Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1186/s13041-022-00901-2.

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.physbeh.2021.113623

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrep.2021.101152

Language：English   Publishing type：Research paper (scientific journal)  

Wasting marmoset syndrome (WMS) is a serious disease in captive common marmoset (Callithrix jacchus) colonies. Because of the high mortality rates, elucidation of the underlying mechanisms is essential. In this study, we compared the histopathology, the number of each epithelial cell in the jejunum and colon, and the expression patterns of some molecular markers between healthy and WMS-affected marmosets. Atrophy of villi in the jejunum and mononuclear cell infiltration in the lamina propria were observed in the intestinal tract of WMS-affected marmosets. Although the numbers of transient amplifying cells and tuft cells were increased, the number of goblet cells was obviously decreased in the jejunum and colon of WMS-affected marmosets compared to healthy marmosets. In addition, the number of enterocytes in the jejunum was decreased in WMS animals. There was no apparent difference in the numbers of stem cells, enteroendocrine cells, or Paneth cells. The expression of β-catenin and Tcf7l2 was increased in WMS, and the co-existence of β-catenin and Tcf7l2/Cyclin D1 was observed around the crypts in WMS-affected marmosets. These findings suggest that cell proliferation continues, but cell differentiation is halted in the intestinal tract due to the enhanced β-catenin/Tcf7l2/Cyclin D1signaling pathway in WMS, which results in malfunction of the villus and mucosa.

DOI： 10.1292/jvms.20-0532

Language：English   Publishing type：Research paper (scientific journal)  

The tooth is mainly composed of dentin and enamel. Identification of dentin-producing odontoblasts and enamel-producing ameloblasts using reporter techniques is useful to study tooth development and regeneration with tissue engineering. Ameloblasts express Amelogenin, Ameloblastin, Enamelin, and Amelotin, whereas odontoblasts express Dentin sialophosphoprotein (Dspp) and Dentin matrix protein1 (Dmp1). Although there are several transgenic lines using promoter elements or bacterial artificial chromosomes (BACs) to label odontoblasts and ameloblasts, there is a possibility that the expression patterns vary from the endogenous genes. Here, we established 2 lines of mice where tdTomato was knocked into the second exon of X-chromosomal Amelogenin (Amelx), and green fluorescent protein (GFP) was knocked into the second exon of Dspp. tdTomato and GFP were highly expressed on secretory ameloblasts and secretory and fully differentiated odontoblasts, respectively. In addition, DSPP and AMELX were not produced in the dentin matrix and enamel matrix of DsppGFP/GFP and AmelxtdTomato male mice (as representative of AmelxtdTomato/Y hemizygous male mice), respectively. Moreover, micro-computed tomography analysis of AmelxtdTomato male mice revealed a notable reduction in enamel volume but increased dentin mineral density. DsppGFP/GFP mice had reduced dentin mineral density. To identify odontoblasts and ameloblasts from developing tooth, we examined the expression of mesenchymal cell surface molecules CD90, CD166 and epithelial cell surface molecules CD49f, Epcam1 with fluorescence on odontoblasts and ameloblasts in these mice. We found that GFP+ odontoblasts and tdTomato+ ameloblasts in tooth germ from 0.5-d-old DsppGFP/+ mice and AmelxtdTomato male mice were enriched in CD45-/Ter119-/Epcam1-/CD90+/Integrin α4+cell fractions and CD45-/Ter119-/Epcam1+/CD49f+/CD147+ cell fractions, respectively. By using antibodies against mesenchymal and epithelial cell surface molecules and fluorescence, we can easily distinguish odontoblasts from ameloblasts and isolate each cell for further studies. These mice would serve as useful models for tooth development and regeneration as well as provide concurrent observation for the differentiation processes of odontoblasts and ameloblasts in vivo and in vitro.

DOI： 10.1177/0022034520974576

Language：English   Publishing type：Research paper (scientific journal)  

Macrophages are innate immune cells that contribute to classical immune functions and tissue homeostasis. Ubiquitin-specific protease 2 (USP2) controls cytokine production in macrophages, but its organ-specific roles are still unknown. In this study, we generated myeloid-selective Usp2 knockout (msUsp2KO) mice and specifically explored the roles of testicular macrophage-derived USP2 in reproduction. The msUsp2KO mice exhibited normal macrophage characteristics in various tissues. In the testis, macrophage Usp2 deficiency negligibly affected testicular macrophage subpopulations, spermatogenesis, and testicular organogenesis. However, frozen-thawed sperm derived from msUsp2KO mice exhibited reduced motility, capacitation, and hyperactivation. In addition, macrophage Usp2 ablation led to a decrease in the sperm population exhibiting high intracellular pH, calcium influx, and mitochondrial membrane potential. Interrupted pronuclei formation in eggs was observed when using frozen-thawed sperm from msUsp2KO mice for in vitro fertilization. Administration of granulocyte macrophage-colony stimulating factor (GM-CSF), whose expression was decreased in testicular macrophages derived from msUsp2KO mice, restored mitochondrial membrane potential and total sperm motility. Our observations demonstrate a distinct role of the deubiquitinating enzyme in organ-specific macrophages that directly affect sperm function.

DOI： 10.1007/s00018-020-03683-9

Language：English   Publishing type：Research paper (scientific journal)  

Sirtuin 1 (SIRT1), is a nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase and a candidate gene for depression. Nicotinamide (NAM), a form of vitamin B3, is reported as a potential inhibitor of SIRT1. Our previous study found that the 24-h-restraint stress could induce long-term depressive-like phenotypes in mice. These mice displayed increased SIRT1 activity. Here, we studied whether NAM was capable of attenuating depressive behaviors through inhibiting SIRT1 activity. Surprisingly, the application of NAM significantly reversed the depressive behaviors but increased SIRT1 activity further. In contrast, the level of adenosine triphosphate (ATP) was reduced in the restraint model for depression, and recovered by the administration of NAM. Furthermore, the Sirt1flox/flox; Nestin-Cre mice exhibited antidepressant behaviors and increased ATP levels. These data suggest that ATP plays an important role in depression pathogenesis, and NAM could be a potential treatment method for depression by regulating ATP independent of SIRT1 activity.

DOI： 10.1186/s13041-020-00703-4

Language：English   Publishing type：Research paper (scientific journal)  

Desbuquois dysplasia (DD) type 1 is a rare skeletal dysplasia characterized by a short stature, round face, progressive scoliosis, and joint laxity. The causative gene has been identified as calcium-activated nucleotidase 1 (CANT1), which encodes a nucleotidase that preferentially hydrolyzes UDP to UMP and phosphate. In this study, we generated Cant1 KO mice using CRISPR/Cas9-mediated genome editing. All F0 mice possessing frameshift deletions at both Cant1 alleles exhibited a dwarf phenotype. Germline transmission of the edited allele was confirmed in an F0 heterozygous mouse, and KO mice were generated by crossing of the heterozygous breeding pairs. Cant1 KO mice exhibited skeletal defects, including short stature, thoracic kyphosis, and delta phalanx, all of which are observed in DD type 1 patients. The glycosaminoglycan (GAG) content and extracellular matrix space were reduced in the growth plate cartilage of mutants, and proliferating chondrocytes lost their typical flat shape and became round. Chondrocyte differentiation, especially terminal differentiation to hypertrophic chondrocytes, was impaired in Cant1 KO mice. These findings indicate that CANT1 is involved in the synthesis of GAG and regulation of chondrocyte differentiation in the cartilage and contribute to a better understanding of the pathogenesis of DD type 1.

DOI： 10.1002/2211-5463.12859

Language：English   Publishing type：Research paper (scientific journal)  

Treatment of epilepsy remains difficult because patients suffer from pharmacoresistant forms of the disease and drug side-effects. Thus, there is an urgent need to identify not only new antiepileptic drug candidates but also novel epileptic animal models. Here, we characterize seizures induced with kainic acid (KA) in the common marmoset (Callithrix jacchus). Adult marmosets received 0.1, 1, or 10 mg/kg of KA subcutaneously. All animals exhibited early convulsive behavior (seizure scores of I and II on the Racine scale). Seizure scores were low at lower KA doses, but the highest dose of KA tested triggered generalized seizures (scores IV and V on the Racine scale). We next performed preliminary evaluation of the efficacy of the antiepileptic drug diazepam. This drug at 1 mg/kg (delivered subcutaneously) prevented 10 mg/kg KA-induced stage V seizures. KA administration to marmosets reliably triggers generalized seizures; therefore, the marmoset is a useful animal model in which to analyze the seizures of a nonhuman primate brain and to develop new treatments for epilepsy.

DOI： 10.1016/j.bbrc.2020.02.121

Language：English   Publishing type：Research paper (scientific journal)  

The most widely used animal models to develop sleep-disorder drugs are rodents, particularly rats and mice. However, unlike humans, these rodents are nocturnal. Thus, diurnal non-human primates represent a valuable and more translational animal model to study sleep. Although sleep-disorder drugs have been screened in non-human primates, the use of a telemetry system is not a desirable method for a rapid drug efficacy assessment system because of the need for expensive equipment, complicated surgery, and the long time before results can be obtained from analysis by inspection. Locomotor activity has traditionally been used as an indicator of the effects of drugs, genes, and disease models. The Nano-Tag, a new device for analyzing activity after an easy implantation surgery, measures locomotor activity without expensive equipment and the need for inspection for data processing, and the overall cost is much lower than that of a telemetry system. In this study, we compared the data obtained from polysomnography and on locomotor activity in telemetry transmitter-embedded cynomolgus monkeys by implanting the Nano-Tag subcutaneously in the forehead and administering sleep-disorder drugs to confirm if sleep-wake states could be measured using the Nano-Tag. When we compared the changes in awake time per unit time measured using polysomnography and locomotor activity counts per unit time measured using the Nano-Tag, cynomolgus monkeys exhibited a diurnal preference, and the correlation coefficients were positive during the 24-h period. Additionally, the correlation coefficients during the 12-h dark period were positive when the hypersomnia treatment drug modafinil was administered. The correlation coefficients during the 12-h light period were also positive when the insomnia treatment drug triazolam was administered. These results suggest that measuring locomotor activity is an effective tool for identifying sleep-wake states and screening sleep-disorder drugs at low cost and with less burden to animal subjects.

DOI： 10.1016/j.heliyon.2020.e03524

Language：English   Publishing type：Research paper (scientific journal)  

Abnormal accumulation of TAR DNA-binding protein 43 (TDP-43), a DNA/RNA binding protein, is a pathological signature of amyotrophic lateral sclerosis (ALS). Missense mutations in the TARDBP gene are also found in inherited and sporadic ALS, indicating that dysfunction in TDP-43 is causative for ALS. To model TDP-43-linked ALS in rodents, we generated TDP-43 knock-in mice with inherited ALS patient-derived TDP-43M337V mutation. Homozygous TDP-43M337V mice developed normally without exhibiting detectable motor dysfunction and neurodegeneration. However, splicing of mRNAs regulated by TDP-43 was deregulated in the spinal cords of TDP-43M337V mice. Together with the recently reported TDP-43 knock-in mice with ALS-linked mutations, our finding indicates that ALS patient-derived mutations in the TARDBP gene at a carboxyl-terminal domain of TDP-43 may cause a gain of splicing function by TDP-43, however, were insufficient to induce robust neurodegeneration in mice.

DOI： 10.1186/s13041-020-0550-4

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/asj.13366

Language：English   Publishing type：Research paper (scientific journal)  

Germ-free (GF) mice are useful models for the examination of host-microbe interactions in health and disease. We recently reported on the maintenance of individual GF mice for more than 1 year in a sealed positive-pressure cage. However, no useful system exists to automatically record basic behavioral patterns, such as activity and the intake of water and food, under GF status. In this study, we examined basic behavior by combining the sealed positive-pressure cage with a behavioral monitoring system and observed the gross morphology of GF mice at 4 weeks and 8 months of age. GF mice exhibited cecal enlargement and had lower body and adipose tissue weights compared with age-matched specific pathogen-free (SPF) mice. Although both strains had similar circadian rhythms, GF mice exhibited decreased activity compared with age-matched SPF mice. GF mice also exhibited increased levels of water intake compared with age-matched SPF mice. Although GF mice demonstrated decreased food intake levels at the age of 4 weeks, they exhibited increased food intake levels compared with age-matched SPF mice at the age of 8 months. The present research indicates that automated measurement systems that record the basic behaviors of GF mice for long periods are useful for the acceleration of the study of metabolic functions and host-microbe interactions.

