Yoshio Takashima

Diversity in the neural circuitry of cold sensing revealed by genetic axonal labeling of transient receptor potential melastatin 8 neurons.

Sensory nerves detect an extensive array of somatosensory stimuli, including environmental temperatures. Despite activating only a small cohort of sensory neurons, cold temperatures generate a variety of distinct sensations that range from pleasantly cool to painfully aching, prickling, and burning. Psychophysical and functional data show that cold responses are mediated by both C- and Aδ-fibers with separate peripheral receptive zones, each of which likely provides one or more of these distinct cold sensations. With this diversity in the neural basis for cold, it is remarkable that the majority of cold responses in vivo are dependent on the cold and menthol receptor transient receptor potential melastatin 8 (TRPM8). TRPM8-null mice are deficient in temperature discrimination, detection of noxious cold temperatures, injury-evoked hypersensitivity to cold, and nocifensive responses to cooling compounds. To determine how TRPM8 plays such a critical yet diverse role in cold signaling, we generated mice expressing a genetically encoded axonal tracer in TRPM8 neurons. Based on tracer expression, we show that TRPM8 neurons bear the neurochemical hallmarks of both C- and Aδ-fibers, and presumptive nociceptors and non-nociceptors. More strikingly, TRPM8 axons diffusely innervate the skin and oral cavity, terminating in peripheral zones that contain nerve endings mediating distinct perceptions of innocuous cool, noxious cold, and first- and second-cold pain. These results further demonstrate that the peripheral neural circuitry of cold sensing is cellularly and anatomically complex, yet suggests that cold fibers, caused by the diverse neuronal context of TRPM8 expression, use a single molecular sensor to convey a wide range of cold sensations.

Activity of the Neuronal Cold Sensor TRPM8 Is Regulated by Phospholipase C via the Phospholipid Phosphoinositol 4,5-Bisphosphate

Cold temperatures robustly activate a small cohort of somatosensory nerves, yet during a prolonged cold stimulus their activity will decrease, or adapt, over time. This process allows for the discrimination of subtle changes in temperature. At the molecular level, cold is detected by transient receptor potential melastatin 8 (TRPM8), a nonselective cation channel expressed on a subset of peripheral afferent fibers. We and others have reported that TRPM8 channels also adapt in a calcium-dependent manner when activated by the cooling compound menthol. Additionally, TRPM8 activity is sensitive to the phospholipid phosphoinositol 4,5-bisphosphate (PIP2), a substrate for the enzyme phospholipase C (PLC). These results suggest an adaptation model whereby TRPM8-mediated Ca2+ influx activates PLC, thereby decreasing PIP2 levels and resulting in reduced TRPM8 activity. Here we tested this model using pharmacological activation of PLC and by manipulating PIP2 levels independent of both PLC and Ca2+. PLC activation leads to adaptation-like reductions in cold- or menthol-evoked TRPM8 currents in both heterologous and native cells. Moreover, PLC-independent reductions in PIP2 had a similar effect on cold- and menthol-evoked currents. Mechanistically, either form of adaptation does not alter temperature sensitivity of TRPM8 but does lead to a change in channel gating. Our results show that adaptation is a shift in voltage dependence toward more positive potentials, reversing the trend toward negative potentials caused by agonist. These data suggest that PLC activity not only mediates adaptation to thermal stimuli, but likely underlies a more general mechanism that establishes the temperature sensitivity of somatosensory neurons.

WHO 1st Consultation on the Development of a Global Biodosimetry Laboratories Network for Radiation Emergencies (BioDoseNet)

Abstract Blakely, W. F., Carr, Z., Chu, M. C-M., Dayal-Drager, R., Fujimoto, K., Hopmeir, M., Kulka, U., Lillis-Hearne, P., Livingston, G. K., Lloyd, D. C., Maznyk, N., Perez, M. D. R., Romm, H., Takashima, Y., Voisin, P., Wilkins, R. C. and Yoshida, M. A. WHO 1st Consultation on the Development of a Global Biodosimetry Laboratories Network for Radiation Emergencies (BioDoseNet). Radiat. Res. 171, 127–139 (2009). The World Health Organization (WHO) held a consultation meeting at WHO Headquarters, Geneva, Switzerland, December 17–18, 2007, to develop the framework for a global biodosimetry network. The WHO network is envisioned to enable dose assessment using multiple methods [cytogenetics, electron paramagnetic resonance (EPR), radionuclide bioassays, etc.]; however, the initial discussion focused on the cytogenetic bioassay (i.e., metaphase-spread dicentric assay). Few regional cytogenetic biodosimetry networks have been established so far. The roles and resources available from United Nations (UN) agencies that provide international cooperation in biological dosimetry after radiological emergencies were reviewed. In addition, extensive reliance on the use of the relevant International Standards Organization (ISO) standards was emphasized. The results of a WHO survey of global cytogenetic biological dosimetry capability were reported, and while the survey indicates robust global capability, there was also a clear lack of global leadership and coordination. The expert group, which had a concentrated focus on cytogenetic biodosimetry, formulated the general scope and concept of operations for the development of a WHO global biodosimetry laboratory network for radiation emergencies (BioDoseNet). Follow-on meetings are planned to further develop technical details for this network.

