Inhibitory neurons are crucial for shaping and regulating the dynamics of the entire network, and disturbances in these neurons contribute to brain disorders. Despite the recent progress in genetic labeling techniques, the heterogeneity of inhibitory neurons requires the development of highly characterized tools that allow accurate, convenient, and versatile visualization of inhibitory neurons in the mouse brain. Here, we report a novel genetic technique to visualize the vast majority and/or sparse subsets of inhibitory neurons in the mouse brain without using techniques that require advanced skills. We developed several lines of Cre-dependent tdTomato reporter mice based on the vesicular GABA transporter (VGAT)-BAC, named VGAT-stop-tdTomato mice. The most useful line (line #54) was selected for further analysis based on two characteristics: the inhibitory neuron-specificity of tdTomato expression and the transgene integration site, which confers efficient breeding and fewer adverse effects resulting from transgene integration-related genomic disruption. Robust and inhibitory neuron-specific expression of tdTomato was observed in a wide range of developmental and cellular contexts. By breeding the VGAT-stop-tdTomato mouse (line #54) with a novel Cre driver mouse line, Galntl4-CreER, sparse labeling of inhibitory neurons was achieved following tamoxifen administration. Furthermore, another interesting line (line #58) was generated through the unexpected integration of the transgene into the X-chromosome and will be used to map X-chromosome inactivation of inhibitory neurons. Taken together, our studies provide new, well-characterized tools with which multiple aspects of inhibitory neurons can be studied in the mouse.

We investigated whether in utero or lactational exposure to 4-hydroxy-2',3,3',4',5'-pentachlorobiphenyl (OH-PCB 106) affects spontaneous locomotor activity and motor coordination in young adult male mice. For in utero exposure, pregnant C57BL/6J mice received 0.05 or 0.5 mg/kg body weight of OH-PCB 106 or corn oil vehicle via gavage every second day from gestational day 10 to 18. For lactational exposure, the different groups of dams received 0.05 or 0.5 mg/kg body weight of OH-PCB 106 or corn oil vehicle via gavage every second day from postpartum day 3 to 13. At 6-7 weeks of age, the spontaneous locomotor activities of male offspring were evaluated for a 24-hr continuous session in a home cage and in an open field for 30-min. Motor coordination function on an accelerating rotarod was also measured. Mice exposed prenatally to OH-PCB 106 showed increased spontaneous locomotor activities during the dark phase in the home cage and during the first 10-min in the open field compared with control mice. Mice exposed lactationally to OH-PCB 106, however, did not show a time-dependent decrease in locomotor activity in the open field. Instead, their locomotor activity increased significantly during the second 10-min block. In addition, mice exposed lactationally to OH-PCB 106 displayed impairments in motor coordination in the rotarod test. These results suggest that perinatal exposure to OH-PCB 106 affects motor behaviors in young adult male mice. Depending on the period of exposure, OH-PCB 106 may have different effects on neurobehavioral development.

Background: Thyroid hormone (TH) plays a key role in the developing brain, including the cerebellum. TH deficiency induces organizational changes of the cerebellum, causing cerebellar ataxia. However, the mechanisms causing these abnormalities are poorly understood. Various animal models have been used to study the mechanism. Lacking dual oxidase (DUOX) and its maturation factor (DUOXA) are major inducers of congenital hypothyroidism. Thus, this study examined the organizational changes of the cerebellum using knockout mice of the Duoxa gene (Duoxa-/-).
Methods: The morphological, behavioral, and electrophysiological changes were analyzed in wild type (Wt) and Duoxa-deficient (Duoxa-/-) mice frompostnatal day (P) 10 to P30. To detect the changes in the expression levels of presynaptic proteins, Western blot analysis was performed.
Results: The proliferation and migration of granule cells was delayed after P15 in Duoxa-/-mice. However, these changes disappeared by P25. Although the cerebellar structure of Duoxa-/-mice was not significantly different from that of Wt mice at P25, motor coordination was impaired. It was also found that the amplitude of paired-pulse facilitation at parallel fiber-Purkinje cell synapses decreased in Duoxa-/-mice, particularly at P15. There were no differences between expression levels of presynaptic proteins regulating neurotransmitter release at P25.
Conclusions: These results indicate that the anatomical catch-up growth of the cerebellum did not normalize its function because of the disturbance of neuronal circuits by the combined effect of hypothyroidism and functional disruption of the DUOX/DUOXA complex.

