Toyota is undoubtedly one of the leaders when it comes to electrification of the model range The Japanese manufacturer holds about 12 percent of the U.S new car market and approximately four percent of the European but already sells roughly 43 percent of all electrified vehicles on the planet it’s not a surprise that it expects the internal combustion engine to die by 2050 the head of advanced R&D and engineering at the automaker who believes that manufacturers will continue facing tighter emission regulations forcing them to accelerate the development of electric cars this will lead to a global sales termination of ICE-powered vehicles “We expect that by 2050 we will have reduced CO2 emissions from vehicles by 90 percent compared to the figure in 2010,” Kuzumaki told Autocar in a recent interview from 2040 simple internal combustion engined cars will not be made but they may be the basis of some hybrid or plug-in hybrid cars.” Toyota will have its first fully electric cars on sale by the end of the decade, using lithium ion batteries and offering a range of about 300 miles at a single charge. However, by the early 2020s, the automaker expects to be able to introduce production solid state batteries This technology is believed to be a breakthrough in the electric vehicle segment providing significantly better performance than today’s batteries combined with smaller dimensions “We hold more patents on solid state batteries than any other company,” Kuzumaki explained “We are getting close to developing cars using the technology and we believe that we will be ahead of our rivals in achieving that.” Source: Autocar 2026 Toyota Camry Nightshade: Better Looks Lotus Emira With Yellow Exhaust Tips Pays Tribute to an F1 GOAT Toyota Chairman Says a Sports Car Must Have a Gas Engine The Cadillac Celestiq Will Be as Rare as a Bugatti The Toyota bZ7 Is a Chinese Electric Flagship Sedan Toyota RAV4 Production Might Stay In America Over Tariffs: Report Metrics details Reelin is a protein encoded by the RELN gene that controls neuronal migration in the developing brain Human genetic studies suggest that rare RELN variants confer susceptibility to mental disorders such as schizophrenia it remains unknown what effects rare RELN variants have on human neuronal cells the analysis of human neuronal dynamics at the single-cell level is necessary we generated human-induced pluripotent stem cells carrying a rare RELN variant (RELN-del) using targeted genome editing; cells were further differentiated into highly homogeneous dopaminergic neurons Our results indicated that RELN-del triggered an impaired reelin signal and decreased the expression levels of genes relevant for cell movement in human neurons Single-cell trajectory analysis revealed that control neurons possessed directional migration even in vitro while RELN-del neurons demonstrated a wandering type of migration We further confirmed these phenotypes in neurons derived from a patient carrying the congenital RELN-del this is the first report of the biological significance of a rare RELN variant in human neurons based on individual neuron dynamics our approach should be useful for studying reelin function and evaluating mental disorder susceptibility focusing on individual human neuronal migration the biological significance of rare RELN variants in human neurons remains unknown Similar regulation seems to be present in developing human neurons; however the single-cell dynamics of neuronal migration remains unexamined Considering that sequential events occur in the developing brain the analysis of live neurons is required for understanding neuronal dynamics relevant to neurodevelopmental events in humans we differentiated iPSCs into homogeneous dopaminergic neurons Our single-cell analysis using live neurons revealed that healthy human neurons had controlled directional migration even at the single-cell level while RELN-del neurons lost migration ability particularly in directionality under the impaired reelin signal We obtained a similar phenotype using neurons derived from subjects carrying congenital RELN-del our automatic evaluation system of the migration of individual neurons confirmed that RELN-del triggers sequential disruption of directional migration All subjects provided written informed consent The given ages of the subjects are those at the time of the blood sampling for iPSC generation Neuronal differentiation was induced as previously reported20 and dorsomorphin (3 μM) for 1 week (days 0–7) were dissociated using TrypLE select and cultured in neurosphere medium consisting of MHM (DMEM/F12 supplemented with 1× N2 and 1 μM purmorphamine for 2 weeks (days 7–21) secondary neurospheres were collected 1 week after passage (day 21) and plated onto Matrigel (BD)- or poly-l-ornithine/laminin-coated dishes in dopaminergic neuron medium (MHM supplemented with B27 Cells were cultured in a 5% CO2/18–22% O2 atmosphere during all experiments the position of each cell in the XY coordinate was converted to the movement angle from the reference position as an inverse tangent (tan−1) of X and Y using MATLAB (Mathworks The cell position in image n was termed Celln Cell0 represented the cell position at the starting time (defined as 48 h after the beginning of induction) and set as the origin The reference direction of the cell was obtained by averaging the movement angles over the 4 h The movement angle of a cell at each time point thereafter was expressed as the angle from the coordinate axis Migration in a direction close to the reference direction (small angle) implies orientated migration while veering from the reference direction (larger angle) implies disorientated migration When a cell did not move between two sequential images the data at that time point were omitted from the analysis which could be detected at all-time points GFP signals in aggregates were omitted from the analysis Two means were compared using the Student’s or Welch’s t-test (unpaired Three or more means were analyzed by analysis of variance followed by Dunnett’s test for pairwise comparisons A p < 0.05 was accepted as a significant difference Frequencies and ratios were compared by Pearson’s chi-squared test with p < 0.05 accepted as significant a The target sites of the CRISPR-sgRNAs used in this study CRISPR-sgRNA#1 and #2 target the sense strand whereas sgRNA#3 and #4 target the antisense strand b Schematic illustration of the differentiation of dopaminergic (DA) neurons c Quantitative real-time PCR was performed to measure RELN expression in neurospheres (day 21) and dopaminergic neurons (day 28) ***p < 0.001 vs respective parental neurospheres ††p < 0.01 vs respective parental DA neurons d Analysis of dopaminergic neuron differentiation efficiency by quantifying the ratio of TH+ to TUJ1+ cells at day 23 (n = 180 cells) e Representative images of early dopaminergic neurons (day 23 = 48 h after the start of induction) immunostained for TH and reelin The white bar in the image represents 200 μm f Immunoblotting for phosphorylated tyrosine DAB1 (p-YDAB1) The values of p-YDAB1/total DAB1 are as follows: CON1 = 100 g The venn diagram containing the altered gene expression in IgCON2(+/−) (blue) or IgCON2(−/−) (red) h GO analysis identified enriched categories for the altered genes in both IgCON2 neurospheres A list of the top 10 categories showing the smallest p-values the amount of phosphorylated DAB1 tyrosine (p-YDAB1) was decreased in the isogenic RELN-del cell lines compared with that in parental lines indicating that the reelin signal is impaired in isogenic RELN-del neurons a Plots of 10-cell trajectories emanating from the origin b Quantification of total migration distance over 4 h (48–52 h after plating) c Upper: Definition of directionality ratio Lower: Directionality ratio over elapsed time d Directionality ratio of the last point trajectory