DOI： 10.1016/j.heliyon.2019.e02176

Language：English   Publishing type：Research paper (scientific journal)  

Although wasting marmoset syndrome (WMS) is one of the biggest problems facing captive marmoset colonies, the mechanisms underlying its pathogenesis remain unclear. In our clinical experience, it is difficult to cure WMS-affected marmosets with severe hypoalbuminemia. Thus, the mechanisms underlying hypoalbuminemia in WMS must be understood. In the present study, we investigated whether intestinal protein loss, a known reason for hypoalbuminemia, occurs in this disease. Fecal α1-proteinase inhibitor (α1-PI, also known as α1-antitrypsin) has been used to diagnose intestinal protein loss in other species. To develop an assay system for this protein, marmoset α1-PI was purified from plasma and antibodies against it were developed using the purified protein. Using the antibodies, a sandwich enzyme-linked immunosorbent assay (ELISA) to measure marmoset α1-PI was developed, and its detection sensitivity for fecal samples was ∼20-fold higher than that of a commercial kit for human α1-PI. From this ELISA, the reference intervals for serum and feces of healthy marmosets were 0.87-1.85 mg/ml and 0.53-395.58 μg/g, respectively. The average concentrations of α1-PI in serum and feces of seven WMS-affected marmosets were 1.17 mg/ml and 1357.58 μg/g, respectively. Although there were no significant differences in the serum concentrations between healthy and WMS-affected marmosets, the fecal concentrations were significantly higher in WMS-affected marmosets than in healthy individuals, suggesting that intestinal protein loss occurs in WMS. Intestinal protein loss of WMS-affected marmosets was significantly attenuated with treatment, suggesting that it is one of the mechanisms involved in the hypoalbuminemia observed in WMS.

DOI： 10.1042/BSR20190562

Language：Others  

Language：English   Publishing type：Research paper (scientific journal)  

Nanomaterial morphology is important for the targeted delivery of drugs to tissues as well as subsequent cellular uptake. Hollow nanotubes composed of peptides, with a diameter of 80 nm and various lengths (100, 200, 300, 600 nm), were successfully capped and sealed with a peptide hemisphere to encapsulate the anticancer drug, cisplatin. The torpedo-shaped nanocapsules with an aspect ratio (length/diameter) of 2.4 showed more rapid cellular uptake and accumulation at the tumor site compared with spherical analogues. Successful delivery of cisplatin to tumors was achieved in a mouse model and tumor growth was efficiently suppressed.

DOI： 10.1021/acsnano.8b06189

Language：English   Publishing type：Research paper (scientific journal)  

Chronic diarrhea in laboratory-bred marmosets poses a serious health problem during experiments. Despite a growing demand for laboratory-bred experimental marmosets, the mechanisms underlying the development of diarrhea and measures for its treatment and prevention remain unclear. To explore the factors affecting development of chronic diarrhea in laboratory-bred marmosets, the gut microbiota composition (GMC) of 58 laboratory-bred marmosets, including 19 animals with chronic diarrhea, was analyzed using terminal restriction fragment length polymorphism. We found that the GMCs in these animals cluster into two groups that differ significantly in rate of chronic diarrhea (56.5% in one group, Cluster 1, and 17.1% in Cluster 2). Additionally, a higher α-diversity and a lower proportion of Bifidobacterium spp. according to quantitative PCR was found the animals in the Cluster 1 than in those in Cluster 2. Taken together, our findings indicate that there is a relationship between GMC and development of chronic diarrhea in laboratory-bred marmosets. This is the first study to highlight the potential of assessing GMC in relation to development of chronic diarrhea in laboratory-bred marmosets.

DOI： 10.1111/1348-0421.12655

Language：Others  

Language：English   Publishing type：Research paper (scientific journal)  

The scavenger receptor CD163 is widely used as a cell signature for alternatively active "M2" macrophages in mammals. In this study, we generated two monoclonal antibodies, ABM-1A9 and ABM-2D6, against the extracellular region of bovine CD163. Conventional Western blotting using the antibodies yielded immunoreactive bands of bovine CD163 at 130 and 150 kDa in non-reduced and reduced spleen lysates, respectively. The minimum limit of detectable concentration of both antibodies was relatively lower (5.0 ng/mL) than that of the anti-human CD163 monoclonal antibody AM-3 K (>1.0 μg/mL), which has been used previously for the detection of bovine CD163. An immunohistochemical study using formalin-fixed paraffin-embedded sections revealed that ABM-1A9 and ABM-2D6 clearly stained some Iba1+ macrophages in the lymph nodes of cattle with mastitis. Moreover, the CD163-stained macrophages were frequently observed engulfing leukocytes. ELISA using ABM-2D6 distinguished levels of circulating soluble CD163 in healthy cattle (less than 16.9 pmol/mL) and cattle with mastitis (more than 33.7 pmol/mL). These new monoclonal antibodies can be used in the diagnosis and evaluation of inflammatory disease prognosis in cattle with immunohistological analyses and blood test applications.

DOI： 10.1016/j.vetimm.2018.02.004

Language：English   Publishing type：Research paper (scientific journal)  

The provision of adequate space for laboratory animals is essential not only for good welfare but accurate studies. For example, housing conditions for primates used in biomedical research may negatively affect welfare and thus the reliability of findings. In common marmosets (Callithrix jacchus), an appropriate cage size enables a socially harmonious family environment and optimizes reproductive potential. In this study, we investigated the effects of cage size on body weight (BW), behavior, and nursing succession in the common marmoset. Large cages (LCs) with environment enrichment led to an increase in BW while small cages (SCs) caused stereotypic behaviors that were not observed in LCs. In addition, the BW of infants increased with aging in LCs. Our findings indicate that the welfare of marmosets was enhanced by living in LCs. Research on non-human primates is essential for understanding the human brain and developing knowledge-based strategies for the diagnosis and treatment of psychiatric and neurological disorders. Thus, the present findings are important because they indicate that different cages may influence emotional and behavioral phenotypes.

DOI： 10.1538/expanim.17-0058

Language：English   Publishing type：Research paper (scientific journal)  

Epigenomic abnormalities caused by genetic mutation in epigenetic regulators can result in neurodevelopmental disorders, deficiency in neural plasticity and mental retardation. As a histone demethylase, plant homeodomain finger protein 8 (Phf8) is a candidate gene for syndromal and non-specific forms of X-chromosome-linked intellectual disability (XLID). Here we report that Phf8 knockout mice displayed impaired learning and memory, and impaired hippocampal long-term potentiation (LTP) without gross morphological defects. We also show that mTOR signaling pathway is hyperactive in hippocampus in Phf8 knockout mouse. Mechanistically, we show that demethylation of H4K20me1 by Phf8 results in transcriptional suppression of RSK1 and homeostasis of mTOR signaling. Pharmacological suppression of mTOR signaling with rapamycin in Phf8 knockout mice recovers the weakened LTP and cognitive deficits. Together, our results indicate that loss of Phf8 in animals causes deficient learning and memory by epigenetic disruption of mTOR signaling, and provides a potential therapeutic drug target to treat XLID.

DOI： 10.1038/s41467-017-02531-y

Language：English  

[This corrects the article DOI: 10.1371/journal.pgen.1006940.].

DOI： 10.1371/journal.pgen.1007035

Language：Others   Publishing type：Research paper (scientific journal)  

© The Author(s) 2017. The risk of schizophrenia is increased in offspring whose mothers experience malnutrition during pregnancy. Polyunsaturated fatty acids (PUFAs) are dietary components that are crucial for the structural and functional integrity of neural cells, and PUFA deficiency has been shown to be a risk factor for schizophrenia. Here, we show that gestational and early postnatal dietary deprivation of two PUFAs - arachidonic acid (AA) and docosahexaenoic acid (DHA) - elicited schizophrenia-like phenotypes in mouse offspring at adulthood. In the PUFA-deprived mouse group, we observed lower motivation and higher sensitivity to a hallucinogenic drug resembling the prodromal symptoms in schizophrenia. Furthermore, a working-memory task-evoked hyper-neuronal activity in the medial prefrontal cortex was also observed, along with the downregulation of genes in the prefrontal cortex involved in oligodendrocyte integrity and the gamma-aminobutyric acid (GABA)-ergic system. Regulation of these genes was mediated by the nuclear receptor genes Rxr and Ppar, whose promoters were hyper-methylated by the deprivation of dietary AA and DHA. In addition, the RXR agonist bexarotene upregulated oligodendrocyte- and GABA-related gene expression and suppressed the sensitivity of mice to the hallucinogenic drug. Notably, the expression of these nuclear receptor genes were also downregulated in hair-follicle cells from schizophrenia patients. These results suggest that PUFA deficiency during the early neurodevelopmental period in mice could model the prodromal state of schizophrenia through changes in the epigenetic regulation of nuclear receptor genes.

DOI： 10.1038/tp.2017.182

Language：English   Publishing type：Research paper (scientific journal)  

We investigated the regulatory roles of USP2 in mRNA accumulation of proinflammatory cytokines in macrophage-like cells after stimulation with a toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS). Human macrophage-like HL-60 cells, mouse macrophage-like J774.1 cells, and mouse peritoneal macrophages demonstrated negative feedback to USP2 mRNA levels after LPS stimulation, suggesting that USP2 plays a significant role in LPS-stimulated macrophages. USP2 knockdown (KD) by short hairpin RNA in HL-60 cells promoted the accumulation of transcripts for 25 of 104 cytokines after LPS stimulation. In contrast, limited induction of cytokines was observed in cells forcibly expressing the longer splice variant of USP2 (USP2A), or in peritoneal macrophages isolated from Usp2a transgenic mice. An ubiquitin isopeptidase-deficient USP2A mutant failed to suppress LPS-induced cytokine expression, suggesting that protein ubiquitination contributes to USP2-mediated cytokine repression. Although USP2 deficiency did not accelerate TNF receptor-associated factor (TRAF) 6-nuclear factor-κB (NF-κB) signaling, it increased the DNA binding ratio of the octamer binding transcription factor (Oct)-1 to Oct-2 in TNF, CXCL8, CCL4, and IL6 promoters. USP2 decreased nuclear Oct-2 protein levels in addition to decreasing the polyubiquitination of Oct-1. In summary, USP2 modulates proinflammatory cytokine induction, possibly through modification of Oct proteins, in macrophages following TLR4 activation.

DOI： 10.1155/2017/6909415

Language：English   Publishing type：Research paper (scientific journal)  

Genetic mutations contribute to the etiology of autism spectrum disorder (ASD), a common, heterogeneous neurodevelopmental disorder characterized by impairments in social interaction, communication, and repetitive and restricted patterns of behavior. Since neuroligin3 (NLGN3), a cell adhesion molecule at the neuronal synapse, was first identified as a risk gene for ASD, several additional variants in NLGN3 and NLGN4 were found in ASD patients. Moreover, synaptopathies are now known to cause several neuropsychiatric disorders including ASD. In humans, NLGNs consist of five family members, and neuroligin1 (NLGN1) is a major component forming a complex on excitatory glutamatergic synapses. However, the significance of NLGN1 in neuropsychiatric disorders remains unknown. Here, we systematically examine five missense variants of NLGN1 that were detected in ASD patients, and show molecular and cellular alterations caused by these variants. We show that a novel NLGN1 Pro89Leu (P89L) missense variant found in two ASD siblings leads to changes in cellular localization, protein degradation, and to the impairment of spine formation. Furthermore, we generated the knock-in P89L mice, and we show that the P89L heterozygote mice display abnormal social behavior, a core feature of ASD. These results, for the first time, implicate rare variants in NLGN1 as functionally significant and support that the NLGN synaptic pathway is of importance in the etiology of neuropsychiatric disorders.

DOI： 10.1371/journal.pgen.1006940

Language：English   Publishing type：Research paper (scientific journal)  

We previously reported that ubiquitin-specific protease (USP) 2 in macrophages down-regulates genes associated with metabolic diseases, suggesting a putative anti-diabetic role for USP2 in macrophages. In this study, we evaluate this role at both cellular and individual levels. Isolated macrophages forcibly expressing Usp2a, a longer splicing variant of USP2, failed to modulate the insulin sensitivity of 3T3-L1 adipocytes. Similarly, macrophage-selective overexpression of Usp2a in mice (Usp2a transgenic mice) had a negligible effect on insulin sensitivity relative to wild type littermates following a three-month high-fat diet. However, Usp2a transgenic mice exhibited fewer M1 macrophages in their mesenteric adipose tissue. Following a six-month high-fat diet, Usp2a transgenic mice exhibited a retarded progression of insulin resistance in their skeletal muscle and liver, and an improvement in insulin sensitivity at an individual level. Although conditioned media from Usp2a-overexpressing macrophages did not directly affect the insulin sensitivity of C2C12 myotubes compared to media from control macrophages, they did increase the insulin sensitivity of C2C12 cells after subsequent conditioning with 3T3-L1 cells. These results indicate that macrophage USP2A hampers obesity-elicited insulin resistance via an adipocyte-dependent mechanism.

DOI： 10.1016/j.bbrep.2017.01.009

Language：English   Publishing type：Research paper (scientific journal)  

Voltage-gated Ca2+ channels (VGCCs) are comprised of α1, α2/δ, β, and γ subunits. The pore-forming α1 subunit is essential for the proper functioning of Ca2+ channels, while the α2/δ subunit interacts with components of the extracellular matrix. The α2/δ subunit is related in many neuropathological symptoms, including epilepsy and cerebellar ataxia. We previously reported that the mutant Cav.2.1α1 subunit has protective effects following brain injury. The present study aimed to investigate the effects of the α2/δ subunit inhibition alone and in combination with the inhibition of the Cav.2.1α1 subunit following brain injury by injecting Gabapentin using Cav.2.1α1 mutant heterozygous rolling Nagoya (rol/+) and wild-type (+/+) mice. Gabapentin binds to the α2/δ subunit and leads to Ca2+ flow disturbance. A cryogenic method was used to induce brain injury. The mice pretreated with 100mg/kg Gabapentin exhibited a decrease in lesion size, while the 40mg/kg Gabapentin injection was effective in rol/+ mice but not +/+ mice. The administration of 100mg/kg Gabapentin also attenuated reactive astrocyte activity and neuronal degeneration; the pattern of results was similar to that for lesion size. An analysis of phosphorylated p38 (pp38) expression revealed that Gabapentin suppressed the p38 mitogen-activated protein kinase (MAPK) signaling cascade by interrupting glutamate-signaling induced by the inhibition of VGCCs. The present findings demonstrated that the administration of the α2/δ subunit inhibitor, Gabapentin, had neuroprotective effects following brain injury.