Thyroid hormones play crucial roles in the development and functional maintenance of the central nervous system. Despite extensive studies of the neural function of thyroid hormones, little is known about the effects of hypothyroidism on behavioural traits and the mechanisms underlying such effects. In the present study, we report an investigation of congenitally hypothyroid mutant rdw rats, revealing a novel function of thyroid hormones in the central nervous system. The rdw rats were subjected to behavioural analyses such as the rotarod test, open field test and circadian activity measurement. To determine the cause of behavioural disorders, cerebellar morphogenesis was examined by immunohistochemical analysis, and the axonal transport of dopamine in the nigrostriatal pathway was analysed by high-performance liquid chromatography and western blotting. The effects of thyroxine administration to the rdw rats were examined by behavioural analysis. The rdw rats showed severe impairment of motor coordination and balance. This could be explained by the fact that the rats showed severe retardation of cerebellar morphogenesis, which correlates with the small somata and poor dendritic arborisation of Purkinje cells and retarded migration of granule cells particularly during the first two postnatal weeks. Moreover, the rdw rats showed hypoactivity, characterised by decreased circadian locomotor activity. After weaning, thyroxine administration improved the dwarfism in rdw rats but had no effect on cerebellar function. In addition, the rdw rats showed anxiety and depression intrinsically to novel surroundings. Interestingly, the rdw rats showed high levels of dopamine in the substantia nigra and low levels in the striatum, an important centre for the coordination of behaviour. Furthermore, low levels of tubulin in the striatum were detected, indicating the aberrant axonal transport of dopamine in the nigrostriatal pathway as a result of the reduced delivery of microtubules. These findings indicate an important function of thyroid hormones in cerebellar formation and in the regulation of axonal transport of dopamine. Moreover, rdw rats will be useful for studies of brain function and behavioural disorders in congenital hypothyroidism.

発達期の脳は非常に脆弱であるため，この時期の環境化学物質の曝露によって，子どもの脳発達に影響を及ぼし，さまざまな異常を引き起こすことが示されてきた．初代培養系を用いたこれまでの研究から，4-hydroxy-2',3,3',4',5'-pentachlorobiphenyl（水酸化PCB）曝露が小脳プルキンエ細胞の樹状突起の伸長に影響を及ぼすことが報告されている（Kimura-Kuroda et al., 2007）．しかし低用量水酸化PCBの周産期曝露によって仔の小脳機能の発達にどのような影響を及ぼすかはあまり分かっていない．本研究では，胎仔期または授乳期に水酸化PCBを曝露したマウスを用いて，ロータロッド試験によって運動協調機能を検証し，水酸化PCB曝露が小脳機能に影響を及ぼすかについて検討した．胎仔期曝露条件では，妊娠したC57BL/6Jマウスに，コーン油に溶解した水酸化PCBを0.05または0.5 mg/kg b.w.の濃度で，妊娠10日目から18日目まで1日おきに投与した．授乳期曝露条件では，母獣にOH-PCB 106を0.05または0.5 mg/kg b.w.の濃度で，出産後3日目から13日目まで1日おきに投与した．これらの母獣の雌雄仔が成熟後にロータロッド試験を行なった．その結果，いずれの曝露条件においても，母獣の体重変化には影響がなかった．胎仔期曝露条件における産仔数や授乳期曝露による仔の死亡率も対照群と比べて有意な差はなかった．ロータロッド試験では，授乳期0.05 および0.5 mg/kg b.w.曝露群の雄仔において，試験の成績が対照群と比べて有意に低下した．このことから，水酸化PCBの曝露は，仔の小脳機能発達に影響を及ぼすことが示された．また水酸化PCBの曝露影響は，曝露時期や性によって異なることが示唆された．

A histoanatomical context is imperative in an analysis of gene expression in a cell in a tissue to elucidate physiological function of the cell. In this study, we made technical advances in fluorescence laser microdissection (LMD) in combination with the absolute quantification of small amounts of mRNAs from a region of interest (ROI) in fluorescence-labeled tissue sections. We demonstrate that our fluorescence LMD-RTqPCR method has three orders of dynamic range, with the lower limit of ROI-size corresponding to a single cell. The absolute quantification of the expression levels of the immediate early genes in an ROI equivalent to a few hundred neurons in the hippocampus revealed that mice transferred from their home cage to a novel environment have distinct activation profiles in the hippocampal regions (CA1, CA3, and DG) and that the gene expression pattern in CA1, but not in the other regions, follows a power law distribution.

The amnesic effects of excitotoxic lesions of the rat retrosplenial cortex (RS) and hippocampus (HPC) in the spontaneous object recognition (SOR) performance were investigated. The SOR test consisted of the sample-exposure session(s) and a test session. First, to test retrograde amnesia, rats received four sample-exposure sessions within a day at 4 weeks and 1 day before the surgery, respectively. In the test sessions conducted 1 week after the surgery, both lesion groups showed a temporally ungraded retrograde amnesia. Second, to test anterograde amnesia, 1- and 4-week retention intervals were inserted between the four sample-exposure sessions and the test session. The RS-lesioned rats showed a retention interval-dependent impairment in the test sessions, while the HPC-lesioned rats showed an impairment regardless of the retention interval. Finally, to test short-term recognition memory, 5- or 30-min delay was interposed between the single sample-exposure session and the test session. Both lesion groups performed normally irrespective of the delay length. These results suggest that both the RS and HPC are important for long-term object recognition memory, but these areas have different roles in it. (c) 2012 Wiley Periodicals, Inc.