e Schematic diagram showing the cell movement angle at each time point n relative to the coordinate axis f Distribution of the cell movement angles for each genotype plotted on circular histograms Each histogram shows a representative cell for each genotype g Summary of 10 cells from each genotype shown as a histogram h The distribution of cells based on their movement angles Angles are absolute values relative to the origin (negative is clockwise) 0°−20° include both αCelln = 0°−20° and −20°–0° Each bar represents the proportion of the total analysis points according to the angular movement range 0°–20° e Measurement of dopamine concentration in the culture supernatant of dopaminergic neurons at day 28 f Immunoblotting for phosphorylated DAB1 tyrosine (p-YDAB1) The relative intensities of p-YDAB1/total DAB1 are as follows: CON1 = 100 To address whether each congenital RELN-del TH+ neuron showed an impaired reelin signal similar to isogenic RELN-del line neurons, we performed immunoblotting experiments for p-YDAB1 and total DAB1. The amount of p-YDAB1 was decreased in congenital RELN-del lines compared with control lines (Fig. 3f) These findings confirm that this rare RELN variant causes decreased reelin expression and impaired reelin signaling in human neurons a Upper: Schematic illustration of the automatic detection system Lower: A representative result of automatic detection b Left: Images of GFP signal at 48 h after plating neurospheres c Plots of cell trajectories emanating from the origin e Directionality ratio of the last point trajectory in d Considering that reelin functions well in the developing brain the establishment of RELN-del iPSCs is an ideal strategy to assess the significance of rare RELN variants in human neurons our findings may suggest the importance of endogenous reelin expression for directional migration in human neurons Further studies are warranted to confirm our findings While we identified a common phenotype in both congenital and isogenic RELN-del neurons we also found some differences between them decreased dopamine production was observed only in congenital RELN-del neurons Considering that the differentiation efficiency into TH+ neurons was similar (>85%) among all RELN-del iPSCs the intracellular degradation of dopamine may be promoted in congenital RELN-del neurons We have not identified the additional RELN-del subjects in more than 2000 SCZ patients The small number of subjects carrying rare RELN variant is the limitation of our study To elucidate a direct causal link between neuronal phenotypes and rare genetic variants such as RELN the derivation of both isogenic and patient-specific iPSCs Although it should be noted that iPSC-derived neurons do not completely recapitulate the physiology of the human brain they are undoubtedly full of novel findings about human brain dynamics migrating neurons are aligned along the radial glia while we observed that iPSC-derived neurons could independently migrate from the radial glia Our findings suggest that reelin widely plays a role in brain development and may be useful for studying the mechanisms underlying neuronal migration independent of the radial glia in humans and cellular mechanisms underlying disease susceptibility is essential for the development of effective treatments we demonstrated that a rare RELN variant causes an impaired reelin signal resulting in a loss of directionality during individual human neuronal migration using both isogenic and congenital RELN-del iPSCs made it possible to come to this conclusion our automatic detection system demonstrated the utility of RELN-del iPSCs for screening and evaluating human neurodevelopmental impairment focusing on the directionality of individual cell migration provides a foundation for the elucidation of reelin function in neural development and may contribute to novel therapeutic strategies for mitigating mental disorder susceptibility High-resolution copy number variation analysis of schizophrenia in Japan transcriptional and chromatin genes disrupted in autism Autosomal recessive lissencephaly with cerebellar hypoplasia is associated with human RELN mutations The involvement of Reelin in neurodevelopmental disorders A decrease of reelin expression as a putative vulnerability factor in schizophrenia RELN mutations in autism spectrum disorder Gene-environment interaction during early development in the heterozygous reeler mouse: clues for modelling of major neurobehavioral syndromes Cellullar insights into cerebral cortical development: focusing on the locomotion mode of neuronal migration Integrative mechanisms of oriented neuronal migration in the developing brain Neuronal migration mechanisms in development and disease Localization of reelin signaling pathway components in murine midbrain and striatum The dopamine hypothesis of schizophrenia: version III—the final common pathway Dopamine in schizophrenia: a review and reconceptualization Induction of pluripotent stem cells from adult human fibroblasts by defined factors Identification and classification of chromosomal aberrations in human induced pluripotent stem cells Recurrent copy number variations in human induced pluripotent stem cells Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage Modeling neurological diseases with induced pluripotent cells reprogrammed from immortalized lymphoblastoid cell lines Phenotypic differences in hiPSC NPCs derived from patients with schizophrenia SHANK proteins limit integrin activation by directly interacting with Rap1 and R-Ras Quantitative and unbiased analysis of directional persistence in cell migration Feature point tracking and trajectory analysis for video imaging in cell biology Efficient genomic correction methods in human iPS cells using CRISPR-Cas9 system flexible and reliable CRISPR/Cas9 target prediction tool Canonical and non-canonical Reelin signaling Human iPSC neurons display activity-dependent neurotransmitter secretion: aberrant catecholamine levels in schizophrenia neurons Abnormal neuronal differentiation and mitochondrial dysfunction in hair follicle-derived induced pluripotent stem cells of schizophrenia patients Modeling human cortical development in vitro using induced pluripotent stem cells FOXG1-dependent dysregulation of GABA/Glutamate neuron differentiation in autism spectrum disorders A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells Controlling the regional identity of hPSC-derived neurons to uncover neuronal subtype specificity of neurological disease phenotypes New insights into Reelin-mediated signaling pathways De novo mutations in schizophrenia implicate synaptic networks Identification of RELN variation p.Thr3192Ser in a Chinese family with schizophrenia Pathogenic rare copy number variants in community-based schizophrenia suggest a potential role for clinical microarrays Dorsal forebrain-specific deficiency of Reelin-Dab1 signal causes behavioral abnormalities related to psychiatric disorders Reelin function in neural stem cell biology Reelin binds alpha3beta1 integrin and inhibits neuronal migration Visualization of migration of human cortical neurons generated from induced pluripotent stem cells Fusion of regionally specified hPSC-derived organoids models human brain development and interneuron migration Migration of dopaminergic neurons in the embryonic mesencephalon of mice Reelin and CXCL12 regulate distinct migratory behaviors during the development of the dopaminergic system Development and evolution of the human neocortex Download references We thank the patient and his family for participating in this study Hattori for gifting plasmids of reelin expression This study was supported by AMED under grant no JP18dm0107087 and JP18dm0207005; Innovative Areas “Glial assembly: a new regulatory machinery of brain function and disorders”; Nagoya University Hospital Funding