DOI： 10.1016/j.brainres.2016.11.009

Language：English   Publishing type：Research paper (scientific journal)  

To understand sleep mechanisms and develop treatments for sleep disorders, investigations using animal models are essential. The sleep architecture of rodents differs from that of diurnal mammals including humans and non-human primates. Sleep studies have been conducted in non-human primates; however, these sleep assessments were performed on animals placed in a restraint chair connected via the umbilical area to the recording apparatus. To avoid restraints, cables, and other stressful apparatuses and manipulations, telemetry systems have been developed. In the present study, sleep recordings in unrestrained cynomolgus monkeys (Macaca fascicularis) and common marmoset monkeys (Callithrix jacchus) were conducted to characterize normal sleep. For the analysis of sleep-wake rhythms in cynomolgus monkeys, telemetry electroencephalography (EEG), electromyography (EMG), and electrooculography (EOG) signals were used. For the analysis of sleep-wake rhythms in marmosets, telemetry EEG and EOG signals were used. Both monkey species showed monophasic sleep patterns during the dark phase. Although non-rapid eye movement (NREM) deep sleep showed higher levels at the beginning of the dark phase in cynomolgus monkeys, NREM deep sleep rarely occurred during the dark phase in marmosets. Our results indicate that the use of telemetry in non-human primate models is useful for sleep studies, and that the different NREM deep sleep activities between cynomolgus monkeys and common marmoset monkeys are useful to examine sleep functions.

DOI： 10.1538/expanim.16-0073

Language：English   Publishing type：Research paper (scientific journal)  

Wasting marmoset syndrome (WMS) has high incidence and mortality rates and is one of the most important problems in captive common marmoset (Callithrix jacchus) colonies. Despite several reports on WMS, little information is available regarding its reliable treatment. We previously reported that marmosets with WMS had high serum levels of matrix metalloproteinase 9 (MMP9). MMP9 is thought to be a key enzyme in the pathogenesis of inflammatory bowel disease, the main disease state of WMS, and is activated by plasmin, a fibrinolytic factor. In a previous study, treating mice with an antibody to inhibit plasmin prevented the progression of inflammatory bowel disease. Here we examined the efficacy of tranexamic acid, a commonly used plasmin inhibitor, for the treatment of WMS, with supportive measures including amino acid and iron formulations. Six colony marmosets with WMS received tranexamic acid therapy with supportive measures for 8 wk. The body weight, Hct, and serum albumin levels of these 6 marmosets were increased and serum MMP9 levels decreased after this regimen. Therefore, tranexamic acid therapy may be a new and useful treatment for WMS.

Language：English   Publishing type：Research paper (scientific journal)  

The common marmoset is a non-human primate that has increasingly employed in the biomedical research including the fields of neuroscience and behavioral studies. Cytochrome P450 (CYP) 2D has been speculated to be involved in psycho-neurologic actions in the human brain. In the present study, to clarify the role of CYP2D in the marmoset brain, we investigated the expression patterns of CYP2D mRNA in the brain using in situ hybridization (ISH). In addition, to identify the gene location of CYP2D19, a well-studied CYP2D isoform in the common marmoset, a fluorescence in situ hybridization (FISH) study was performed. Consistent with findings for the human brain, CYP2D mRNA was localized in the neuronal cells of different brain regions; e.g., the cerebral cortex, hippocampus, substantia nigra, and cerebellum. FISH analysis showed that the CYP2D19 gene was located on chromosome 1q, which is homologous to human chromosome 22 on which the CYP2D6 gene exists. These results suggest that CYP2D in the marmoset brain may play the same role as human CYP2D6 in terms of brain actions, and that the CYP2D19 gene is conserved in a syntenic manner. Taken together, these findings suggest that the common marmoset is a useful model for studying psychiatric disorders related to CYP2D dysfunction in the brain.

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1089/thy.2015.0293

Language：English   Publishing type：Research paper (scientific journal)  

Neuronal voltage-gated Cav2.1 channel controls a broad array of functions, including neurotransmitter release, neuronal excitability, activity-dependent gene expression, and neuronal survival. The Cav2.1 channel is molecular complexes consisting of several subunits: α1, α2/δ, β, and γ. The pore-forming subunit, α1, is encoded by the Cacna1a gene. Tottering-6j mice, generated by the Neuroscience Mutagenesis Facility at The Jackson Laboratory, are a recessive mutant strain in which the mutation has been chemically induced by ethylnitrosourea. In tottering-6j mice, mutation in the Cacna1a gene results in a base substitution (C-to-A) in the consensus splice acceptor sequence, which results in deletion of a part of the S4-S5 linker, S5, and a part of S5-S6 linker domain I in the α1 subunit of Cav2.1 channel. The mice display motor dysfunctions and absence-like seizures. However, protein expression in the cerebellum of tottering-6j mice has not been investigated. Real-time quantitative reverse transcription polymerase chain reaction and histological analyses of the cerebellum of tottering-6j mice revealed high expression levels of tyrosine hydroxylase, zebrin II, and ryanodine receptor 3 compared with those of wild-type mice. Conversely, a low level of calretinin expression was found compared with wild-type mice. These results indicate that Cacna1a mutation plays a significant role in protein expression patterns and that the tottering-6j mouse is a useful model for understanding protein expression mechanisms.

DOI： 10.1538/expanim.15-0120

Language：English   Publishing type：Research paper (scientific journal)  

Use of the common marmoset (Callithrix jacchus) as a non-human primate experimental animal has increased in recent years. Although wasting marmoset syndrome (WMS) is one of the biggest problems in captive marmoset colonies, the molecular mechanisms, biochemical markers for accurate diagnosis and a reliable treatment remain unknown. In this study, as a first step to finding biochemical marker(s) for the accurate diagnosis of WMS, we conducted blood cell counts, including hematocrit, hemoglobin and platelets, and examined serum chemistry values, including albumin, calcium and levels of serum matrix metalloproteinase 9 (MMP9), using a colony of marmosets with and without weight loss. MMP9 is thought to be an enzyme responsible for the degradation of extracellular matrix components and participates in the pathogenesis of inflammatory conditions, such as human and murine inflammatory bowel disease, which, like WMS, are characterized histologically by inflammatory cell infiltrations in the intestines. The values of hematocrit and hemoglobin and levels of serum albumin and calcium in the WMS group were significantly decreased versus the control group. The platelet values and serum MMP9 concentrations were increased significantly in the WMS group compared with the control group. MMP9 could be a new and useful marker for the diagnosis of WMS in addition to hematocrit, hemoglobin, serum albumin and calcium. Our results also indicate that MMP9 could be a useful molecular candidate for treatment.

DOI： 10.1292/jvms.15-0675

Language：English   Publishing type：Research paper (scientific journal)  

We previously reported that rolling Nagoya mice carrying a mutation in the α1 subunit of the Cav2.1 channel protective from ischemia- and kainate-induced neuronal damage. However, the protective effect of this mutation and its relationship to brain injury recovery have not been examined. To examine the relationship between Cav2.1 channel function and brain injury, we induced cryogenic brain damage in homozygous rolling Nagoya (rol/rol), control wild-type (+/+), ω-agatoxin IVA-pretreated +/+ (ω-aga +/+), and ω-agatoxin IVA-post-treated +/+ (ω-aga-post-treated +/+) mice. We measured the lesion area, blood brain-barrier permeability and performed immunohistochemistry and western blot analysis. The lesions of rol/rol and ω-aga +/+ mice were significantly smaller than those observed in +/+ mice at both day 1 and day 7 after injury. Similar results were shown in blood-brain barrier permeability. We observed more reactive astrogliosis in +/+ mice than in rol/rol or ω-aga +/+ mice. rol/rol and ω-aga +/+ mice had fewer degenerating cells due to cryogenic injury than did +/+ mice at both day 1 and day 7. ω-Aga-post-treated +/+ mice 24h after injury were sacrificed on day 7. The lesions were smaller in ω-aga-post-treated +/+ mice than those in vehicle-treated +/+ mice. We also examined phosphorylated p38 (pp38) at the injured site. ω-Aga-post-treated +/+ mouse brain slices showed weak pp38 signal; vehicle-treated +/+ mouse brain slices were pp38-positive. These findings demonstrate that the mutant Cav2.1 channel exerts a protective effect against cryogenic brain injury in rolling Nagoya mice. Our results indicate that inhibitors of the Cav2.1-dependent p38 signaling cascade would be useful as therapeutic agents in the treatment of brain injury.

DOI： 10.1016/j.neuroscience.2015.11.035

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrc.2015.05.050

Language：English   Publishing type：Research paper (scientific journal)  

The biological activity of cell-derived substrates to maintain undifferentiated murine-induced pluripotent stem (iPS) cells was correlated to membrane fluidity as a new parameter of cell culture substrates. Murine embryonic fibroblasts (MEFs) were employed as feeder cells and their membrane fluidity was tuned by chemical fixation using formaldehyde (FA). Membrane fluidity was evaluated by real-time single-molecule observations of green fluorescent protein-labeled epidermal growth factor receptors on chemically fixed MEFs. Biological activity was monitored by colony formation of iPS cells. Treatment with a low concentration of FA sustained the membrane fluidity and biological activity, which were comparable to those of mitomycin C-treated MEFs. The biological activity was further confirmed by sustained expression of alkaline phosphatase, SSEA-1, and other pluripotency markers in iPS cells after 3-5 days of culture on FA-fixed MEFs. Chemical fixation of feeder cells has several advantages such as providing ready-to-use culture substrates without contamination by proliferating feeder cells. Therefore, our results provide an important basis for the development of chemically fixed culture substrates for pluripotent stem cell culture as an alternative to conventional treatment by mitomycin C or x-ray irradiation.

DOI： 10.1038/srep11386

Language：English   Publishing type：Research paper (scientific journal)  

Genetic analyses have revealed an important association between P/Q-type calcium channel activities and hereditary neurological disorders. The P/Q-type channels are composed principally of heterologous multimeric subunits including CaV2.1 and CaVβ4. Of these, the β4 subunit is thought to play a significant role in channel physiology, because a mouse line mutant in that subunit (the lethargic mouse: lh) exhibits a severe ataxic phenotype. The aim of the present study was to elucidate the physiological importance of the β4 subunit. ECG analysis showed that the T wave was high in 8-week-old lh mutants; this may be associated with hyperkalemia. Upon pharmacological ECG analysis, 2-3-week-old lh mutants exhibited reduced responses to a β-blocker and a muscarinic receptor antagonist. Analysis of heart rate variability revealed that the R-R interval was unstable in lh mutants and that both the low- and high-frequency components had increased in extent, indicating that the tonus of both the sympathetic and parasympathetic nervous systems was modified. Thus, our present study revealed that the β4 subunit played a significant role in regulation of sympathetic and parasympathetic nerve activities.

DOI： 10.1016/j.bbrc.2015.03.112

Language：English   Publishing type：Research paper (scientific journal)  

The representative DNA-labeling agent 5-ethynyl-2'-deoxyuridine (EdU) was chemically modified to improve its function. Chemical monophosphorylation was expected to enhance the efficiency of the substrate in DNA polymerization by circumventing the enzymatic monophosphorylation step that consumes energy. In addition, to enhance cell permeability, the phosphates were protected with bis-pivaloyloxymethyl that is stable in buffer and plasma, and degradable inside various cell types. The phosphorylated EdU (PEdU) was less toxic than EdU, and had the same or a slightly higher DNA-labeling ability in vitro. PEdU was also successfully applied to DNA labeling in vivo. In conclusion, PEdU can be used as a less toxic DNA-labeling agent for studies that require long-term cell survival or very sensitive cell lines.

DOI： 10.1039/c5ob00199d

Language：English   Publishing type：Research paper (scientific journal)  

The following is a report on a congenital vaginal malformation, imperforate vagina, in the common marmoset (Callithrix jacchus). This anomaly was observed for the first time in an adult female in our research colony. There was no uterine and vaginal aplasia or atresia in her grossly normal genital tract. The plasma progesterone concentration suggested that the ovarian cycle had ceased. However, this may not be related to a functional anomaly, but rather to suppressed ovulation resulting from subordination to cagemates considering the various stages of follicular development observed.