2012/12/13共同通信ニュース、東京新聞、信濃毎日新聞他、12/14東奥日報、熊本日日新聞、長崎新聞、ヤフーニュース・ヘッドライン他、12/18日刊工業新聞他、12/20ナショナル・ジオグラフィック・ニュース他報道

There has been a long-standing need to develop efficient and standardized behavioral test methods for evaluating higher-order brain functions in mice. Here, we developed and validated a behavioral flexibility test in mice using IntelliCage, a fully automated behavioral analysis system for mice in a group-housed environment. We first developed a "behavioral sequencing task" in the IntelliCage that enables us to assess the learning ability of place discrimination and behavioral sequence for reward acquisition. In the serial reversal learning using the task, the discriminated spatial patterns of the rewarded and never-rewarded places were serially reversed, and thus, mice were accordingly expected to realign the previously acquired behavioral sequence. In general, the tested mice showed rapid acquisition of the behavioral sequencing task and behavioral flexibility in the subsequent serial reversal stages both in intra- and inter-session analyses. It was found that essentially the same results were obtained among three different laboratories, which confirm the high stability of the present test protocol in different strains of mice (C57BL/6, DBA/2, and ICR). In particular, the most trained cohort of C578L/6 mice achieved a markedly rapid adaptation to the reversal task in the final phase of the long-term serial reversal test, which possibly indicated that the mice adapted to the "reversal rule" itself. In conclusion, the newly developed behavioral test was shown to be a valid assay of behavioral flexibility in mice, and is expected to be utilized in tests of mouse models of cognitive deficits. (C) 2011 Elsevier B.V. All rights reserved.

We here introduce characteristic features of the novel behavioral tests, named Flavor Map and IntelliCage, recently developed to elucidate how maternal exposure to environmental chemicals affects executive brain functions in rats and mice. Flavor Map test requires rats to learn the existence of a schema of paired-associate of flavor and location, and enables to examine the short-term memory, long-term memory and paired-associative learning ability in adult rats. We have found that maternal exposure to a low dose of dioxin causes deficit in the paired-associative memory formation. Using the IntelliCage system, we have developed our own test protocol in mice. IntelliCage is versatile in terms of accommodation of as many as 16 mice at a time to monitor and record their basic physical activities and learning function. Our protocol successfully increases the efficiency of detection of the learning behavior with minimal variations between individual mice. We found that male mice born to dioxin-administered dams develop less behavioral flexibility. We believe that these behavioral tests can be used to study the executive functions in rats and mice, in biomedical research fields not only in toxicological study but also in animal psychology.

Environmental-level in utero and lactational exposures to dioxins have been considered to affect brain functions of offspring. Here, we determined whether in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD), at the dose that does not harm the dams, affects the acquisition and retention of fear memory in mouse offspring. Pregnant C57BL/6J mice were administered by gavages TCDD or TBDD at a dose of 0 or 3.0 mu g/kg body weight on gestation day 12.5, and their male offspring were examined for their behavior in adulthood. In the fear conditioning, a paired presentation of tone and foot shock was repeated three times, and retention tests for contextual and auditory fear memory were carried out 1 and 24 h after the fear conditioning. Groups of mice that were exposed to TCDD and TBDD in utero and via lactation showed deficits in the contextual and auditory retention tests at 1 and 24 h retention intervals. The present results suggest that maternal exposure to a low dose of TCDD or TBDD disrupts the functions of memory and emotion in male mouse offspring, and that the developmental toxicities of these chemicals are similar to each other. (C) 2010 Elsevier Inc. All rights reserved.

Retrograde and anterograde amnesic effects of excitotoxic lesions of the rat retrosplenial cortex (RS) and hippocampus (HPC) were investigated. To test retrograde amnesia, rats were trained with two-arm place discrimination in a radial maze 4 wk and 1 d before surgery with a different arm pair, respectively. In the retention test 1 wk after surgery, both lesion groups showed temporally ungraded retrograde amnesia. To test anterograde amnesia, animals were trained after surgery to discriminate three arm pairs successively within a day, and then after interposition of 1- to 4- wk intervals, one of these pairs, respectively, was tested for retention. RS-lesioned rats could acquire these pairs of place discriminations rapidly but showed a retention interval-dependent impairment in the retention test. Conversely, HPC-lesioned rats took more sessions to acquire these pairs than did the control group, and their retention was similar to 70% of correct performance regardless of retention interval. Results suggest that RS and HPC have different roles in spatial memory and that RS is important for remote memory process.