for Clinical Research; the MEXT KAKENHI (grants-in-Aid for Scientific Research) Nagoya University Graduate School of Medicine Center for Advanced Medicine and Clinical Research School of Pharmacy and Pharmaceutical Sciences The remaining authors declare that they have no conflict of interest Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41398-018-0177-8 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Phillip Martinez is a game and culture reporter for Newsweek he was a reporter and editor for iDigitalTimes and Player.One he graduated with his Bachelor’s degree in Journalism from St he’s always looking to become a Pokémon Master while fantasy booking this year’s WrestleMania You can contact him at p.martinez@newsweek.com either observed and verified firsthand by the reporter or reported and verified from knowledgeable sources Translations may contain inaccuracies—please refer to the original content There's been plenty of speculation and rumors surrounding the next Super Smash Bros but it seems one of the game's higher-ups is feeding into it with an Instagram post Super Smash Bros. Ultimate game supervisor Kuzumaki Shinya posted a photo of Kirby in front of a shield from the Dragon Quest series. A recent and reliable data mine of the game revealed some interesting code names to DLC characters, and one of them points to Erdrick one of the main protagonists of the series. A post shared by Shinya Kumazaki／熊崎信也 (@kumazaki_shinya) on Feb 3 jam1garner on Twitter found code for three new roster additions in Smash Ultimate They believe "Jack" is referring to Joker from Persona 5 (he is the "jack of all trades") "Packu" is the Japanese name for the recently-released Piranha Plant but "Brave" remained up for interpretation one of the class of heroes in Dragon Quest is called Yuusha which directly translates to "brave" in English the names were removed from the game's code after the version 2.0 update The connections to a representative from the Dragon Quest series coming to Super Smash Bros and the game's supervisor posting a photo of Kirby with a shield from that series will get fans talking even more If you're not familiar with the Kirby significance the pink puffball was created by Super Smash Bros Ultimate director Masahiro Sakurai and is known to be his favorite character Kirby is also the only survivor in Smash Ultimate's World of Light Adventure Mode Sakurai announced that Smash Ultimate will include Piranha Plant as a free DLC fighter for purchasing the game early Five additional DLC characters will be released as part of the game's Fighters Pass Joker is the first confirmed DLC character but the release of the fighter has yet to be announced Rumors about the other four fighters include characters from Minecraft and even Doom Ultimate is available now for Nintendo Switch Piranha Plant is available to purchase and download now on the Nintendo eShop Do you think this Instagram post is a troll or a hint as to Dragon Quest coming to Smash Ultimate Newsweek is committed to challenging conventional wisdom and finding connections in the search for common ground Newsletters in your inbox See all “We expect that by 2050 we will have reduced CO2 emissions from vehicles by 90% compared to the figure in 2010 To achieve that from 2040 simple internal combustion engined cars will not be made but they may be the basis of some hybrid or plug-in hybrid cars,” he said One important part in Toyota’s long-term EV plans will be its adoption of advanced solid-state batteries The brand’s first all-electric vehicle will arrive in 2020 with traditional lithium-ion batteries Toyota says it will be able to productionize solid-state batteries by the early 2020s allowing its EVs to offer exceptional range and fast-charging “We hold more patents on solid-state batteries than any other company We are getting close to developing cars using the technology and we believe that we will be ahead of our rivals in achieving that,” he revealed Today's print edition Home Delivery Nicol has been named co-winner of the Green Culture Prize for his outstanding work on woodlands restoration A longtime contributor to The Japan Times Nicol received the annual award Saturday from the National Land Afforestation Promotion Organization awarded for outstanding efforts in greenery and forestry which actively promotes green tourism.googletag.cmd.push(function() { googletag.display('div-gpt-ad-1499653692894-0'); }); The 75-year-old Wales native visited Japan for the first time in 1962 he developed a passionate interest in ecology and chose in 1980 to reside in the mountainous Kurohime area of Nagano Prefecture where he became ardently engaged in forest restoration efforts setting up the Afan Woodland Trust in 1986 "He has advocated the positive effects of forestry on human body and soul and has invited children with disabilities and scarred by abuse to the forest," the organization said in a press release "He has also contributed to turning elementary schools damaged by the Great East Japan Earthquake into 'forestry schools.' "It is highly commendable that he has also been conveying the importance of forests through his writing and speaking engagements nationwide." In a time of both misinformation and too much information quality journalism is more crucial than ever.By subscribing Your subscription plan doesn't allow commenting. To learn more see our FAQ Sponsored contents planned and edited by JT Media Enterprise Division Toyota's research and development boss has predicted that the internal combustion engine will very much be a thing of the past by 2050 Seigo Kuzumaki is also predicting that by 2040 only 10 percent of cars will be hybrids as environmental concerns and legislation continues to clamp down on more traditionally powered vehicles 'We expect that by 2050 we will have reduced CO2 emissions from vehicles by 90 percent compared with the figure in 2010,' said Kuzumaki recently 'To achieve that from 2040 simple internal combustion-engined cars will not be made but they may be the basis of some hybrid or plug-in hybrid cars.' Toyota's 'green' strategy has mainly centred around hybrid cars for two decades now but the company is also investing in and developing battery technology for electric vehicles The Japanese car maker is shortly to introduce its first fully electric car which uses now-traditional lithium ion tech but the company says that it is working on getting innovative solid-state batteries on to the market by the early 2020s The Japanese manufacturer has also been a major proponent of hydrogen fuel cell cars being one of the first to get the technology into production Although Toyota doesn't yet sell an all-electric car the firm has gradually warmed up the world to electrification with its Prius hybrid representing the best selling electrified vehicle across the world – the car has shifted more than 11 million units since it was first introduced back in 1997 in July environment secretary Michael Gove announced that the UK government plans to introduce legislation to stop the sale of new purely petrol and diesel-powered cars by 2040 – in line with Toyota's prediction There was some worry around the initial announcement which appeared to suggest that only electric cars would be legal after 2040 but the government then clarified the situation to make it clear that certain hybrid setups would be allowed Metrics details exhibits extraordinary resistance to cancer Here we report that NMR somatic cells exhibit a unique tumour-suppressor response to reprogramming induction we generate NMR-induced pluripotent stem cells (NMR-iPSCs) and find that NMR-iPSCs do not exhibit teratoma-forming tumorigenicity due to the species-specific activation of tumour-suppressor alternative reading frame (ARF) and a disruption mutation of the oncogene ES cell-expressed Ras (ERAS) The forced expression of Arf