DOI： 10.1292/jvms.14-0378

Language：English   Publishing type：Research paper (scientific journal)  

The classic piebald mutation in the endothelin receptor type B (Ednrb) gene was found on rolling Nagoya genetic background (PROD-s/s) mice with white coat spotting. To examine whether genetic background influenced the phenotype in the piebald mutant mice, we generated a congenic strain (B6.PROD-s/s), produced by repeated backcrosses to the C57BL/6J (B6) strain. Although B6.PROD-s/s mice showed white coat spotting, 7% of B6.PROD-s/s mice died between 2 and 5 weeks after birth due to megacolon. The PROD-s/s, s/s and Japanese fancy mouse 1 (JF1) strains, which also have piebald mutations on different genetic backgrounds with B6, showed only pigmentation defects without megacolon. In expression analyses, rectums of B6.PROD-s/s with megacolon mice showed ~5% of the level of Ednrb gene expression versus B6 mice. In histological analyses, aganglionosis was detected in the rectum of megacolon animals. The aganglionic rectum was thought to lead to severe constipation and intestinal blockage, resulting in megacolon. We also observed an abnormal intestinal flora, including a marked increase in Bacteroidaceae and Erysipelotrichaceae and a marked decrease in Lactobacillus and Clostridiales, likely inducing endotoxin production and a failure of the mucosal barrier system, leading ultimately to death. These results indicate that the genetic background plays a key role in the development of enteric ganglion neurons, controlled by the Ednrb gene, and that B6 has modifier gene (s) regarding aganglionosis.

DOI： 10.1292/jvms.14-0408

Language：Others   Publishing type：Research paper (scientific journal)  

<p>A newer method of hiPS culture on feeder cell-derived niche is reported in this study.</p>

DOI： 10.1039/c4tb01635a

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1177/153567601401900404

Language：English   Publishing type：Research paper (scientific journal)  

Neat1 is a non-protein-coding RNA that serves as an architectural component of the nuclear bodies known as paraspeckles. Although cell-based studies indicate that Neat1 is a crucial regulator of gene expression, its physiological relevance remains unclear. Here, we find that Neat1 knockout (KO) mice stochastically fail to become pregnant despite normal ovulation. Unilateral transplantation of wild-type ovaries or the administration of progesterone partially rescued the phenotype, suggesting that corpus luteum dysfunction and concomitant low progesterone were the primary causes of the decreased fertility. In contrast to the faint expression observed in most of the adult tissues, Neat1 was highly expressed in the corpus luteum, and the formation of luteal tissue was severely impaired in nearly half of the Neat1 KO mice. These observations suggest that Neat1 is essential for the formation of the corpus luteum and for the subsequent establishment of pregnancy under a suboptimal condition that has not yet been identified.

DOI： 10.1242/dev.110544

Language：English   Publishing type：Research paper (scientific journal)  

Cav2.1α1 is involved in glutamate release. The kainate-induced intensive firing of neurons via glutamate receptors causes seizure and neuronal damage, especially in the hippocampus. Cav2.1α1 mutation in homozygous rolling Nagoya (rol/rol) mice caused reduced Ca(2+) permeability compared to wild-type mice. The rol/rol mice exhibited ataxia approximately after 2 weeks of age. Although we have reported that heterozygous rolling Nagoya (rol/+) mice show age-dependent behavioral changes, sensitivity to kainate has not been examined. To examine the relationship between Cav2.1 function and neurological disease, we investigated how Cav2.1 is related to kainate-induced seizure and neuronal damage using 2- and 18-month-old rol/+ mice. The seizure scores of 18-month-old rol/+ mice that received 20mg/kg kainate intraperitoneally were significantly lower than those of wild-type mice. As a consequence of seizure, kainate induced delayed neuronal damage along with astrocytic growth in the hippocampus in wild-type mice, with a moderate effect observed in rol/+ mice. In the hippocampus of 18-month-old rol/+ mice, the levels of mutant-type Cav2.1α1 were increased compared to +/+ mice. The phosphorylation of p38, a mitogen-activated protein kinase (MAPK) activated by kainate, was not increased after kainate injection compared to +/+ mice. No difference was observed between 2-month-old rol/+ and wild-type mice intraperitoneally injected with 20mg/kg kainate in these analyses. These findings suggest that rol/+ mice experience age-related changes in sensitivity to kainate due to changes in the p38 MAPK signaling pathway via a mutant Cav2.1 channel. Hence, rol/+ mice may represent a novel model to delineate the association between Cav2.1 function, synaptic transmission, and the postsynaptic signaling cascade.

DOI： 10.1016/j.pbb.2014.06.022

Language：English   Publishing type：Research paper (scientific journal)  

Ataxic rolling Nagoya (PROD-rol/rol) mice, which carry a mutation in the α1 subunit of the Cav2.1 channel (Cacna1a) gene, were discovered in 1969. They show white spots on agouti coat and have a mutation in the piebald spotting (s) locus. However, mutation analysis of the s locus encoding the endothelin receptor type B (Ednrb) gene in PROD-rol/rol mice had not been performed. Here, we examined the genomic and mRNA sequences of the Ednrb gene in PROD-rol/rol and wild-type rolling Nagoya (PROD-s/s) and studied the expression patterns of Ednrb and Cacna1a genes in these mice in comparison with C57BL/6J mice. Polymerase chain reaction analyses revealed two silent nucleotide substitutions in the coding region and insertion of a retroposon-like element in intron 1 of the Ednrb gene. Expression analyses demonstrated similar localizations and levels of Ednrb and Cacna1a expression in the colon between PROD-rol/rol and PROD-s/s mice, but the expression levels of both genes were diminished compared with C57BL/6J mice. Microsatellite genotyping showed that at least particular regions of chromosome 14 proximal to the Ednrb locus of the PROD strain were derived from Japanese fancy piebald mice. These results indicated that PROD-rol/rol mice have two mutant genes, Ednrb and Cacna1a. As no PROD strain had an intact Ednrb gene, using congenic rolling mice would better serve to examine rolling Nagoya-type Cav2.1 channel dysfunctions.

Language：English   Publishing type：Research paper (scientific journal)  

The senescence-accelerated mouse (SAM) is used as an animal model of senescence acceleration and age-associated disorders. SAM is derived from unexpected crosses between the AKR/J and unknown mouse strains. There are nine senescence-prone (SAMP) strains and three senescence-resistant (SAMR) strains. Although SAMP strains exhibit strain-specific and age-related pathological changes, the genes responsible for the pathologic changes in SAMP strains have not been comprehensively identified. In the present study, we evaluated sweet taste perception using the two-bottle test. We compared genotypes of the taste related gene, Tas1r3, using SAM strains and the parental AKR/J strain. The two-bottle test revealed that SAMR1 (R1), SAMP6 (P6), SAMP8 (P8), and SAMP10 (P10) mice were saccharin-preferring strains, whereas AKR/J did not prefer saccharin. All genotypes of the R1, P6, P8, and P10 strains at the polymorphic sites in Tas1r3, which is known to influence saccharin preference, were identical to those of C57BL6/J, a well-known saccharin-preferring strain, and were completely different from those of the parental AKR/J strain. These genetic alterations in SAM strains appear to arise from an unknown strain that is thought to have been crossed with AKR/J initially.

DOI： 10.1016/j.physbeh.2014.04.005

Language：English   Publishing type：Research paper (scientific journal)  

Although fear extinction requires N-methyl-d-aspartate (NMDA) receptor signaling, Cav2.1-regulated synaptic function in extinction remains unknown. This study examined whether Cav2.1-mediated signaling plays role in consolidation of extinction. Wild-type mice received intracerebroventricular injection of Cav2.1 blocker (ω-agatoxin IVA, 4.0 pg/side) showed impaired extinction behavior and increased expression of CREB-dependent gene Arc in medial prefrontal cortex (mPFC). Intra-mPFC injections of NMDA receptor antagonist (MK-801, 0.5 μg/midline), which was ineffective in wild-type controls, blocked extinction in heterozygous rolling Nagoya (rol/+) mice carrying Cav2.1α1 gene mutation rol/+ mice. These results indicate that Cav2.1-mediated NMDA receptor signaling is functional pathway in mPFC-dependent fear extinction. Our results also indicate that the combination of pharmacological and genetic approaches can be used to study functional signaling pathways in neuronal circuits.

DOI： 10.1016/j.bbr.2013.10.033

Language：English   Publishing type：Research paper (scientific journal)  

The common marmoset (Callithrix jacchus) is used as a non-human primate laboratory animal. Marmoset wasting syndrome (MWS) is a disease endemic to captive colonies, and the pathogenesis is unclear. In the present study, marmosets with chronic bloody high-viscosity diarrhea, which is a contributing factor to MWS, were evaluated, and inflammation in the colon was found. Calprotectin is a surrogate marker of intestinal inflammation and induces apoptosis. Marmosets with chronic diarrhea exhibited higher levels of fecal calprotectin. Histochemical analyses showed high expression of calprotectin in the extravascular neutrophils and apoptosis in the chronic colitis lesions. No internal microbiological diseases were identified. Although the cause of chronic colitis was not identified, the marmoset could be a useful model of inflammatory bowel disease.

Language：English   Publishing type：Research paper (scientific journal)  

The common marmoset (Callithrix jacchus) is increasingly being used as a non-human primate animal model in biomedical research. To perform accurate quantitative analysis of gene expression by quantitative reverse transcription polymerase chain reaction, reliable reference genes should be selected. In this study, we evaluated the expressions of 11 widely used reference genes: ACTB, ATP5F1, B2M, GAPDH, HPRT1, PGK1, PPIA, RN18S1, RPLP0, TBP and UBC in 12 tissues and five brain areas of healthy common marmosets. NormFinder and geNorm indicated that the most suitable reference genes for cross-sectional studies of the 17 tissues were RN18S1 and RPLP0. Conversely, ACTB and PPIA were the most suitable for analyzing brain samples; however, the expression of PGK1 fluctuated among brain areas. These results indicate that suitable reference genes differ between the tissues examined. This study provides fundamental information for gene expression studies of the common marmoset and highlights the importance of validating reference genes before quantification of target mRNAs.

DOI： 10.1007/s11033-013-2791-0

Language：English   Publishing type：Research paper (scientific journal)  

The majority of mammalian somatic cells maintain a diploid genome. However, some mammalian cell types undergo multiple rounds of genome replication (endoreplication) as part of normal development and differentiation. For example, trophoblast giant cells (TGCs) in the placenta become polyploid through endoreduplication (bypassed mitosis), and megakaryocytes (MKCs) in the bone marrow become polyploid through endomitosis (abortive mitosis). During the normal mitotic cell cycle, geminin and Cdt1 are involved in 'licensing' of replication origins, which ensures that replication occurs only once in a cell cycle. Their protein accumulation is directly regulated by two E3 ubiquitin ligase activities, APC(Cdh1) and SCF(Skp2), which oscillate reciprocally during the cell cycle. Although proteolysis-mediated, oscillatory accumulation of proteins has been documented in endoreplicating Drosophila cells, it is not known whether the ubiquitin oscillators that control normal cell cycle transitions also function during mammalian endoreplication. In this study, we used transgenic mice expressing Fucci fluorescent cell-cycle probes that report the activity of APC(Cdh1) and SCF(Skp2). By performing long-term, high temporal-resolution Fucci imaging, we were able to visualize reciprocal activation of APC(Cdh1) and SCF(Skp2) in differentiating TGCs and MKCs grown in our custom-designed culture wells. We found that TGCs and MKCs both skip cytokinesis, but in different ways, and that the reciprocal activation of the ubiquitin oscillators in MKCs varies with the polyploidy level. We also obtained three-dimensional reconstructions of highly polyploid TGCs in whole, fixed mouse placentas. Thus, the Fucci technique is able to reveal the spatiotemporal regulation of the endoreplicative cell cycle during differentiation.

DOI： 10.1242/dev.099226

Language：English   Publishing type：Research paper (scientific journal)  

We have reported that in ganglioside GM3-deficient (GM3(-/-)) mice, spontaneous alternation behavior assessed by a Y-maze task was significantly lower, and total arm entries were significantly higher than in wild-type mice. The objective of the present study was to examine the role of nicotinic acetylcholine receptor (nAChR) signaling in impairment of spontaneous alternation behavior of GM3(-/-) mice. Nicotine treatment (0.3, 1.0 mg/kg, s.c.) dose dependently improved the spontaneous alternation deficit without affecting total arm entries in GM3(-/-) mice. The nicotine-induced (1.0 mg/kg, s.c.) improvement was significantly abolished by the nAChR antagonist mecamylamine (1.0 mg/kg, i.p.). The α4β2 nAChR antagonist dihydro-β-erythroidine (2.5, 10.0 mg/kg, i.p.) dose dependently counteracted the nicotine-induced improvement of spontaneous alternation in GM3(-/-) mice, whereas the α7 nAChR antagonist methyllycaconitine (2.5, 10.0 mg/kg, i.p.) did not. In addition, the α4β2 nAChR agonist RJR-2403 (5.0, 10.0 mg/kg, s.c.) dose dependently and significantly improved the spontaneous alternation deficit, whereas the α7 nAChR agonist PNU120596 (0.3, 1.0, 3.0 mg/kg, i.p.) did not. These findings revealed that nicotine improved spontaneous alternation behavior of GM3(-/-) mice via the activation of α4β2, but not α7, nAChR. Thus, the ganglioside GM3 might be responsible for α4β2 nAChR signaling in the spontaneous alternation behavior.

