Y. Wada

Implementation of the modified-SHIRPA protocol for screening of dominant phenotypes in a large-scale ENU mutagenesis program

SHIRPA is a three-stage protocol for the comprehensive assessment of primarily mouse behavior. The first stage consists of high-throughput phenotyping of 33 behavioral observations and 7 metabolic or disease observations. We modified this part of the protocol by integrating new morphologic observations into the initial phenotype assay of behavior and dysmorphology. Behavioral observations assessed by this protocol, now referred to as the “modified-SHIRPA,” are compatible with the original “SHIRPA” protocol. Using modified-SHIRPA, we screened dominant phenotypes of more than 10,000 G1 progeny generated by crossing DBA/2J females with ENU-treated C57BL/6J males. To date, we have obtained 136 hereditary-confirmed mutants that exhibit behavioral and morphologic defects. Some independent mutant lines exhibited similar phenotypes, suggesting that they may represent alleles of the same gene or mutations in the same genetic pathway. They could hold great potential for the unraveling of the molecular mechanisms of certain phenotypes.

Development and implementation of a database system to manage a large-scale mouse ENU-mutagenesis program.

A mouse ENU-mutagenesis program at RIKEN GSC has been initiated to conduct a large-scale, genome-wide, early- and late-onset phenotypic screen of mutant mice. We screened about a hundred mice every week with a comprehensive set of phenotype assays including behavioral tests based on a modified SHIRPA protocol, blood tests (both clinical biochemical testing and hemogram), and measurement of locomotor activity in their home cages. To manage the entire program, we developed a client/server architecture database system and named it MUSDB (Mutagenesis Universal Support DataBase). It manages mouse husbandry, mating protocols, procedures for ENU injection and phenotypic screens, phenotype inheritance tests, preservation of sperm and organs, and other materials generated during the program. We have implemented MUSDB in quite a large-scale system that includes 150 client computers. It has, helped reduce typographical errors and provided simple and efficient operation via its front-end user interface. It significantly contributed to the communication within and between workgroups in the program and in the accumulation of various phenotypic and inheritance data.

Phenotypic characterization of a new Grin1 mutant mouse generated by ENU mutagenesis

In the RIKEN large‐scale N‐ethyl‐N‐nitrosourea (ENU) mutagenesis project we screened mice with a dominant mutation that exhibited abnormal behavior in the open‐field test, passive avoidance test and home‐cage activity test. We tested 2045 progeny of C57BL/6J males treated with ENU and untreated DBA/2J females in the open‐field test and isolated behavioral mutant M100174, which exhibited a significant increase in spontaneous locomotor activity. We identified a missense mutation in the Grin1 gene, which encodes NMDA receptor subunit 1, and designated the mutant gene Grin1Rgsc174. This mutation results in an arginine to cysteine substitution in the C0 domain of the protein. Detailed analyses revealed that Grin1Rgsc174 heterozygote exhibited increased novelty‐seeking behavior and slight social isolation in comparison with the wild type. In contrast to other Grin1 mutant mice, this mutant exhibited no evidence of heightened anxiety. These results indicate that this is a unique behavioral Grin1 gene mutant mouse that differs from the known Grin1 mutant mice. The results of immunohistochemical and biochemical analyses suggested that impaired interaction between the glutamatergic pathway and dopaminergic pathway may underlie the behavioral phenotypes of the Grin1Rgsc174 mutant.