in mouse iPSCs markedly reduces tumorigenicity we identify an NMR-specific tumour-suppression phenotype—ARF suppression-induced senescence (ASIS)—that may protect iPSCs and somatic cells from ARF suppression and NMR-specific ARF regulation and the disruption of ERAS regulate tumour resistance in NMR-iPSCs Our findings obtained from studies of NMR-iPSCs provide new insight into the mechanisms of tumorigenicity in iPSCs and cancer resistance in the NMR (e) Karyotype of NMR-iPSCs (clone 24) at passage 10 (f) RNA-seq of expression levels of selected pluripotency and fibroblast markers in NMR-iPSCs and NMR-fibroblasts Y axis: ratio of the average value of fragments per kilobase of transcript per million mapped reads (FPKM) of four NMR-iPSC clones to the average of NMR-fibroblast lines (g) Immunocytochemical analyses of the expression of differentiated EBs is shown as follows: mesoderm (DESMIN endoderm (FOXA2 and VIMENTIN) and ectoderm (NESTIN and GFAP) markers (h) Tumours or testes after transplantation of human-iPSCs (10 weeks) Ms-iPSCs (4 weeks) or NMR-iPSCs (10 or 20 weeks) into the testes of NOD/SCID mice 20 weeks (n=16) or 28 weeks (n=10) for NMR; 10 weeks (n=8) for human not significant; Kruskal–Wallis test followed by the Dunn’s test These observations raise the question of whether somatic cell reprogramming conferring pluripotency and tumorigenicity can be induced in cancer-resistant animals we identify a mechanism that we have termed ‘ARF suppression-induced senescence’ (ASIS) which appears to be a NMR-specific tumour-suppression mechanism This study provides novel insights into NMR cancer resistance and methods for generating safe iPSCs These results suggest that reprogramming to a pluripotent state can be induced in cancer-resistant NMRs without inducing tumorigenicity (a) The qRT–PCR analysis of the expression of INK4a INK4b and p21 in NMR- and Ms-iPSCs (n=3 clones) (b) Alignment of the coding sequences of ERAS of 11 species mERas- or shARF/mERas-expressing NMR-iPSCs were transplanted into the testes of NOD/SCID mice Representative tumours 10 weeks after transplantation (c) ARF activation and disruption of ERAS regulates the tumour resistance of NMR-iPSCs (a) Nude mice transplanted subcutaneously with Ms-iPSCs expressing red fluorescent protein (RFP; left) or Arf (right) 5 weeks after transplantation (b) Comparison of tumour weights 3 weeks after transplantation (c) Kaplan–Meier curve of tumour-free mice transplanted with Low-Arf-group Ms-iPSC clones (clone 5 and 6 blue line) or control Ms-iPSCs expressing RFP (n=8 mice (a–d) Co-transduction of NMR-fibroblasts with shARF or shINK4a with the OSKM; cell morphology (a) (e–g) Transduction of NMR-fibroblasts overexpressing c-Myc with shARF or shINK4a; cell morphology (e) (h–j) Transduction of shARF of serially-passaged NMR-fibroblasts; cell morphology (h) (k) Role of ARF and ERAS in reprogramming without acquisition of tumorigenicity in NMR-iPSCs *P<0.05 between the indicated groups (one-way analysis of variance (ANOVA) for b–d,f suggesting that unique cancer-resistance mechanisms mediated by INK4a and ARF evolved in NMRs We conclude that the tumour resistance in NMR-iPSCs is based on NMR-specific ARF regulation and disruption of ERAS Further research into the detailed mechanisms underlying ASIS in NMRs may contribute to the generation of non-tumorigenic human-iPSCs enabling safer cell-based therapeutics and shed new light on cancer resistance in the naked mole-rat The Ethics Committees of Hokkaido University (Approval no which were in accordance with the Guide for the Care and Use of Laboratory Animals (United States National Institutes of Health The NMR colonies are maintained at Hokkaido University C57BL/6 mice and BALB/c nude mice were purchased from CLEA Japan The NOD/SCID mice were purchased from Charles River The cells and tissues were obtained from at least two animals The data were analysed using one-way analysis of variance followed by the analysis of variance test or the Kruskal–Wallis nonparametric test followed by Dunn’s test for multiple comparisons or the unpaired t-test for two groups Graphpad Prism was used for statistical analysis NMR- or Ms-skin fibroblasts were isolated from 1- to 2-year-old adult male NMRs or 6-week-old adult male C57BL/6 mice The skin including the epidermis was washed with phosphate-buffered saline (PBS) containing 1% penicillin/streptomycin (Wako) and amphotericin B (Wako) and then treated with 0.25% trypsin/EDTA (Wako) and 5 mg ml−1 collagenase (GIBCO) at 32 °C for 30 min The reaction was stopped by adding 15% fetal bovine serum (FBS) medium (contents described below) The cell clumps and minced tissues were collected by centrifugation (180g plated on gelatin-coated 10-cm cell culture dishes and cultured at 32 °C (NMR-fibroblasts) or 37 °C (Ms-fibroblasts) in a humidified atmosphere containing 5% CO2 The cells were cultured in 15% FBS medium composed of Dulbecco’s Modified Eagle’s Medium (DMEM Sigma Aldrich) supplemented with 15% FBS (JRH or BioWest) 2 mM L-glutamine (Nakalai Tesque) and 0.1 mM non-essential amino acids (NEAA pMXs-based retroviral vectors were introduced into Plat-E cells with Fugene 6 transfection reagent (Roche) according to the manufacturer’s instructions the conditioned medium containing virus particles derived from these Plat-E cultures was used for viral transduction A second retroviral infection was performed 4 days after the first because of the low infection efficiency of NMR-fibroblasts fibroblasts were plated at 1.5 × 105 cells per 10-cm dish on a mitomycin C-treated SNL-STO (MSTO) feeder layer The medium was replaced with standard human ES medium containing DMEM/F12 (Sigma Aldrich) supplemented with 20% knockout serum replacement (Life Technologies) Sigma Aldrich) and 4 ng ml−1 fibroblast growth factor 2 (PeproTech) and we isolated iPSC-like colonies 30 days after reseeding on feeder layers Chromosomal G-band analyses were performed at Nihon Gene Research Laboratories The karyotypes of NMR-iPSC clones 24 and 27 were normal and that of clone 12 was tetraploid DNA fragments were inserted into the pENTR/D-TOPO entry vector and sequenced using the Sanger method The NMR-ERAS sequence (NCBI Reference Sequence: XM_004921208) previously deposited has one base deletion to adjust frame by automated computational analysis using gene prediction method (Gnomon) AP activity was measured using an Alkaline Phosphatase Detection Kit (System Biosciences) according to the manufacturer’s instructions NMR-iPSCs and differentiated cells were fixed with 4% paraformaldehyde in PBS for 5 min at room temperature washed with PBS and then treated with 0.3% Triton X-100 in Tris-NaCl-blocking buffer (PerkinElmer) for 60 min at room temperature The cells were incubated with primary antibodies against Oct4 (Millipore; 7F9.2; 1:200) and E-cadherin (BD Pharmingen; clone 36; 1:500) for 12 h at 4 °C the cells were incubated with secondary antibody Alexa Fluor 555 anti-Ms IgG (Cell Signaling Technology (CST); A21424; 1:1,000) and Alexa Fluor 555 anti-rabbit IgG (CST; A21429; 1:1,000) and nuclei were counterstained with 1 μg ml−1 Hoechst 33,258 (Sigma Aldrich) for 60 min at room temperature DESMIN (Thermo Scientific; RB-9014; 1:1,000) FOXA2 (ABNOVA; 7E6; 1:1,000) and VIMENTIN (abcam; EPR3776; 1:1,000) the cells were fixed with 4% paraformaldehyde and analysed using the antibodies against the proteins as follows: αSMA (Sigma Aldrich; 1A4; 1:1,000) Telomerase activity was detected using the TRAPeze Telomerase Detection Kit (Chemicon) according to the manufacturer’s instructions Telomere repeat additions were performed at 30 °C for 120 min The samples were electrophoretically separated through 20% polyacrylamide gels in 0.5 × TBE The gel was stained with SYBR Gold (Invitrogen; 1:10,000) Total RNA was extracted from NMR-fibroblasts and NMR-iPSCs using TRIzol reagent (Invitrogen) followed by Qiagen RNeasy column purification The quality and quantity of the RNA preparations were assessed using a 2100 Bioanalyzer with an RNA 6000 Nano LabChip Kit (Agilent Technologies) Poly(A)+ RNA was selected and converted to a library of cDNA fragments (200–250 bp) with adaptors attached to both ends for sequencing using a TruSeq RNA Sample Prep Kit v2 (Illumina) Libraries were quantified using a Bioanalyzer DNA High Sensitivity Kit (Agilent Technologies) and Kapa Library Quantification Kit (Kapa Biosystems) using an Applied Biosystems StepOne Real-Time PCR System The libraries were then loaded into a flow cell for cluster generation using the TruSeq Rapid SR Cluster Kit (Illumina) and sequenced using an Illumina HiSeq2500 to obtain 100-nucleotide sequences (single-end) NMR-iPSCs and -fibroblasts were passaged every 7 days and replated at 2 × 105 per six-well plate and 3 × 105 per 10-cm dish Reseeded cells were counted using a Coulter Counter (Beckman Coulter) including cell cycle arrest and cell death was calculated from the slopes of growth curve The conditioned medium containing virus particles was concentrated and used for viral transduction The transfected cells were enriched by 2 days of puromycin selection starting 24 h after transfection and colonies were picked up DNA was extracted from each colony and coding sequence for ERas was amplified and sequenced using the Sanger method the slides were incubated with secondary antibodies conjugated to horseradish peroxidase (HRP) anti-Ms IgG (CST; 7,076; 1:1,000) and anti-rabbit IgG (CST; 7,074; 1:1,000) for 1 h at room temperature washed with PBS and antigen–antibody complexes were detected using DAB (Vector Laboratories) The tumorigenicity of Ms-iPSCs was assessed by injecting subcutaneously 5 × 105 cells into nude mice The mice were monitored once each week for teratoma formation and general health and killed 3 or 5 weeks later To determine the tumour-free survival rate Scoring criterion: mice with visible tumours were designated ‘tumour-positive’ NMR-iPSCs were suspended to 3 × 103 cells per 3 ml of 0.35% agar in growth medium and poured over a solidified layer of 0.65% agar medium in a six-well plate the cell colonies were stained using toluidine blue and enumerated SA-βGal activity was measured using the Senescence Detection Kit (BioVision) Cells were stained for 48 h at 37 °C according to the manufacturer’s instructions The cells were washed with PBS and stained with Hoechst 33,258 (diluted 1:1,000 with PBS) for 30 min at room temperature in the dark The cells were washed in PBS and analysed by microscopy The cell populations in at least three random fields (≥500 cells) were analysed for perinuclear blue staining indicative of SA-βGal activity and the nuclei were visualized using Hoechst staining Accession codes: RNA-seq data have been deposited in the DNA Data Bank of Japan (DDBJ) database under accession code: DRA003980. The sequence of NMR-ERAS was deposited in the GenBank database under accession code: LC074725 Tumour resistance in induced pluripotent stem cells derived from naked mole-rats Eusociality in a mammal: cooperative breeding in naked mole-rat colonies Negligible senescence in the longest living rodent the naked mole-rat: insights from a successfully aging species Successful aging and sustained good health in the naked mole rat: a long-lived mammalian model for biogerontology and biomedical research Spontaneous histologic lesions of the adult naked mole rat (Heterocephalus glaber): a retrospective survey of lesions in a zoo population Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors Variation in the safety of induced pluripotent stem cell lines Steps toward safe cell therapy using induced pluripotent stem cells The tumorigenicity of human embryonic and induced pluripotent stem cells Premature termination of reprogramming in vivo leads to cancer development through altered epigenetic regulation The Ink4/Arf locus is a barrier for iPS cell reprogramming Senescence impairs successful reprogramming to pluripotent stem cells Immortalization eliminates a roadblock during cellular reprogramming into iPS cells Role of ERas in promoting tumour-like properties in mouse embryonic stem cells The ground state of embryonic stem cell self-renewal High-molecular-mass hyaluronan mediates the cancer resistance of the naked mole rat expresses a functional p15/p16 hybrid isoform Genome sequencing reveals insights into physiology and longevity of the naked mole rat Inhibition of pluripotent stem cell-derived teratoma formation by small molecules Cyclin A1 is essential for setting the pluripotent state and reducing tumorigenicity of induced pluripotent stem cells CDK1 inhibition targets the p53-NOXA-MCL1 axis Increased dosage of tumor suppressors limits the tumorigenicity of iPS cells without affecting their pluripotency Generation of germline-competent induced pluripotent stem cells Differential effects on ARF stability by normal versus oncogenic levels of c-Myc expression Crosstalk between the Rb pathway and AKT signaling forms a quiescence-senescence switch Reversal of human cellular senescence: roles of the p53 and p16 pathways Necessary and sufficient role for a mitosis skip in senescence induction Induced pluripotent stem cells and senescence: learning the biology to improve the technology Molecular cloning and characterization of the INK4a and ARF genes in naked mole-rat CATs and HATs: the SLC7 family of amino acid transporters Purified mesenchymal stem cells are an efficient source for iPS cell induction Efficient selection for high-expression transfectants with a novel eukaryotic vector Expression of tubulin beta II in neural stem/progenitor cells and radial fibers during human fetal brain development Evaluation of in vitro proliferative activity of human fetal neural stem/progenitor cells using indirect measurements of viable cells based on cellular metabolic activity Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells The ability of inner-cell-mass cells to self-renew as embryonic stem cells is acquired following epiblast specification An integrated database of genes responsive to the Myc oncogenic transcription factor: identification of direct genomic targets Development of a self-inactivating lentivirus vector Genome engineering using the CRISPR-Cas9 system Production of mouse pups from germline transmission-failed knockout chimeras Dax1 and Nanog act in parallel to stabilize mouse embryonic stem cells and induced pluripotency Download references Takaoka and professors at the Institute for Genetic Medicine Hokkaido University for their administrative support and scientific discussion; N Fujimura for help with animal maintenance; S laboratories for technical assistance and scientific discussion; S 38C2 Nanog-EGFP Ms-iPSCs and 201B7 human-iPSCs; K Kitamura for PLAT-E and pMXs retroviral vectors; J Naka-Kaneda for pCSII-EF-RfA-TK-hyg vector This work was supported in part by PRESTO of the Japan Science and Technology Agency to K.M.; Grants-in-Aid for Scientific Research from the Japanese Society for the Promotion of Science from the Ministry of Education and Y.K.; Grant-in-Aid for Scientific Research on Innovative Areas ‘Oxygen Biology: a new criterion for integrated understanding of life’ from the MEXT to K.M.; and the Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program) to H.O was supported by the Takeda Science Foundation Sekisui Chemical Innovations Inspired by Nature Research Support Program Astellas Foundation for Research on Metabolic Disorders and Mochida Memorial Foundation for Medical and Pharmaceutical Research were Research Fellows of the Japanese Society for the Promotion of Science Division of Biomedical Information Analysis Iwate Tohoku Medical Megabank Organization Research Organization of Information and Systems Aichi Medical University School of Medicine Yamaguchi University Graduate School of Medicine Hoshi University School of Pharmacy and Pharmaceutical Sciences conducted certain qRT–PCR experiments; Y.Oiwa conducted certain knockdown experiments; A.S. determined the telomerase activity; Y.Okada provided technical support and analyses in this study; S.M The authors declare no competing financial interests Supplementary Figures 1-11 and Supplementary Tables 1-2 (PDF 15443 kb) Download citation Sorry, a shareable link is not currently available for this article. Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily. + 1 PhotosJapan to trial autonomous cars during the 2020 OlympicsSeigo Kuzumaki, head of the Japanese government’s self-driving car program, told Bloomberg it will run an autonomous car trial around competition sites in the week leading up to the 2020 Tokyo Olympics. The government plans to have around 100 self-driving cars in service during the week starting on July 6, 2020. Vehicles will be supplied by Toyota, Nissan, and automotive parts suppliers. In that week vehicles will travel freely in the waterfront district, which is home to many Olympic venues. During the games themselves Toyota is planning on running a dozen or so autonomous e-Palette shuttles (above and below) in a loop around the Olympic and Paralympic village transporting athletes and officials. The automaker will also use one of its self-driving concept cars to accompany runners during the torch relay. It's understood all these self-driving vehicles will still have a driver on-board to monitor events and just in case things go awry. The trials around the Olympics and Paralympics are part of concerted push by the Japanese government to have autonomous vehicles available on the market by 2025. As part of its plan the government will also periodically test self-driving vehicles on public roads from now until 2022. Copyright Drive.com.au 2025ABN: 84 116 608 158 all prices are shown as Manufacturer's Recommended List Price (MRLP) inclusive of GST Metrics details it has been reported that chronic pain induces depression The endogenous opioid system has been implicated in nociception The present study was undertaken to investigate whether chronic pain could induce anxiogenic effects and changes in the opioidergic function in the amygdala in mice We found that either injection of complete Freund's adjuvant (CFA) or neuropathic pain induced by sciatic nerve ligation produced a significant anxiogenic effect at 4 weeks after the injection or surgery the selective μ-opioid receptor agonist [D-Ala2,N-MePhe4,Gly5-ol]-enkephalin (DAMGO)- and the selective δ-opioid receptor agonist (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80)-stimulated [35S]GTPγS binding in membranes of the amygdala was significantly suppressed by CFA injection or nerve ligation CFA injection was associated with a significant increase in the κ-opioid receptor agonist 2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1-phenyl-2-(1-pyrrolidinyl)ethyl]acetamide hydrochloride (ICI199,441)-stimulated [35S]GTPγS binding in membranes of the amygdala The intracerebroventricular administration and microinjection of a selective μ-opioid receptor antagonist and the endogenous κ-opioid receptor ligand dynorphin A caused a significant anxiogenic effect in mice We also found that thermal hyperalgesia induced by sciatic nerve ligation was reversed at 8 weeks after surgery the time spent in the lit compartment was not changed at 8 weeks after surgery the present data constitute the first evidence that chronic pain has an anxiogenic effect in mice This phenomenon may be associated with changes in opioidergic function in the amygdala Based on reports of the comorbidity between chronic pain and depressive illness in human patients it is possible that these disease states are linked The present study was then undertaken to investigate whether chronic pain could induce anxiogenic effects and alter opioidergic functions in the amygdala which is a key structure in the regulation of anxiety and the expression of emotional responses to stress in mice The present study was conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals as adopted by the Committee on Animal Research of Hoshi University which is accredited by Ministry of Education All efforts were made to minimize the number of animals used and their suffering Animals were kept in a room with an ambient temperature of 23±1°C and a 12-h light–dark cycle (lights on 0800 to 2000) All behavioral studies were performed during the light period Control mice were given saline into the plantar surface of the right hind paw To assess the sensitivity to thermal stimulation the right plantar surface of mice was tested individually using a well-focused radiant heat light source (model 33 Analgesia Meter; IITC/Life Science Instruments The intensity of the thermal stimulus was adjusted to achieve an average baseline paw-withdrawal latency of approximately 8–10 s in naive mice The paw-withdrawal latency was determined as the average of three measurements per paw Only quick hind paw movements (with or without licking of hind paws) away from the stimulus were considered to be a withdrawal response Paw movements associated with locomotion or weight shifting were not counted as a response The paws were measured alternating between left and right with an interval of more than 3 min between measurements Before the behavioral responses to the thermal stimulus were tested mice were habituated for at least 30 min in a clear acrylic cylinder (15 cm high and 8 cm in diameter) the latency of paw withdrawal in response to the thermal stimulus was tested The data represent the average value for the paw-withdrawal latency of the right hind paw To ascertain the acute effect of a selective benzodiazepine receptor agonist etizolam (1 mg/kg s.c.) on the increased sensitivity to the thermal stimulation each paw was measured just before and 30 min after etizolam (1 mg/kg s.c.) injection at 7 days after nerve ligation mice were treated repeatedly with etizolam once a day for 3 weeks (from day 7 to day 28) after nerve ligation and the paw withdrawal latency was measured one day after last injection A von Frey filament was applied to the plantar surface of each hind paw for 3 s and this was repeated three times with an inter-trial interval of at least 5 s Each of the hind paws was tested individually Paw withdrawal in response to a tactile stimulus was evaluated by the scoring as follows: 0 a slow and/or slight response to the stimulus; 2 a quick withdrawal response away from the stimulus without flinching or licking; 3 an intense withdrawal response away from the stimulus with brisk flinching and/or licking The paw withdrawal in response to each filament was determined as the average of two scores per paw The paws were measured alternating between left and right with an interval of more than 3 min between the measurements Before the behavioral responses to tactile stimuli were tested mice were habituated for at least 30 min on an elevated nylon mesh floor paw withdrawal in response to a tactile stimulus was tested We used a box consisting of a small (mouse: 18 × 13 × 18 cm3; rat: 40 × 25 × 40 cm3) dimly lit compartment with black walls and a black floor connected by a tunnel (mouse: 5 cm long; rat: 15 cm long) to a large (mouse: 18 × 18 × 18 cm3; rat: 40 × 40 × 40 cm3) intensely lit (500 lux) compartment with a white walls and a white floor Each animal was placed in the dark compartment at the beginning of the observation session Compartment entry and exit were defined as all four paws into and out of an arm The time spent in the lit compartment was recorded for 10 min The elevated plus-maze consisted of two opposing open and two closed arms (30 × 6 cm2 each) joined by a common central platform (9 × 9 cm2) The maze was elevated 40 cm above