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.3109/10799893.2012.756895

Language：English   Publishing type：Research paper (scientific journal)  

Senescence-accelerated mouse prone (SAMP) strains of mice show early onset of senescence, whereas senescence-accelerated mouse resistant (SAMR) strains are resistant to early senescence and serve as controls. Although SAMP6 and SAMP8 are established models of central nervous system alterations, it is unclear whether SAMP1/Sku (SAMP1) is characterized by brain alterations and dysfunction related to behavioral functioning. In the present study, behavioral tests (i.e., locomotor activity, Y-maze, rotating rod, hind-limb extension, and traction), histochemistry, and Western blot analyses were employed to study this mouse model using 2- and 4-month-old SAMP1 and age-matched control SAMR1. Although 2-month-old SAMP1 and SAMR1 showed similar activity, 4-month-old SAMP1 exhibited less activity than age-matched SAMR1 in locomotor activity and Y-maze tests. In rotating rod test, 2- and 4-month-old SAMP1 showed motor-coordination dysfunction. An abnormal extension reflex in the hind-limb test was observed in 2- and 4-month-old SAMP1. There were no significant differences between SAMP1 and SAMR1 with respect to grip strength in the traction test or alternation behavior in the Y-maze test. Histochemistry and Western blot analyses exhibited that cerebellar Purkinje cells in 4-month-old SAMP1 mice persistently expressed tyrosine hydroxylase. These results suggest that SAMP1 is a useful model for examining mechanisms underlying motor dysfunction.

DOI： 10.1016/j.brainres.2013.03.053

Language：English   Publishing type：Research paper (scientific journal)  

One of the main instigators leading to cell death and brain damage following ischemia is Ca(2+) dysregulation. Neuronal membrane depolarization results in the activation of voltage-gated Ca(2+) (CaV) channels and intracellular Ca(2+) influx. We investigated the physiological role of the CaV2.1 (P/Q-type) channel in ischemic neuronal injury using CaV2.1 channel α1 subunit mutant mice, rolling Nagoya and leaner mice. The in vivo ischemia model with a complete occlusion of the middle cerebral artery showed that the infarct area at 24h was significantly smaller in rolling Nagoya (27.1±3.5% of total brain volume) and leaner (20.1±3.5%) mice compared to wild-type (42.9±4.5%) mice. In an in vitro Ca(2+) imaging study, oxygen-glucose deprivation using a hippocampal slice induced a significantly slower rate of increase in intracellular Ca(2+) concentration ([Ca(2+)]i) in rolling Nagoya (0.083±0.007/min) and leaner (0.062±0.006/min) mice compared to wild-type (0.105±0.008/min) mice. These results demonstrate that the mutant CaV2.1 channel in rolling Nagoya and leaner mice plays a different protective role in a ([Ca(2+)]i)-dependent manner in ischemic models and indicate that CaV2.1 channel blockers may be used preventively against ischemic injury.

DOI： 10.1016/j.bbrc.2013.03.066

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1371/journal.pone.0044230

Language：English   Publishing type：Research paper (scientific journal)  

The role of the P/Q-type voltage-gated Ca(2+) channels (VGCCs) in release of neurotransmitters involved in nociception is not fully understood. Rolling mouse Nagoya (tg(rol)), a P/Q-type channel mutant mouse, expresses P/Q-type VGCC whose activation curve has a higher half activation potential and a smaller slope factor than the wild type channel. We previously reported that tg(rol) mice showed hypoalgesic responses to noxious stimuli. In this study, we examined the VGCC current in dorsal root ganglion (DRG) neurons by the whole-cell patch-clamp method. Both ω-agatoxin IVA (0.1 μM) and ω-conotoxin GVIA (1 μM) inhibited the VGCC current by about 40-50% in both the homozygous tg(rol) (tg(rol)/tg(rol)) and wild type (+/+) mice. The voltage-activation relationships of the total VGCC current and the ω-agatoxin IVA-sensitive component in the tg(rol)/tg(rol) mice shifted positively compared to the +/+ mice, whereas that sensitive to the ω-conotoxin GVIA was not different between the two genotypes. The time constant of activation of the VGCC current at -20 mV was longer in the tg(rol)/tg(rol) mice than in the +/+ mice. These changes in the properties of the VGCC in the tg(rol)/tg(rol) mouse may reduce the amount of the released neurotransmitters and account for the hypoalgesic responses.

DOI： 10.1016/j.neures.2012.04.006

Language：English   Publishing type：Research paper (scientific journal)  

Advances in mouse neural circuit genetics, brain atlases, and behavioral assays provide a powerful system for modeling the genetic basis of cognition and psychiatric disease. However, a critical limitation of this approach is how to achieve concordance of mouse neurobiology with the ultimate goal of understanding the human brain. Previously, the common marmoset has shown promise as a genetic model system toward the linking of mouse and human studies. However, the advent of marmoset transgenic approaches will require an understanding of developmental principles in marmoset compared to mouse. In this study, we used gene expression analysis in marmoset brain to pose a series of fundamental questions on cortical development and evolution for direct comparison to existing mouse brain atlas expression data. Most genes showed reliable conservation of expression between marmoset and mouse. However, certain markers had strikingly divergent expression patterns. The lateral geniculate nucleus and pulvinar in the thalamus showed diversification of genetic organization between marmoset and mouse, suggesting they share some similarity. In contrast, gene expression patterns in early visual cortical areas showed marmoset-specific expression. In prefrontal cortex, some markers labeled architectonic areas and layers distinct between mouse and marmoset. Core hippocampus was conserved, while afferent areas showed divergence. Together, these results indicate that existing cortical areas are genetically conserved between marmoset and mouse, while differences in areal parcellation, afferent diversification, and layer complexity are associated with specific genes. Collectively, we propose that gene expression patterns in marmoset brain reveal important clues to the principles underlying the molecular evolution of cortical and cognitive expansion.

DOI： 10.1523/JNEUROSCI.4788-11.2012

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1371/journal.pone.0032707

Language：English   Publishing type：Research paper (scientific journal)  

Heterozygous rolling Nagoya (rol/+) mice with a Ca(V)2.1α₁ mutation show normal Y-maze behavior. Intra-accumbens injection of N-methyl-d-aspartate (NMDA; 0-2.0 μg/side) induced similar spontaneous alternations in wild-type and rol/+ mice; injections of NMDA receptor antagonist MK-801 (0.5 μg/side) or Ca(V)2.1 inhibitor levetiracetam (0.1 μg/side) did not affect controls but decreased spatial cognition in rol/+ mice, suggesting that Ca(V)2.1-mediated NMDA receptor signaling in the nucleus accumbens is involved in short-term spatial learning.

DOI： 10.1016/j.bbr.2010.12.019

Language：English   Publishing type：Research paper (scientific journal)  

Although rolling mouse Nagoya, a Ca(v)2.1α(1) mutant, exhibits ataxia and elevated serotonin concentrations, heterozygous mice have not been examined in detail. Patients with heterozygous mutations in this orthologous gene exhibit neurological disorders. To examine the emotional behavior of heterozygous mice, we used behavioral tasks and examined Ca(v)2.1α(1) message levels, tryptophan hydroxylase expression patterns, and monoamine concentrations in 2- and 22-month-old mice. Reduced anxiety in the elevated plus maze, light-dark exploration, and marble-burying behavioral tests and reduced depression in the forced swimming and tail suspension tests were observed in 22-month-old heterozygous mice compared to aged-matched wild-type mice. The levels of mutant-type Ca(v)2.1α(1) message, phosphorylation of tryptophan hydroxylase, and serotonin increased in the brainstems of 22-month-old heterozygous mice. No difference was observed between 2-month-old heterozygous and wild-type mice in these analyses. These findings suggest that heterozygous mice show age-related emotional changes due to alterations in the serotonin system associated with mutant-type Ca(v)2.1α(1), and that heterozygous mice may represent a novel model to delineate the interaction between Ca(v)2.1 function and synaptic transmission.

DOI： 10.1016/j.neurobiolaging.2009.03.001

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The ganglioside GM3 synthase (SAT-I), encoded by a single-copy gene, is a primary glycosyltransferase for the synthesis of complex gangliosides. Although its expression is tightly controlled during early embryo development and postnatal development and maturation in the brain, the physiological role of ganglioside GM3 in the regulation of neuronal functions has not been elucidated. In the present study, we examined motor activity, cognitive and emotional behaviors, and drug administration in juvenile GM3-knockout (GM3-KO) mice. GM3-KO male and female mice showed hyperactivity in the motor activity test, Y-maze test, and elevated plus maze test. In the Y-maze test, there was significantly less spontaneous alternation behavior in GM3-KO male mice than in wild-type mice. In the elevated plus maze test, the amount of time spent on the open arms by GM3-KO male mice was significantly higher than that of sex-matched wild-type mice. In contrast, there was no significant difference between GM3-KO and wild-type female mice in these tests. Thus, juvenile GM3-KO mice show gender-specific phenotypes resembling attention-deficit hyperactivity disorder (ADHD), namely hyperactivity, reduced attention, and increased impulsive behaviors. However, administration of methylphenidate hydrochloride (MPH) did not ameliorate hyperactivity in either male or female GM3-KO mice. Although these data demonstrate the involvement of ganglioside GM3 in ADHD and the ineffectiveness of MPH, the first-choice psychostimulant for ADHD medication, our studies indicate that juvenile GM3-KO mice are a useful tool for neuropsychological studies.

DOI： 10.1016/j.bbrc.2011.02.071

Language：English   Publishing type：Research paper (scientific journal)  

Heterozygous rolling Nagoya (rol/+) mice carrying Ca(V)2.1 alpha(1) mutation demonstrated normal behavior in Y maze test. Similar spontaneous alternation patterns were noted in wild-type and rol/+ mice injected with N-methyl-D-aspartate (NMDA; 0-50mg/kg, sc). Systemic injection of NMDA receptor blocker (MK-801; 0.05 mg/kg, ip) or intrahippocampal injection of MK-801 (0.5 microg/side), which had no effect in wild-type controls, decreased spatial cognition in rol/+ mice. These results indicate that Ca(V)2.1 alpha(1) mutation probably through decrease in Ca(2+) influx lowers the threshold for learning impairment. The combination subthreshold pharmacological and genetic approach is useful to study functional pathways in neuronal circuits.

DOI： 10.1016/j.bbr.2010.04.030

Language：English   Publishing type：Research paper (scientific journal)  

Ca(V)2.1 is highly expressed in the nervous system and plays an essential role in the presynaptic modulation of neurotransmitter release machinery. Recently, the antiepileptic drug levetiracetam was reported to inhibit presynaptic Ca(V)2.1 functions, reducing glutamate release in the hippocampus, although the precise physiological role of Ca(V)2.1-regulated synaptic functions in cognitive performance at the system level remains unknown. This study examined whether Ca(V)2.1 mediates hippocampus-dependent spatial short-term memory using the object location and Y-maze tests, and perirhinal cortex-dependent nonspatial short-term memory using the object recognition test, via a combined pharmacological and genetic approach. Heterozygous rolling Nagoya (rol/+) mice carrying the Ca(V)2.1alpha(1) mutation had normal spatial and nonspatial short-term memory. A 100mg/kg dose of levetiracetam, which is ineffective in wild-type controls, blocked spatial short-term memory in rol/+ mice. At 5mg/kg, the N-methyl-D-aspartate (NMDA) receptor blocker (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), which is ineffective in wild-type controls, also blocked the spatial short-term memory in rol/+ mice. Furthermore, a combination of subthreshold doses of levetiracetam (25 mg/kg) and CPP (2.5mg/kg) triggered a spatial short-term memory deficit in rol/+ mice, but not in wild-type controls. Similar patterns of nonspatial short-term memory were observed in wild-type and rol/+ mice when injected with levetiracetam (0-300 mg/kg). These results indicate that Ca(V)2.1-mediated NMDA receptor signaling is critical in hippocampus-dependent spatial short-term memory and differs in various regions. The combination subthreshold pharmacological and genetic approach presented here is easily performed and can be used to study functional signaling pathways in neuronal circuits.

DOI： 10.1016/j.ejphar.2010.07.029

Language：English   Publishing type：Research paper (scientific journal)  

Ca(V)2.1, which is highly expressed in the nervous system, plays an essential role in presynaptic neurotransmitter release. Although recent data suggest that the antiepileptic drug levetiracetam (LEV) inhibits presynaptic Ca(V)2.1 activity, the precise physiological role of Ca(V)2.1/LEV-regulated emotional performance has not been elucidated. We examined whether Ca(V)2.1/LEV mediates emotional behavior using a combined pharmacologic and genetic approach. Heterozygous rolling Nagoya (rol/+) mice carrying the Ca(V)2.1alpha(1) mutation demonstrated normal emotional behavior. Exposure to 75mg/kg LEV, which had no effect in wild-type controls, reduced anxiety in elevated plus maze and light-dark exploration tests and reduced depression in forced swimming and tail suspension behavioral tests in rol/+ mice. Similar behavioral patterns in motor activity were noted in wild-type and rol/+ mice injected with 0-150mg/kg LEV. The phosphorylation of tryptophan hydroxylase at serine-58 and serotonin concentration were increased in the brainstems of rol/+ mice injected with 75mg/kg LEV but not in those of wild-type controls. These results indicate that Ca(V)2.1/LEV mediates serotonin signaling leading to alterations in emotion. Our results also indicate that a combination of subthreshold pharmacologic and genetic approaches can be used to study functional signaling pathways in neuronal circuits.