Behavioral and neuromorphological characterization of a novel Tuba1 mutant mouse

As part of the RIKEN large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis project, we screened mice with a dominant mutation that exhibited abnormal behavior using an open-field test and a home-cage activity test. We tested 495 male progeny of C57BL/6J males treated with ENU and untreated C3H/HeJ females using the open-field test and isolated behavioral mutant M101736, which exhibited a significant increase in spontaneous locomotor activity. We identified a missense mutation in the Tuba1 gene, which encodes the TUBA1 protein, and designated the mutant gene Tuba1(Rgsc1736). This mutation results in an aspartic acid to glycine substitution in the TUBA1 protein. Detailed analyses revealed that Tuba1(Rgsc1736) heterozygotes exhibited inattention to novel objects and aberrant patterns of home-cage activity. The results of a behavioral pharmacological analysis using methylphenidate and morphological analyses of embryonic and adult brains suggested that Tuba1(Rgsc1736) is a novel animal model for neurodevelopmental disorders.

The total cross section for the 4He (γ, npp)n reaction in the Δ-resonance region

Abstract This is the first measurement of the total cross section σ( 4 He (γ npp)n) in the range of 135–455 MeV. The σ, which cannot be reproduced by any model, has similar E γ dependence to γ T (γ 4 He → all channels). The averaged ratio σ( 4 He(γ, npp)n)/ σ T (γ 4 He → all channels) = (2.3 ± 0.3)% is compared with a cascade-model calculation in which quasi-free pion production followed by pion reabsorption is assumed.

Probing theΔNNcomponent of3He

The 3He(gamma,pi^+/- p) reactions were measured simultaneously over a tagged photon energy range of 800<E_gamma<1120 MeV, well above the Delta resonance region. An analysis was performed to kinematically isolate Delta knockout events from conventional Delta photoproduction events, and a statistically significant excess of pi+p events was identified, consistent with Delta++ knockout. Two methods were used to estimate the DeltaNN probability in the 3He ground state, corresponding to the observed knockout cross section. The first gave a lower probability limit of 1.5+/-0.6+/-0.5%; the second yielded an upper limit of about 2.6%.

A step-like rise in the 4He(γ,pn)2H cross section near the pion-production threshold

Abstract A kinematically complete 4He(γ,pn)2H measurement was carried out in the photon energy (Eγ) range 145–425 MeV. The total cross section (σα) forms a prominent structure whose peak lies at Eγ ∼ 245 MeV (the pnd invariant mass of 3960 MeV/c2). The ratio σ α σ d , where σd is the deuteron photodisintegration cross section, shows a step-like rise to a value of 6 at Eγ ∼ 140 MeV.

Simultaneous measurement of 3He(γ, pπ±) yields and model implications

Abstract 3 He(γ, pπ ± ) reactions are simultaneously measured for tagged photon energies of 380 ⩽ Eγ ⩽ 700 MeV. The yield ratio for the two channels, under the same kinematical and dynamical conditions, is found to be 0.90±0.07. Implications of this ratio in terms of the delta component in the ground state of 3He are examined. An alternate interpretation of this ratio as due to an admixture of isospin T = 1 2 and 3 2 transition amplitudes in a pure isovector interaction is also presented.

Measurement of the cross section for γd → π0d at 140° ⩽ θCM ⩽ 156°

Abstract The differential cross sections for the reactions γ d → π 0 d have been measured for photons from 500 to 1000 MeV. We discuss the effect of the double scattering term and suggest no indication for the dibaryon resonances of the mass around 2.5 GeV.

P3-n19 Phenotype analyses of a novel GLUT1 deficient syndrome model mouse

in the thalamocortical loops. Involvement of basal ganglia in absence seizures was not shown in mice models, and roles of basal ganglia in the SWD generation were not known in any animal models. To address these issues, we performed in vivo and in vitro experiments using tottering (tg) mice, a well established model of absence epilepsy. In vivo experiments showed the involvement of basal ganglia in the SWD generation. In vitro experiments in the subthalamic nucleus (STN) neurons showed the enhanced membrane excitability in tg mice. This enhancement seemed to result from the decrement of the HCN channel activity. Unilateral blockades of STN HCN channels of tg mice extended the mean duration of SWDs. The results suggested that the basal ganglia play a positive role in the SWD generation through the enhanced membrane excitability caused by decreased HCN channel activity in the STN.