the floor The closed arms were enclosed by 15 cm high walls and the open arms were surrounded by 0.3-cm ledges Illumination of the open and closed arms and the central platform was approximately equal (100 lux) Arm entry and exit were defined as all four paws into and out of an arm The results were calculated as mean ratios of the time spent in the open arms to the total time spent in both the open and closed arms Entries to the open and closed arms were also scored In order to ascertain the anxiolytic effect of etizolam s.c.) was performed 30 min before the light–dark or the elevated-plus maze procedures mice were used for this procedure following repeated treatment with etizolam once a day for 3 weeks day 7 to day 28 after nerve ligation The saline- or CFA-injected mice and sham-operated or sciatic nerve-ligated mice were decapitated at 4 weeks after injection or surgery the brain was quickly removed and dissected including the basolateral amygdala and medial nucleus of the amygdala (about 2 × 2 × 2 mm3 cubic weighting 35 mg per mouse) according to the brain atlas of Paxinos G and Franklin KBJ (The Mouse Brain The tissue was homogenized using a Potter-Elevehjem tissue grinder with a Teflon pestle in 20 vol (w/v) of ice-cold Tris-Mg2+ buffer containing 50 mM Tris-HCl and 1 mM EGTA for the [35S]GTPγS binding assay The homogenate was centrifuged at 4°C for 10 min at 48 000g The pellet was resuspended in ice-cold Tris buffer or [35S]GTPγS binding assay buffer containing 50 mM Tris-HCl and 1 mM EGTA and 100 mM NaCl and centrifuged at 4°C for 10 min at 48 000g The resultant pellet was resuspended on ice-cold [35S]GTPγS binding assay buffer and stored at −70°C until use The membrane homogenate (3–8 μg protein per assay) was incubated at 25°C for 2 h in 1 ml of assay buffer with various concentrations of agonist The reaction was terminated by filtration using a Brandle cell harvester and Whatman GF/B glass filters presoaked in 50 mM Tris-HCl Filters were then washed three times with 5 mM ice-cold Tris-HCl buffer transferred to scintillation-counting vials containing 0.5 ml of Soluene-350 and 4 ml of Hionic Fluor and the radioactivity in the samples was determined with a liquid scintillation analyzer Nonspecific binding was measured in the presence of 10 μM unlabeled GTPγS Comparable results were obtained from at least three independent sets and in each set experiments were performed in triplicate Intracerebroventricular administration was performed as described previously (Haley and McCormick, 1957) A few days before intracerebroventricular injection the mice were lightly anesthetized with diethyl ether and a 2 mm double-needle tip: 27G × 2 mm and base: 22G × 10 mm (Natsume Seisakusho Co Japan) attached to a 25-μl Hamilton microsyringe was inserted into the unilateral injection site; a hole was made in the skull for the injection The solution was injected in a volume of 4 μl per mouse The guide cannula was fixed to the skull with cranioplastic cement the animals were injected with a selective μ-opioid receptor antagonist CTOP a δ-opioid receptor antagonist NTI or dynorphin A into the basolateral amygdala 10 min before testing Eicom Co.) extended beyond the guide cannula by 0.5 mm A stainless steel injection cannula was inserted into that guide cannula for each animal The injection cannula was connected through polyethylene tubing to a 10 μl Hamilton syringe that was preloaded with CTOP The receptor antagonist or vehicle was delivered by motorized syringe pump in a volume of 0.3 μl over 60 s 5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (Tocris Cookson Ltd USA) were dissolved in assay buffer containing 20% ethanol D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP; Sigma Chemical Co. USA) and dynorphin A (Peptide Institute Inc. Japan) were dissolved in saline for in vivo experiments Naltrindole hydrochloride (NTI: Tocris Cookson Inc. USA) was dissolved in saline containing 1% dimethyl sulfoxide Etizolam (Wako Pure Chemical Industries Ltd Japan) was dissolved in distilled water containing 0.5% carboxymethyl cellulose sodium salt The statistical significance of differences between groups was assessed with repeated measures analysis of variance (ANOVA) and one-way ANOVA followed by the Bonferroni–Dunn multiple comparison test or Student's t-test Changes in the latency of paw withdrawal after thermal and mechanical stimulation induced by the injection of complete Freund's adjuvant (CFA) or sciatic nerve ligation in mice The data represent the results of (a) paw-withdrawal latencies after the thermal stimulus and (b) paw withdrawal in response to a tactile stimulus on the ipsilateral side at 1 There was no difference in the basal response before injection or surgery between saline- and CFA-injected or sham-operated and sciatic nerve-ligated mice The tactile stimulus was applied using filaments with a bending force of 0.02g Each point represents the mean±SEM of 6–8 mice **p<0.01 and ***p<0.001: saline group vs CFA group ##p<0.01 and ###p<0.001: sham group vs ligation group Paw-withdrawal latencies and responses on the contralateral side of mice were not changed by injection or surgery (data not shown) Inflammatory pain- or neuropathic pain-induced anxiogenic effect in the elevated plus-maze test in mice The percentage of time spent in the open arms was significantly decreased by CFA injection (a) or sciatic nerve ligation (d) at 4 weeks after injection or surgery The number of entries into the open arms was similar in both groups (b and e) There was no change in the number of entries into the closed arms in both groups (c and f) Each column represents the mean±SEM of 6–8 mice **p<0.01: saline 4 weeks group vs CFA 4 weeks group #p<0.05: sham 4 weeks group vs ligation 4 weeks group Effect of the selective μ-opioid receptor agonist DAMGO (a) and the δ-opioid receptor agonist SNC80 (b) on [35S]GTPγS binding to membranes of the amygdala obtained from sham-operated or sciatic nerve-ligated mice at 1 week after surgery Membranes were incubated with [35S]GTPγS (50 pM) and GDP (30 μM) with or without each agonist The data are shown as the percentage of that in the presence of GDP and the absence of each agonist Anxiogenic effects of the selective μ-opioid receptor antagonist CTOP (a) the selective δ-opioid receptor antagonist naltrindole (b) and dynorphin A (c) in mice The time spent in the lit compartment was significantly decreased at 30 min after intracerebroventricular injection using the light–dark test Each column represents the mean±SEM of 5–11 mice #p<0.05 and ##p<0.01: vs saline treatment group Role of the rat amygdala in the expression of anxiogenic effects induced by the selective μ-opioid receptor antagonist CTOP (a) the selective δ-opioid receptor antagonist naltrindole (b) and dynorphin A (c) The time spent in the lit compartment was a significant decrease at 10 min after each microinjection using the light–dark test Each column represents the mean±SEM of 5–11 rat Recovery from the sciatic nerve ligation-induced-anxiogenic effect at 8 weeks after surgery. (a) The latency of paw withdrawal after exposure to a thermal stimulus was not changed at 8 weeks after surgery. (b) The time spent in the lit compartment was no change at 8 weeks after the surgery using the light–dark test. Each column represents the mean±SEM of eight mice. Effects of the selective μ-opioid receptor agonist DAMGO (a) and the δ-opioid receptor agonist SNC80 (b) on [35S]GTPγS binding to membranes of the amygdala obtained from sham-operated or sciatic nerve-ligated mice at 8 weeks after surgery The data are shown as the percentage of the results in the presence of GDP and the absence of each agonist Effects of the selective benzodiazepine receptor agonist etizolam on anxiogenic (a–d and f–i) and hyperalgesic (e and j) effects induced by sciatic nerve ligation s.c.) produced a significant