BB, 48 h after exposure showed no difference between them with respect to eosinophil accumulation. These findings contribute to our understanding of the role of differential effects of MRT on the tumor immune response.