Involvement of eddy currents in the mutagenicity of ELF magnetic fields.

Possible carcinogenic and/or mutagenic activity of extremely low frequency magnetic fields was examined using somatic mutation and recombination test system of Drosophila melanogaster. An X-linked semi-dominant DNA repair defective mutation mei-41(D5) was introduced into the conventional mwh/flr test system to enhance mutant spot frequency. Virgin females of w mei-41(D5)/FM6; flr/TM6 were crossed with w mei-41(D5)/Y; mwh jv; spa(pol) males. The F(1) third instar larvae were exposed to a 50Hz, 20mT sinusoidal AC magnetic field for 24h. After moulting from pupal cases, their wings were examined under a bright field microscope to detect hair spots with mwh or flr mutant morphology. The exposure caused a statistically significant enhancement in somatic recombination spot frequency. Mutant spots arising due to chromosomal non-disjunction or terminal deletion also increased but the frequency of spots resulting from point mutation was not altered. The enhancement in the recombination spot frequency was suppressed to the control level when a culture medium without electrolytes was used during exposure. When larvae were exposed to a magnetic field in an annular dish, flies from the outer ring showed more mutant spots compared to those from the inner ring. These results suggest that the detected mutagenic activity was that of the induced eddy current, rather than that of the magnetic field itself.

A Threshold Exists in the Dose–Response Relationship for Somatic Mutation Frequency Induced by X Irradiation of Drosophila

Abstract Koana, T., Takashima, Y., Okada, M. O., Ikehata, M., Miyakoshi, J. and Sakai, K. A Threshold Exists in the Dose– Response Relationship for Somatic Mutation Frequency Induced by X Irradiation of Drosophila. Radiat. Res. 161, 391– 396 (2004). The dose–response relationship of ionizing radiation and its stochastic effects has been thought to be linear without any thresholds. The basic data for this model were obtained from mutational assays in the male germ cells of the fruit fly Drosophila melanogaster. However, it is more appropriate to examine carcinogenic activity in somatic cells than in germ cells. Here the dose–response relationship of X irradiation and somatic mutation was examined in Drosophila. A threshold at approximately 1 Gy was observed in DNA repair-proficient flies. In the repair-deficient siblings, the threshold was smaller and the inclination of the dose–response curve was much steeper. These results suggest that the dose–response relationship between X irradiation and somatic mutation has a threshold and that the DNA repair function contributes to its formation.

Dependence of DNA Double Strand Break Repair Pathways on Cell Cycle Phase in Human Lymphoblastoid Cells

DNA double‐strand breaks (DSBs) are usually repaired by nonhomologous end‐joining (NHEJ) or homologous recombination (HR). NHEJ is thought to be the predominant pathway operating in mammalian cells functioning in all phases of the cell cycle, while HR works in the late‐S and G2 phases. However, relative contribution, competition, and dependence on cell cycle phases are not fully understood. We previously developed a system to trace the fate of DSBs in the human genome by introducing the homing endonuclease I‐SceI site into the thymidine kinase (TK) gene of human lymphoblastoid TK6 cells. Here, we use this system to investigate the relative contribution of HR and NHEJ for repairing I‐SceI‐induced DSBs under various conditions. We used a novel transfection system, Amaxa™ nucleofector, which directly introduces the I‐SceI expression vector into cell nuclei. Approximately 65% of transfected cells expressed the I‐SceI enzyme and over 50% of the cells produced a single DSB in the genome. The relative contribution of NHEJ and HR for repairing the DSB was ∼100:1 and did not change with transfection efficiency. Cotransfection with KU80‐siRNA significantly diminished KU80 protein levels and decreased NHEJ activity, but did not increase HR. We also investigated HR and NHEJ in synchronized cells. The HR frequency was 2–3 times higher in late‐S/G2 phases than in G1, whereas NHEJ was unaffected. Even in late‐S/G2 phases, NHEJ remained elevated relative to HR. Therefore, NHEJ is the major pathway for repairing endonuclease‐induced DSBs in mammalian cells even in late‐S/G2 phase, and does not compete with HR. Environ. Mol. Mutagen. 2009.