increase in the percentage of the time spent in open arms (a) and the number of entries into open arms (b) in the elevated plus-maze test (*p<0.05: vehicle vs etizolam) (c) There was no difference in the number of entries into closed arms between the vehicle- and etizolam-treated mice (d) Acute treatment wiith etizolam (1 mg/kg s.c.) caused a significant increase in time spent in the lit compartment using the light–dark test (*p<0.05: vehicle vs etizolam) the thermal threshold was significantly decreased (*** ###p<0.001: pretest vs prevehicle and pre-etizolam After confirming the predrug threshold at 7 days after nerve ligation mice were treated with a single injection of vehicle or etizolam (1 mg/kg s.c.) and the thermal threshold was measured 30 min after the injection (postdrug) The decreased thermal threshold on the ipsilateral side in nerve-ligated mice was not affected by a single injection of etizolam (1 mg/kg (f–i) Neuropathic pain induced the anxiogenic effect in the elevated plus-maze (f–h) and the light–dark (i) tests Groups of mice were operated with or without sciatic nerve ligation and each groups was divided into two groups: chronic vehicle treatment and chronic etizolam treatment Mice were repeatedly injected with vehicle or etizolam (1 mg/kg s.c.) once a day for 3 weeks (from day 7 to day 28) after nerve ligation The assay was performed one day after last injection The decreased percentage of time spent in the open arms was completely recovered by repeatedly s.c treatment with etizolam (f) (fffp<0.001: vs sham-vehicle) There was no change in the number of entries into the open (g) and closed (h) arms in each group The decreased time spent in the lit compartment was significantly recovered by repeatedly s.c treatment with etizolam (i) (fffp<0.001: vs sham-vehicle) ###p<0.001: vehicle 0 week vs vehicle 2 and 4 weeks ###p<0.001: etizolam 0 week vs etizolam 2 and 4 weeks there was no difference in the decreased thermal threshold on the ipsilateral sides between vehicle- and etizolam-treated mice following nerve ligation The relationship between depression and pain is well known Some functional changes in the emotionality-related brain regions must occur in association with chronic pain little is known about the molecular mechanism that underlies pain-induced anxiety The present study was undertaken to investigate whether chronic pain could induce an anxiogenic effect in mice Thermal hyperalgesia and tactile allodynia induced by either CFA injection or sciatic nerve ligation lasted for at least 4 weeks after injection or surgery we found that either an inflammatory or neuropathic pain-like state led to an anxiogenic effect at 4 weeks after CFA injection or surgery thermal hyperalgesia induced by sciatic nerve ligation was reversed at 8 weeks after surgery These findings suggest that pain and anxiety are closely connected At 1 week after the sciatic nerve ligation there were no significant changes in G-protein activation through μ- and δ-opioid receptors in membranes of the amygdala These findings raise the possibility that chronic pain-induced changes in opioidergic function in the amygdala may lead to the anxiogenic effect These data support the idea that the dysfunction of μ- δ-opioid receptors (and the possible enhancement of κ-opioid receptor function) in the amygdala may play a role in the mood altering effect of chronic pain These data support the hypothesis that sustained pain induces a regionally selective release of endogenous opioids interacting with μ-opioid receptors in the amygdala resulting in the internalization and recycling of μ-opioid receptors these findings suggest that a state of pain may cause physiological changes in opioid transmission at supraspinal levels there may be differences in the mechanisms between the neuropathic and inflammatory pain-like states this phenomenon could explain why the κ-opioid receptor function in the amygdala was changed by inflammation Conceptual frameworks for the presumed interaction between chronic pain and anxiety Negative emotions such as anxiety can result from the experience of chronic pain When the anxiolytic drug is treated under a chronic pain-like state it is likely that chronic pain is not always suppressed by the anxiolytic drug anxiety could increase somatic sensitivity which may be associated with changes in opioidergic function in the amygdala mCPP-induced anxiety in the light–dark box in rats—a new method for screening anxiolytic activity Nonpeptidic δ-opioid receptor agonists reduce immobility in the forced swim assay in rats Prolonged morphine treatment targets delta opioid receptors to neuronal plasma membranes and enhances delta-mediated antinociception Molecular cloning and functional expression of a mu-opioid receptor from rat brain Neurotransmission in the rat amygdala related to fear and anxiety Use of the elevated plus maze in the search for novel anxiolytic agents Involvement of mu-opioid receptors in the modulation of pituitary–adrenal axis in normal and stressed rats Clinical aspects of depression in chronic pain patients Cloning of a delta opioid receptor by functional expression Mice deficient for δ- and μ-opioid receptors exhibit opposing alterations of emotional responses Primary structures and expression from cDNAs of rat opioid receptor delta- and mu-subtypes The reliability of depression diagnosis in chronic low back pain Pharmacological effects produced by intracerebral injections of drugs in the conscious mouse Behavioral changes induced by stressful situations: effects of enkephalins The delta-opioid receptor: isolation of a cDNA by expression cloning and pharmacological characterization Effects of mu-opioid receptor stimulation in the hypothalamic paraventricular nucleus on basal and stress-induced catecholamine secretion and cardiovascular responses A lateralized deficit in morphine antinociception after unilateral inactivation of the central amygdala Opioid-receptor mRNA expression in the rat CNS: anatomical and functional implications Cloning and pharmacological characterization of a rat kappa opioid receptor Decreased experimental anxiety and voluntary ethanol consumption in rats following central but not basolateral amygdala lesions Direct evidence for the involvement of the mesolimbic kappa-opioid system in the morphine-induced rewarding effect under an inflammatory pain-like state Up-regulation of the TrkB receptor in mice injured by the partial ligation of the sciatic nerve Involvement of spinal protein kinase C in thermal hyperalgesia evoked by partial sciatic nerve ligation Suppression of the morphine-induced rewarding effect and G-protein activation in the lower midbrain following nerve injury in the mouse: involvement of G-protein-coupled receptor kinase 2 Suppression of the morphine-induced rewarding effect in the rat with neuropathic pain: implication of the reduction in μ-opioid receptor functions in the ventral tegmental area Psychotomimesis mediated by kappa opiate receptors The alpha(2a)-adrenergic receptor plays a protective role in mouse behavioral models of depression and anxiety Role of the kappa-opioid system in the attenuation of the morphine-induced place preference under chronic pain The relationship between pain and depression Mu opiate receptor: cDNA cloning and expression Direct evidence for the involvement of brain-derived neurotrophic factor in the development of neuropathic pain-like state in mice Cloning and functional comparison of kappa and delta opioid receptors from mouse brain Regional mu opioid receptor regulation of sensory and affective dimensions of pain Download references This work was supported by a Research Grant from the Ministry of Education We thank Ms Saori Kitaya for her technical assistance Toyama Medical and Pharmaceutical University Download citation DOI: https://doi.org/10.1038/sj.npp.1300858