DOI： 10.1016/j.pbb.2010.05.020

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1538/expanim.59.441

Language：English   Publishing type：Research paper (scientific journal)  

It has been reported earlier that interactions between Ca(v)2.1alpha(1) and calcium/calmodulin-dependent protein kinase II (CaMKII) in the presynaptic fraction and between the NMDA receptor subunit NR2B and CaMKII in the postsynaptic density (PSD) fraction are important for neuronal function. Ca(v)2.1alpha(1), CaMKII, and NR2B are predominantly expressed in the hippocampus. To examine the above interactions and CaMKII activity in the hippocampal presynapse and PSD of Rolling Nagoya mice carrying a mutation in Ca(v)2.1alpha(1) subunit, we performed immunoprecipitation and Western blot analyses. In the presynapse, the interaction between Ca(v)2.1alpha(1) and CaMKII and the phosphorylation of CaMKII (at Thr286) and its substrate Synapsin I (at Ser603) were decreased in mutant mice compared to wild-type mice. In the PSD, a similar pattern was observed for the interaction between NR2B and CaMKII and the phosphorylation of CaMKII (at Thr286) and its substrate AMPA receptor subunit glutamate receptor 1 (at Ser831) between mutant and wild-type mice. Our data indicate that disruption of the interaction between Ca(v)2.1alpha(1) and CaMKII may down-regulate presynaptic CaMKII activity and that Rolling Nagoya mice would be a useful model for examining presynaptic function.

DOI： 10.1007/s12031-009-9216-5

Language：English   Publishing type：Research paper (scientific journal)  

N-type voltage-dependent calcium channels (VDCCs) play an important role in neurotransmission, synaptic plasticity, and brain development. They are composed of several subunits named alpha(1), alpha(2), delta, beta and gamma. The alpha(1) subunit is essential for channel functions and determines fundamental channel properties. Since N-type VDCC are critically involved in the release of neurotransmitters and clinical relevance, we predicted that alpha(1) subunit KO mice would show several alterations in behavior. In the present study, we investigated neuronal functions in mice lacking the alpha(1B) (Ca(V)2.2) subunit of the N-type calcium channels. Ca(V)2.2(-/-) mice exhibited a significant increase in locomotion on an activity wheel during the dark phase. Furthermore, when challenged with apomorphine, mutant mice showed enhanced locomotor activity. Cognitive functions were examined using a Y-maze task for short-term memory and a passive avoidance task for long-term memory. The Y-maze revealed no differences in spontaneous alternation behavior between mutant and wild-type mice. The passive avoidance test revealed that the latency time in mutant mice was significantly decreased. The mutant mice showed prepulse inhibition deficits reminiscent of the sensorimotor gating deficits observed in a large majority of schizophrenic patients. Decreases in baseline levels of dopamine and serotonin within the striata and frontal cortices of mutant mice were also observed. These results suggest that Ca(2+) in the central nervous system modulates various neurophysiological functions, such as locomotor activity, long-term memory, and sensorimotor gating through the alpha(1B) subunit of the N-type calcium channels.

DOI： 10.1016/j.bbr.2009.11.042

Language：English   Publishing type：Research paper (scientific journal)  

Rolling Nagoya mice show ataxia and carry a mutation in the Cacna1a gene, which encodes the pore-forming alpha1 subunit of the Cav2.1 channels. Because an impaired motor function has not been examined during neonatal stages in detail, we employed a battery of tests including assessments of body weight gain, righting reflex, negative geotaxis, hind-limb suspension, and tail suspension using neonatal wild-type, heterozygous, and homozygous rolling mice. We found deterioration of body weight gain after postnatal day 8 (P8) in the homozygous mice, as well as a longer latency time to complete the righting reflex and the negative geotaxis tests after P8. Additionally, the homozygous rolling mice exhibited lower pulling and holding attempts after P8 in the hind-limb suspension test. The mice heterozygous and homozygous for the rolling mutation exhibited muscle fatigue after P10 and P8, respectively, following movement execution tests administered immediately after the first trial, suggesting that gene dosage plays an important role in determining when muscle weakness occurs. The homozygous rolling mice showed hind-limb clasping or touching after P14 during the hind-limb and tail suspension tests. Our results indicate that the gait abnormality of neonatal rolling Nagoya would be due to the combination of muscle weakness and neuronal dysfunction and that the rolling mice could be a useful model for delineating neonatal motor deficiencies.

DOI： 10.1016/j.bbr.2009.10.017

Language：English   Publishing type：Research paper (scientific journal)  

Aged heterozygous Rolling Nagoya mice carrying Cav2.1alpha1 mutation show deficits with regard to spatial short-term memory using hippocampus-related object location test, but not with regard to nonspatial memory using perirhinal cortex-related object recognition test. In hippocampus, wild-type Cav2.1alpha1 mRNA exhibited lower expression and mutant-type expression was higher in aged heterozygous mice. In perirhinal cortex, there were no significantly different expressions. Alteration of age-dependent expressions of Cav2.1 channels differs in different regions with related effects on behavioral performances.

DOI： 10.1016/j.bbr.2009.05.020

Language：English   Publishing type：Research paper (scientific journal)  

Senescence-accelerated mouse prone 6 (SAMP6) is a model of senile osteoporosis. From 10 to 22 wk of age, SAMP6 mice were heavier than age-matched AKR/J and SAMR1 mice. Body mass indices of 10- and 25-wk-old SAMP6 mice were higher than those of age-matched AKR/J and SAMR1 mice, indicating obesity in the SAMP6 animals. We compared the blood biochemical values among SAMP6, SAMR1, and AKR/J mice to assess whether the SAMP6 strain has abnormal obesity-related parameters. Plasma glucose, triglyceride, insulin, and leptin levels were higher in 10-wk-old SAMP6 mice than in age-matched SAMR1 and AKR/J mice, whereas plasma glucagon and adiponectin levels in 25-wk-old SAMP6 were lower compared with those in age-matched SAMR1 and AKR/J. Total cholesterol levels in SAMR1 and SAMP6 mice at 10 and 25 wk of age were higher than those in AKR/J mice. Hepatic lipid levels were higher in 10- and 25-wk-old SAMP6 mice compared with age-matched AKR/J and SAMR1 animals. These results indicate that SAMP6 mice exhibit obesity and hyperlipidemia, suggesting that the SAMP6 strain is a potential tool for the study of hyperlipidemia.

Language：English   Publishing type：Research paper (scientific journal)  

The senescence-accelerated mouse (SAM) is a murine model of aging that was developed from the AKR/J strain. We examined whether there are behavioral differences among SAM prone 6 (SAMP6; an established model of senile osteoporosis), SAM resistant 1 (SAMR1), and AKR/J, using a modified SmithKline/Harwell/Imperial College/Royal Hospital/Phenotype Assessment (SHIRPA) procedure and pharmacological tests. The modified SHIRPA, which is suitable for rapid and comprehensive phenotyping of transgenic and gene-targeted mice, revealed increased rearing, spontaneous activity, locomotor activity, tail elevation, head bobbing, and tail rattling behaviors of SAMP6 compared with SAMR1 and AKR/J. These phenotypes are consistent with alteration of the dopamine system in SAMP6. Adopting a pharmacological approach to examine dopamine signaling, we evaluated the locomotor activity of the mice after intraperitoneal administration of apomorphine, a subtype non-selective dopamine receptor agonist. Apomorphine at 1mg/kg significantly increased the locomotor activity of SAMP6, but not SAMR1 or AKR/J. At 3mg/kg, apomorphine significantly increased the locomotor activities of all three strains, but the increase in SAMP6 was still significantly greater than that in SAMR1 or AKR/J. These results indicate increased sensitivity of the dopamine receptor signaling pathway in SAMP6. Thus, alteration of dopamine receptor signal transduction appears to be one of the underlying mechanisms of the increased locomotor activity of SAMP6. The combination of modified SHIRPA and examination of drug threshold dose differences between strains appears to be an effective approach to extend the applicability of existing mouse models.

DOI： 10.1016/j.neulet.2009.04.042

Language：English   Publishing type：Research paper (scientific journal)  

Although rolling Nagoya mice exhibit ataxia and carry a mutation in the alpha1 subunit of the Cav2.1 channel regulating neurotransmitter release, heterozygous mice have not received a great deal of attention. Given the pivotal role of Cav2.1 channels in controlling neurotransmitter release, age-dependent alterations in Cav2.1 channel function may result in aberrant synaptic signaling, leading to motor dysfunction. To examine age-related motor alterations in heterozygous mice, we used a battery of tests (e.g., motor activity, footprint, traction, wire suspension, balance beam, rotating rod, hind-limb extension analysis) in 2- and 22-month-old mice and examined expression patterns of the alpha1 gene in their cerebellum. No significant difference was observed between 2-month-old heterozygous and wild-type mice in the any of the behavioral tests or in the alpha1 expression levels. Although 22-month-old heterozygous and wild-type mice exhibited no significant difference in motor activity, footprint, or traction tests, 22-month-old heterozygous mice showed deficits in the wire hanging, balance beam, and rotating rod tests. Additionally, 22-month-old heterozygous mice displayed clasping behavior in the hind-limb extension test. Expression analysis showed that wild-type Cav2.1alpha(1) mRNA was lower in aged mice than in young mice and that mutant-type Cav2.1alpha(1) mRNA was higher in aged mice than in young mice. These findings suggest that heterozygous mice show age-related motor changes due to mutant-type Cav2.1 and that heterozygous mice may represent a new model for examining motor function.

DOI： 10.1016/j.brainres.2009.05.016

Language：English   Publishing type：Research paper (scientific journal)  

Voltage-dependent Ca(2+) channels (VDCCs) play important roles in physiological functions and pathological processes of the nervous system. Given that the precise regulation of Ca(2+) signaling is important for neuronal processes such as action potential generation, transmitter release, and synaptic plasticity, alterations in Ca(2+) current through VDCCs affect the functions of neurons and circuits. Central nervous system (CNS) diseases, including pain, epilepsy, seizure, anxiety, depression, dementia, and stroke, are characterized by an altered balance between excitatory and inhibitory neuronal functions. An efficient way of controlling such diseases is to block or modulate VDCC function. An effective strategy to reduce the likelihood of adverse effects is to develop agents that selectively control the VDCC isoform/subunit involved in the mechanism of the disease in question. This review provides an overview of knowledge on VDCCs, traditional and newly developed therapeutic fields, clinical fields, and the diverse medicinal chemistry of traditional and newly developed VDCC blockers in the CNS based on the scientific and patent literature.

Language：English   Publishing type：Research paper (scientific journal)  

Senescence-accelerated mouse prone 6 (SAMP6) mice exhibit increased expression of NMDA receptor NR2B subunit (NR2B) and improved short-term memory compared with senescence-accelerated mouse resistance 1 (SAMR1) mice. The Thr286 phosphorylation of alpha calcium/calmodulin-dependent protein kinase II (CaMKII) has a crucial role in plasticity and learning among multiple downstream signaling pathways linked to the NMDA receptor. To examine the relationship between CaMKII activity and spatial learning in SAMP6, the authors employed western blot analysis and behavioral analyses (object location and delayed spatial win-shift eight-arm radial-maze tests). The levels of Thr286 and Ser831 phosphorylation of CaMKII and AMPA receptor subunit glutamate receptor 1 (CaMKII substrate), respectively, were increased in hippocampus of SAMP6 compared with SAMR1. SAMP6 showed faster hippocampal-dependent spatial memory formation than SAMR1 in both the object location and win-shift eight-arm radial-maze tests. Our results indicate that increased CaMKII activity influences the NR2B/CaMKII signal pathway and cognitive function in SAMP6.

DOI： 10.1037/a0015119

Language：English   Publishing type：Research paper (scientific journal)  

Rolling Nagoya mice carrying Ca(v)2.1alpha1 gene mutation show ataxia, whereas heterozygous mice show no apparently abnormal behavior. It has been reported that Ca(v)2.1 regulates neurotransmitter release and that Ca(2+) influx through Ca(v)2.1 decreases with aging. Age-related decline in cognitive function could be at least partly attributable to decreases in Ca(v)2.1-related neurotransmission. In this study to examine age-related cognitive alterations in heterozygous mice, we used Y-maze and delayed spatial win-shift eight-arm radial-maze tests, and 2- and 22-month-old mice. Although there was no difference between 2-month-old heterozygous and wild-type mice, 22-month-old heterozygous mice showed decreased memory formation versus 2-month-old heterozygous mice in both tests. Expression analysis in forebrain showed that total Ca(v)2.1alpha1 mRNA, including wild-type and mutant-type Ca(v)2.1alpha1 mRNA, in 2-month-old heterozygous mice was expressed at a level similar to that in 22-month-old heterozygous mice. However, wild-type Ca(v)2.1alpha1 mRNA was expressed at a lower level in 22-month-old mice than in 2-month-old mice, and mutant-type Ca(v)2.1alpha1 mRNA was expressed at a higher level in 22-month-old versus 2-month-old mice. Our results suggest that aged heterozygous mice show deficits in spatial learning due to Ca(v)2.1 channel dysfunction and that heterozygous mice may be a useful model for examining mechanisms underlying age-related cognitive dysfunction.