Live cell imaging of micronucleus formation and development

The micronucleus (MN) test is widely used to biomonitor humans exposed to clastogens and aneugens, but little is known about MN development. Here we used confocal time-lapse imaging and a fluorescent human lymphoblastoid cell line (T105GTCH), in which histone H3 and α-tubulin stained differentially, to record the emergence and behavior of micronuclei (MNi) in cells exposed to MN-inducing agents. In mitomycin C (MMC)-treated cells, MNi originated in early anaphase from lagging chromosome fragments just after chromosome segregation. In γ-ray-treated cells showing multipolar cell division, MN originated in late anaphase from lagging chromosome fragments generated by the abnormal cell division associated with supernumerary centrosomes. In vincristine(VC)-treated cells, MN formation was similar to that in MMC-treated cells, but MNi were also derived from whole chromosomes that did not align properly on the metaphase plate. Thus, the MN formation process induced by MMC, γ-rays, and VC, were strikingly different, suggesting that different mechanisms were involved. MN stability, however, was similar regardless of the treatment and unrelated to MN formation mechanisms. MNi were stable in daughter cells, and MN-harboring cells tended to die during cell cycle progression with greater frequency than cells without MN. Because of their persistence, MN may have significant impact on cells, causing genomic instability and abnormally transcribed genes.

Differential Wiring of Layer 2/3 Neurons Drives Sparse and Reliable Firing During Neocortical Development

Sensory information is transmitted with high fidelity across multiple synapses until it reaches the neocortex. There, individual neurons exhibit enormous variability in responses. The source of this diversity in output has been debated. Using transgenic mice expressing the green fluorescent protein coupled to the activity-dependent gene c-fos, we identified neurons with a history of elevated activity in vivo. Focusing on layer 4 to layer 2/3 connections, a site of strong excitatory drive at an initial stage of cortical processing, we find that fluorescently tagged neurons receive significantly greater excitatory and reduced inhibitory input compared with neighboring, unlabeled cells. Differential wiring of layer 2/3 neurons arises early in development and requires sensory input to be established. Stronger connection strength is not associated with evidence for recent synaptic plasticity, suggesting that these more active ensembles may not be generated over short time scales. Paired recordings show fosGFP+ neurons spike at lower stimulus thresholds than neighboring, fosGFP- neurons. These data indicate that differences in circuit construction can underlie response heterogeneity amongst neocortical neurons.

Thalamocortical Projections onto Behaviorally Relevant Neurons Exhibit Plasticity during Adult Motor Learning

Layer 5 neurons of the neocortex receive direct and relatively strong input from the thalamus. However, the intralaminar distribution of these inputs and their capacity for plasticity in adult animals are largely unknown. In slices of the primary motor cortex (M1), we simultaneously recorded from pairs of corticospinal neurons associated with control of distinct motor outputs: distal forelimb versus proximal forelimb. Activation of ChR2-expressing thalamocortical afferents in M1 before motor learning produced equivalent responses in monosynaptic excitation of neurons controlling the distal and proximal forelimb, suggesting balanced thalamic input at baseline. Following skilled grasp training, however, thalamocortical input shifted to bias activation of corticospinal neurons associated with control of the distal forelimb. This increase was associated with a cell-specific increase in mEPSC amplitude but not presynaptic release probability. These findings demonstrate distinct and highly segregated plasticity of thalamocortical projections during adult learning.

The Dose Response of Chromosome Aberrations in Human Lymphocytes Induced In Vitro by Very Low-Dose γ Rays

Abstract This paper considers the dose–effect relationship for unstable chromosome aberration yields in human lymphocytes in very low-dose range. Data are presented for 60Co γ-ray doses of 0, 10, 20, 40 and 1000 mGy. More than 5,000 metaphases were scored for each data point at the very low doses, and each cell was double-checked using a semi-automated metaphase finding/relocation system. Aberration yields of dicentrics plus centric rings followed an excellent linear dose response down to zero dose; the yields were significantly above the control frequency from 20 mGy.