DOI： 10.1016/j.exger.2008.11.006

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.neuroscience.2009.02.032

Language：English   Publishing type：Research paper (scientific journal)  

The motor function of senescence-accelerated mouse prone 6 (SAMP6) was evaluated with a battery of behavioral tests: locomotor activity test, traction test, wire hanging test, and rotating rod test. SAMP6 exhibited increased locomotor activity compared with senescence-accelerated mouse resistant 1 (SAMR1). There was no difference between SAMP6 and SAMR1 in the traction and wire hanging tests. In the rotating rod test, shorter retention times at each day in the accelerating version of the test were observed in SAMP6 compared with SAMR1, indicating a motor coordination deficit of SAMP6. To understand the mechanism involved, we focused on the dopamine system. Measurement of dopamine and its metabolites with HPLC revealed that the concentrations of dopamine in nucleus accumbens (NAcs) and cerebellum and of one or more dopamine metabolites in all tissues assayed were significantly higher in SAMP6 compared with SAMR1. Increases of dopamine transporter and dopamine receptor 1 (D1) in striatum, of dopamine receptor 3 (D3) in NAc, and of D1 and D3 in cerebellum in SAMP6 were observed. These results indicate that increased dopamine concentration in NAc and increased expression of D1 in striatum are possible cause(s) of the increased locomotor activity of SAMP6, and that increased D3 expression in cerebellum contributes to the motor coordination deficit of SAMP6.

DOI： 10.1016/j.physbeh.2008.11.012

Language：English   Publishing type：Research paper (scientific journal)  

Recent genetic analyses revealed an important association between P/Q-type channels and hereditary neurological disorders. The alpha1 subunit of P/Q-type channels is coded by a single CaV2.1 gene. Since calcium entry via neuronal calcium channels is essential for neurotransmission, P/Q-type channels may play an important role in cardiac autonomic neurotransmission. To elucidate the physiological importance of P/Q-type channels in autonomic nerve control, we used rolling Nagoya (tg(rol)) mice, which have a mutation in the CaV2.1 gene and decreased P/Q-type channel currents with reduced voltage sensitivity. The tg(rol) mice demonstrated unmodified expression of other calcium channel subunits. Electrocardiogram and echocardiographic analyses revealed decreased heart rate. Furthermore, omega-agatoxin IVA, a P/Q-type channel inhibitor, decreased heart rate and ejection fraction only in wild-type mice, thus suggesting a significant involvement of P/Q-type channels in chronotropic regulation. Atrium contraction analyses revealed a minor but significant role for P/Q-type channels in sympathetic and parasympathetic nerve regulation.

DOI： 10.1016/j.bbrc.2009.01.184

Language：English   Publishing type：Research paper (scientific journal)  

Using gene knockout mice of particular genes is one of the most effective methods in conducting successful study on the mode of action of target gene products in targeted organs. So called the knockout technology is now a powerful tool that can lead us to find clear understanding on difficult questions such as the effects of full antagonist against target molecules. Cacna1b (alpha(1B)) gene knockout mouse was generated to study mechanisms of N-type calcium (Ca(2+)) channel. The model was able to overcome physiological obstacles in studies of N-type Ca(2+) channel selective blockers, such as unspecific binding to structurally similar molecules, and failed distribution to targeted organs. In the case of N-type Ca(2+) channel studies, knockout technology was successfully applied to various cardiovascular, sympathetic, nociceptive, sleep-awake cycles, metabolic and neurodegenerative experiments using homozygous mutants of the alpha(1B) gene that turned out to be viable. These studies were able to confirm not only the predicted phenotypes, but were able to present completely unexpected phenotypes that are great interest for future study. Thus the outputs from the knockout mouse studies lead to gain the proof of concept as a drug for specific inhibitors of the gene products and enabled us to make further prediction of side-effects of these inhibitors in the drug discovery and development process.

Language：English   Publishing type：Research paper (scientific journal)  

Motor activity is a key component in many behavioral tests. To assess the relationship between aging and activity, we recorded motor activity for 72 consecutive hours for C57BL/6J (B6J), BALB/c, AKR/J, senescence-accelerated mouse prone 6 (SAMP6), and senescence-accelerated mouse resistant 1 (SAMR1) strains at the ages of 6 and 12 months. Further, to examine whether the dopamine receptor 1 (D1) signaling system is associated with the age-related alteration of activity, we evaluated the motor activity of the mice treated with SKF82958 (6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1-phenyl-1H-3-benzazepine hydrobromide), a D1 agonist. Twelve-month-old B6J showed higher activity on day 1 and higher D1 sensitivity than 6-month-old mice. Twelve-month-old BALB/c showed higher activity on day 3 and a slightly lower threshold of D1 than 6-month-old mice. Twelve-month-old AKR/J, SAMR1 and SAMP6 strains showed lower motor activity than 6-month-old mice. The D1 sensitivities in 12-month-old AKR/J and SAMR1 were similar to those of corresponding 6-month-old mice, whereas the D1 sensitivity of 12-month-old SAMP6 was significantly lower than that of 6-month-old SAMP6. SKF82958 significantly increased the motor activity of 6-month-old SAMP6 compared with age-matched, AKR/J and SAMR1. Our results indicate that D1 contributes substantially to the age-related increase of activity in B6J, but not to that in BALB/c. In AKR/J, SAMR1, and SAMP6, an age-related decrease of activity was observed. The contribution of D1 to this appeared to be small in AKR/J and SAMR1, but substantial in SAMP6. Thus, the contribution of D1 to age-related changes of motor activity is strongly strain-dependent.

DOI： 10.1016/j.brainres.2008.10.043

Language：English   Publishing type：Research paper (scientific journal)  

Senescence accelerated prone mouse 6 (SAMP6) mice have been known to be a model for accelerated aging. Compared with the normal control senescence accelerated resistant mouse 1 (SAMR1) mice, although the SAMP6 mice have normal bone mass at 4 months, they exhibit a significantly lower bone mass at 8 months. It was recently reported that SAMP6 has memory deficit at 4 months of age, indicating that the change of nervous function might be already detected at 4 months of age. To assess whether SAMP6 mice exhibit an age-related abnormality of nociceptive transmission, we examined a battery of tests using the von Frey test for mechanically induced response, the hot plate test for thermally induced response, and the formalin paw test for chemically induced response. SAMP6 and SAMR1 showed similar response patterns in the von Frey test and the hot plate test. In the formalin paw test, 1-month-old SAMP6 and SAMR1 had similar responses, while 4-month-old SAMP6 exhibited attenuated phase 2 response, but normal phase 1 response. These findings indicate that onset of age-related phenotypes in SAMP6 differs in different tissues. SAMP6 could be useful to delineate the involvement of age-related nociceptive mechanisms.

DOI： 10.1016/j.neulet.2008.08.003

Language：English   Publishing type：Research paper (scientific journal)  

The increased dopamine and serotonin were suggested [Niimi et al., 2008. Emotional behavior and expression pattern of tyrosine hydroxylase and tryptophan hydroxylase in senescence-accelerated mouse (SAM) P6 mice. Behav. Brain Res. 188, 329-336], and as these monoamines are well known to influence working memory processes, SAMP6 may show improved working memory. We found that spatial Y-maze memory and non-spatial novel object recognition memory of SAMP6 were improved compared with those of senescence-accelerated mouse resistant 1 (SAMR1). Among molecules known to be related with memory processes other than dopamine and serotonin, we focused on N-methyl-D-aspartate (NMDA) receptors. Animals treated with (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), a NMDA receptor antagonist, were subjected to the Y-maze and novel object recognition tests to examine whether NMDA receptors are associated with the improved short-term memory of SAMP6. CPP (10 mg/kg) significantly impaired the spontaneous alternation behavior and the exploratory preference of SAMR1, whereas no significant effect was seen in SAMP6 in either of these behavioral tests. Western blot analyses revealed increased expression of NMDA receptor (NR) subunit 2B in forebrain of SAMP6 compared with SAMR1, while there was no difference in the levels of NR1 and NR2A between SAMR1 and SAMP6. Our results indicate that increased expression of NR2B in forebrain of SAMP6 is one of the causes of the improved short-term memory of SAMP6.

DOI： 10.1016/j.exger.2008.06.010

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.m802319200

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.ejphar.2008.05.020

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbr.2007.11.014

Language：Others  

Effect of psychosocial stress on spatial memory in C57BL/6J and C57BL/6N mice. Journal of experimental animal technology

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1523/jneurosci.4486-07.2007

Language：English   Publishing type：Research paper (scientific journal)  

To elucidate the physiological importance of neuronal (N)-type calcium channels in sympathetic controls, we analyzed N-type channel-deficient (NKO) mice. Immunoprecipitation analysis revealed increased interaction between beta3 (a major accessory subunit of N-type channels) and R-type channel-forming CaV2.3 in NKO mice. R-R intervals in NKO ECG recordings were elongated and fluctuating, suggesting disturbed sympathetic tonus. N-type channel inhibitors elongated the R-R interval in control mice, whereas R-type channel blocking with SNX-482 significantly affected NKO but not control mice, indicating a compensatory role for R-type channels. Echocardiography and Langendorff heart analysis confirmed a major role for R-type channels in NKO mice. Combined, our biochemical and physiological analyses strongly suggest that the remaining sympathetic tonus in NKO mice is dependent on R-type calcium channels.

Language：Others   Publishing type：Research paper (scientific journal)  

<title>Abstract</title>
<sec>
<title>Background</title>
ADAM11 is a member of the ADAM gene family and is mainly expressed in the nervous system. It is thought to be an adhesion molecule, since it has a disintegrin-like domain related to cell-cell or cell-matrix interactions. To elucidate the physiological functions of ADAM11, we generated ADAM11-deficient mice by means of gene targeting.


</sec>
<sec>
<title>Results</title>
ADAM11-deficient mice were apparently normal, and survived more than one year with no major histological abnormalities in the brain or spinal cord. Because ADAM11 is highly expressed in the hippocampus and cerebellum, we have examined ADAM11 mutant mice for learning using visual and hidden water maze tasks, and their motor coordination using a rotating rod task. Our results showed that their visual water maze task results are normal, but the hidden water maze and rotating rod task skills are impaired in ADAM11-deficient mice.


</sec>
<sec>
<title>Conclusion</title>
Our results indicate that ADAM11 mutation does not affect cell migration and differentiation during development, but affects learning and motor coordination. Thus, ADAM11 might play an important signalling or structural role as a cell adhesion molecule at the synapse, and may thus participate in synaptic regulation underlying behavioural changes.


</sec>

DOI： 10.1186/1471-2202-7-19

Language：English   Publishing type：Research paper (scientific journal)  

N-type Ca2+ channel alpha1B-deficient mice have increased activity (ambulation, repetitive behavior, and rearing combined), suggesting contribution by the N-type Ca2+ channel, localized in the plasma membrane and essential for neurotransmitter release, on motor activity. We evaluated the effect of a 6-wk postweaning period of either individual or group housing on the activity displayed in a novel environment with or without previous habituation. Without habituation, male homozygous alpha1B-deficient mice showed significantly higher activity than wild-type controls, with no influence of the housing condition. When habituated, hyperactivity was seen in individually housed but not group-housed homozygous alpha1B-deficient mice. The results indicate that controlling for housing condition can be important when phenotypically analyzing mutant mice.

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1101/lm.183406

Language：English   Publishing type：Research paper (scientific journal)  

Electrophysiologic studies have demonstrated that adrenal medulla chromaffin cells express voltage-dependent P/Q-, N-, L-, and R-type Ca2+ channels and that these channels regulate release of norepinephrine and epinephrine. However, N-type Ca2+ channel alpha1B-deficient mice with a CBA/JN background show normal plasma norepinephrine and epinephrine levels, presumably owing to compensation by other gene(s). To examine the expression patterns of the P/Q-type alpha1A, L-type alpha1C/alpha1D, and R-type alpha1E, beta1, beta2, beta3, and beta4 subunits, as well as of tyrosine hydroxylase (Th), dopamine beta hydroxylase (Dbh), and phenylethanolamine-N-methyltransferase (Pnmt) in the adrenal gland of alpha1B-deficient mice, we used real-time quantitative reverse transcription-polymerase chain reaction and Western blot analyses. The expression levels of alpha1A, beta4, Th, and Th phosphorylated at serine 40 were higher in homozygous mice than in wild-type and heterozygous mice, but the expression levels of alpha1C, alpha1D, alpha1E, beta1, beta2, beta3, Dbh, and Pnmt did not differ among wild-type, heterozygous, and homozygous mice. These results suggest that the compensatory mechanisms to maintain normal levels of epinephrine and norepinephrine in the adrenal gland of N-type Ca2+ channel alpha1B-deficient mice include increased expression of alpha1A and beta4 subunits and increased catecholamine biosynthetic activity.

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.brainres.2006.04.043

Language：English   Publishing type：Research paper (scientific journal)  

Pheromones affect gonadal functions and sexual behaviors. Information in regard to pheromones is received by the vomeronasal organ (VNO) and transmitted to the accessory olfactory bulb (AOB). We investigated the physiological role of the alpha1B and beta3 subunits of the N (neuronal)-type voltage-dependent Ca2+ channel in the neurotransduction in the accessory olfactory (vomeronasal) system using alpha1B-deficient mice and beta3-deficient mice. RT-PCR studies showed the existence of beta1, beta2, beta3, beta4, alpha1A, alpha1B, and alpha1C subunits of voltage-dependent Ca2+ channels in the mouse VNO. Immunohistochemical studies showed that the alpha1A, alpha1B, and alpha1C subunits of voltage-dependent Ca2+ channels exist in the sensory neurons and supporting cells of the mouse VNO. Exposure of the VNO to urine samples excreted from male mice induced lower Fos-immunoreactivity in the periglomerular (PG) cells of the AOBs in alpha1B-deficient female mice than in those of wild mice. The density of Fos-immunoreactive (Fos-ir) cells after exposure to female urine samples at the periglomerular cell layer of alpha1B-deficient male mice was lower than that of wild mice. Exposure of the VNO of beta3-deficient female mice to male urine samples also induced low Fos-ir cells in the periglomerular cell layer of the AOB. These data suggest the importance of the alpha1B and beta3 subunits of the N-type voltage-dependent Ca2+ channel for the pheromone signal transduction system.

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1080/10799890600623373

Language：Others  

Language：English   Publishing type：Research paper (scientific journal)  

The voltage-dependent N-type Ca2+ channel is localized in the plasma membrane of insulin-releasing beta-cells and glucagon-releasing alpha-cells in the islets of Langerhans in the pancreas. To examine the contribution of N-type Ca2+ channel to glucose homeostasis, we performed glucose tolerance and insulin tolerance tests with N-type Ca2+ channel alpha(1B)-subunit-deficient mice on a normal or high-fat diet. The fasting glucose level in homozygous mice on the normal diet was significantly lower than those in wild and heterozygous mice. In glucose tolerance tests, the homozygous mice showed a higher glucose clearance rate and a similar pattern of insulin levels to those of wild and heterozygous mice. In insulin tolerance tests, glucose clearance rates showed no significant difference among wild, heterozygous and homozygous mice. In animals on the high-fat diet, food consumption was the same among wild, heterozygous and homozygous mice, but body weight gain was reduced in homozygous mice. After 8 weeks of the high-fat diet, homozygous mice showed lower fasting glucose levels and exhibited higher glucose clearance and lower insulin levels than wild or heterozygous mice in glucose tolerance tests. Glucose clearance rates showed no significant difference among wild, heterozygous and homozygous mice in insulin tolerance tests. After 10 weeks of the high-fat diet, the alpha(1B)-deficient homozygous mice showed lower lipid deposition in liver and lower plasma glucagon, leptin and triglyceride levels than wild or heterozygous mice. These results suggest that N-type Ca2+ channels play a role in insulin and glucagon release, and that N-type Ca2+ channel alpha(1B)-subunit deficient mice show improved glucose tolerance without any change in insulin sensitivity. Thus, N-type Ca2+ channel blockers might be candidate anti-diabetic/anti-obesity agents.

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DOI： 10.1007/s10528-005-5220-9

Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: ADAM22 is a member of the ADAM gene family, but the fact that it is expressed only in the nervous systems makes it unique. ADAM22's sequence similarity to other ADAMs suggests it to be an integrin binder and thus to have a role in cell-cell or cell-matrix interactions. To elucidate the physiological functions of ADAM22, we employed gene targeting to generate ADAM22 knockout mice. RESULTS: ADAM22-deficient mice were produced in a good accordance with the Mendelian ratio and appeared normal at birth. After one week, severe ataxia was observed, and all homozygotes died before weaning, probably due to convulsions. No major histological abnormalities were detected in the cerebral cortex or cerebellum of the homozygous mutants; however, marked hypomyelination of the peripheral nerves was observed. CONCLUSION: The results of our study demonstrate that ADAM22 is closely involved in the correct functioning of the nervous system. Further analysis of ADAM22 will provide clues to understanding the mechanisms of human diseases such as epileptic seizures and peripheral neuropathy.

Language：English   Publishing type：Research paper (scientific journal)  

The Ca2+ channel alpha1B subunit is a pore-forming component capable of generating N-type Ca2+ channel activity. Although N-type Ca2+ channel plays a role in a variety of neuronal functions, alpha1B-deficient mice exhibit normal life span without apparent abnormalities of behavior, histology or plasma norepinephrine level, presumably owing to compensation by some other Ca2+ channel alpha1 or beta subunit. In this study, we studied the levels of alpha1A, alpha1C, alpha1D, C1E, beta1, beta2, beta3 and beta4 mRNAs in adrenal gland of alpha1B-deficient mice. The alpha1A mRNA in homozygous mice was expressed at higher level than in wild or heterozygous mice, but no difference in the expression levels of alpha1c, alpha1D, alpha1E, beta1, beta2, beta3 and beta4 was found among wild, heterozygous and homozygous mice. The protein level of alpha1A in homozygous mice was also expressed at higher level than in wild or heterozygous mice. To examine whether increased expression is induced by cis-regulatory element within 5'-upstream region of alpha1A gene, we examined lacZ expression in alpha1B-deficient x alpha1A6.3-lacZ mice (carrying a 6.3-kb 5'-upstream fragment of alpha1A gene fused to E. coli lacZ reporter gene), which express lacZ in medullar chromaffin cells, but not in cortex. The levels of lacZ expression in homozygous alpha1B-deficient x alpha1A6.3-lacZ mice were higher than in wild or heterozygous mice. Therefore, a possible explanation of the normal behavior and plasma norepinephrine level of alpha1B-deficient mice is that compensation by alpha1A subunit occurs and that 6.3-kb 5'-upstream region of alpha1A gene contains enhancer cis-element(s) for compensation in adrenal medulla chromaffin cells.

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The Ca(2+) channel alpha(1B) subunit is a pore-forming component capable of generating N-type Ca(2+) channel activity. Although the N-type Ca(2+) channel plays a role in a variety of neuronal functions, alpha(1B)-deficient mice with a CBA/JN genetic background show no apparent behavioral or anatomical-histological abnormality, presumably owing to compensation by other Ca(2+) channels. In this study, we examined the mRNA expression of the alpha(1A), alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2), beta(3) and beta(4) subunits in the olfactory bulb, cerebral cortex, hippocampus and cerebellum of alpha(1B)-deficient mice. We found that the mRNA expression levels of the alpha(1A), alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2), beta(3) and beta(4) subunits were the same in the olfactory bulbs of wild, heterozygous and homozygous alpha(1B)-deficient mice. In the cerebral cortex, alpha(1A) mRNA in homozygous alpha(1B)-deficient mice was expressed at a higher level than in wild or heterozygous mice, but no difference in the expression levels of the alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2), beta(3) and beta(4) subunits was found among wild, heterozygous and homozygous mice. In hippocampus and cerebellum, beta(4) mRNA in homozygous alpha(1B)-deficient mice was expressed at a higher level than in wild or heterozygous mice, but no difference in the expression levels of the alpha(1A), alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2) and beta(3) subunits was found among wild, heterozygous and homozygous mice. These results suggest that the compensatory mechanisms differ in different brain regions of alpha(1B)-deficient mice with a CBA/JN genetic background.

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Although the N-type Ca2+ channel plays a role in a variety of neuronal functions, N-type Ca2+ channel alpha1B-deficient mice exhibit normal life span without apparent behavioral or histologic abnormalities. To examine whether the reason for their normal behavior is compensation by other Cav2 channel alpha1 or beta subunit genes and to analyze whether genetic background influences the subunit expression pattern, we studied the alpha1A, alpha1E, beta1b, beta2, beta3 and beta4 subunit mRNA levels in cerebellum of alpha1B-deficient mice with CBA x C57BL/6 or CBA/JN background. In cerebellum of the mice with a CBA x C57BL/6 background, alpha1A mRNA was expressed at a higher level than that in wild-type or heterozygous mice, but difference in the expression levels of alpha1E, beta1b, beta2, beta3 and beta4 subunits was not found among wild-type, heterozygous, and homozygous mice. In cerebellum of alpha1B-deficient mice with CBA/JN background, beta4 mRNA was expressed at a higher level than that in wild-type or heterozygous mice, but alpha1A, alpha1E, beta1b, alpha2, beta3 and transcripts were expressed at similar levels in all genotypes. Therefore, a possible explanation of the normal behavior of alpha1B-deficient mice is that Cav2 channel family members compensate for the deficiency, and that the change of functional subunit expression pattern for compensation differs in animals with different genetic backgrounds.

Language：English   Publishing type：Research paper (scientific journal)  

N-type and P/Q-type Ca2+ channels play an important role in the processing of olfactory information. However, N-type Ca2+ channel alpha1B-deficient mice show normal behavior, presumably owing to compensation by other Ca2+ channels. P/Q-type Ca2+ channel alpha1A mRNA was expressed at a higher level in olfactory bulb of homozygous alpha1B-deficient mice than wild-type or heterozygous mice. LacZ expression in olfactory mitral cells of homozygous alpha1B-deficient x alpha1A1.5-lacZ mice, carrying a 1.5-kb 5'-upstream fragment of the alpha1A gene fused to the lacZ reporter gene, was increased compared to that in wild-type or heterozygous mice. Therefore, a possible explanation for the normal behavior of alpha1B-deficient mice is compensation by the alpha1A gene and that the 1.5-kb 5'-upstream region of this gene contains an enhancer cis-element for compensation in olfactory mitral cells.

Language：English   Publishing type：Research paper (scientific journal)  

The Ca(2+) channel alpha(1B) subunit is a pore-forming component capable of generating N-type Ca(2+) channel activity. Although the N-type Ca(2+) channel plays a role in a variety of neuronal functions, alpha(1B)-deficient mice show normal behavior, presumably owing to compensation by the other Ca(2+) channels. In this study, we examined the mRNA expression of the P/Q-type Ca(2+) channel alpha(1A) subunit in cerebellum of alpha(1B)-deficient mice. The alpha(1A) subunit mRNA in homozygous alpha(1B)-deficient mice was expressed at a significantly higher level than in wild or heterozygous mice. To examine whether the increased expression is induced by a cis-regulatory element within the 5'-upstream region of the alpha(1A) subunit gene, we examined lacZ expression in alpha(1B)-deficient x alpha(1A)3.0-lacZ mice (carrying a 3.0-kb 5'-upstream fragment of the alpha(1A) subunit gene fused to Escherichia coli lacZ reporter gene), which express lacZ in granule but not in Purkinje cells, and in alpha(1B)-deficient x alpha(1A)6.3-lacZ mice (carrying a 6.3-kb 5'-upstream region fused to lacZ gene), which express lacZ in Purkinje but not in granule cells. The levels of lacZ expression in homozygous alpha(1B)-deficient x alpha(1A)3.0-lacZ mice were significantly higher than in wild or heterozygous mice, but no difference in lacZ expression level was found among wild, heterozygous and homozygous alpha(1B)-deficient x alpha(1A)6.3-lacZ mice. Therefore, a possible explanation of the normal behavior of alpha(1B)-deficient mice is that compensation by alpha(1A) subunit gene occurs and that the 3.0-kb 5'-upstream region of alpha(1A) subunit gene contains an enhancer cis-element(s) for compensation in cerebellar granule cells.

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DOI： 10.1002/hipo.10208

Language：Others   Publishing type：Research paper (scientific journal)  

Analysis of Gene Expression in Peripheral Blood Eosinophils from Patients with Atopic Dermatitis by Differential Display

DOI： 10.1159/000070478

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DOI： 10.1097/00001756-200108080-00027

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DOI： 10.1073/pnas.081089398

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Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/s1385-299x(00)00009-x

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DOI： 10.1016/s0304-3940(00)00964-2

Language：Others   Publishing type：Research paper (scientific journal)  

The omega-agatoxin-IVA-sensitive P/Q-type Ca(2+) channel plays a role in insulin release from the pancreatic islets of beta cells. To dissect the molecular mechanisms underlying beta cell expression of the P/Q-type channel, we characterized the 5'-upstream region of the mouse alpha(1A) subunit gene using transgenic mice and HIT insulinoma cells. The E. coli lacZ reporter gene was expressed in pancreatic acini and islets in transgenic mice carrying the 6.3 kb or 3.0 kb of the 5'-upstream region, although those with 1.5 kb or 0. 5 kb of the 5'-upstream region failed to show reporter expression on histological examination. As the expression of alpha(1A)subunit gene could not be detected in acini using RT-PCR analysis, the reporter expression in acini might have been ectopic expression. When linked to the placental alkaline phosphatase reporter gene to examine promoter activity for beta cell expression, the 6.3 kb and 3.0 kb fragment of the 5'-upstream region, but not the smaller 1.5 kb fragment, were able to drive reporter gene expression in HIT cells. The sequence between 3.0 and 1.5 kb upstream of the start codon enhanced thymidine kinase promoter activity in HIT cells, but not in fibroblast NIH3T3 cells. These results suggested that the beta cell-specific elements of the alpha(1A) subunit gene are likely to be located in the distal upstream region (-3021 to-1563) of the 5'-upstream sequence and that the 6.3 kb fragment of the 5'-upstream region alone might be a lack of a negative cis-regulatory element(s) to suppress the alpha(1A) subunit gene expression in acini.

DOI： 10.1677/jme.0.0240225

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DOI： 10.1016/s0006-8993(99)02077-6

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DOI： 10.1006/bbrc.1999.0857

Language：Others   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/0303-7207(94)90164-3

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Responsible for pages：総ページ数：219   Language：Japanese  

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