Genetic analysis for learning and instinctive behaviors by gene targeting mice.

通过基因靶向小鼠对学习和本能行为进行遗传分析。

• 批准号: 06454674
• 负责人: YAGI Takeshi
• 金额: $ 4.48万
• 依托单位: (National Institute of Physiological Sciences) Okazaki National Research Institutes
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06454674/
• 关键词: Gene targeting; Fyn; NMDA; Emotion; RNP; GABA; Seizure; behavior; ピューロマイシン; 行動; ターゲティング; チロシンキナーゼ

The mammalian nervous system constitutes an enormous neural network and regulates various behaviors such as locomotion, sense, recognition, emotion, memory and learning. The question is how these neural networks are systematically formed during development and how they regulate behaviors. Fyn is a member of the Src family kinases, a non-receptor-type tyrosine kinase and its deficiencies causes impairments of spatial learning in mice. In this reserch project, firstly we have confirmed that gene disruption of Fyn causes several other behavioral abnormalities. They have suckling problem and higher fear-response scores in the novelty preferance and passive avoidance tests, exhibit stronger light aversion in the light-dark choice test, and are hyperresponsive to fear-inducing environments and to acoustically primed audiogenic seizures. Anatomical defects in the neural cell layr of the hippocampal formation and in the glomeruli of the olfactory bulb were also observed in the mutants. Thus, t … More he Fyn signaling pathway should offer insights into the morphological, electrophysiological, and behavioral events in the mammalian nervous system. Second we focused to molecular function of Fyn during brain formation and on several behavior formation. We examined the susceptibility to seizures induced by various convulsive drugs, including, pentylenetetrazol, picrotoxin, bicuculline, kainic acid, N-methyl-D-aspartate (NMDA) and strychine. Fyn-deficient mice were significantly more likely to show myoclonic convulsions, when pentylenetetrazol, picrotoxin, bicuculline, kainic acid, or NMDA were administered. On the other hand, no difference in seizure susceptibility was found in Fyn-deficient mice, when strychinine was administered. These results provide evidence of abnormal susceptibility to seizures induced by anti-GABAergic agents and agonists for central excitatory amino acids in Fyn-deficient mice. And we isolated five cDNA clones that directly associate with Fyn in neonatal mouse brain by using a two hybrid yeast system. Sequence analyzes revealed that three of them are previously reported molecules, SON,tctex-1 and hnRNP K,and two clones encode novel sequences. The hnRNP K has been reported to asssociate with Fyn. A full-length cDNA of novel clone 82 was obtained and its deduced amino acid sequence was homologous to the RNA-binding proteins. Isolation of many Fyn-binding molecules suggest that, in the mouse brain, Fyn mediates multiple signaling patheways by binding to multiple molecules and that some of these pathways play critical roles in determing a certain type of behavior. Third we newly producted gene-knocked out mice for NMDA receptor subtypes ; epsilon1, epsilon2, epsilon4, glutamate receptor ; delta2, and GAD65. These gene-dificient mice have impaired of spatial learning, defects of suckling behavior, reduced spontaneous activity, malformation of synapse and different susceptibility to seizure-induced drug, respectivelity. We think to be successful in Less

哺乳动物的神经系统构成了一个庞大的神经网络，调节着运动、感觉、识别、情感、记忆和学习等各种行为。问题是这些神经网络是如何在发展过程中系统形成的，以及它们是如何规范行为的。Fyn是Src家族的成员，是一种非受体类型的酪氨酸激酶，它的缺陷会导致小鼠空间学习的障碍。在这个研究项目中，我们首先证实了Fyn基因的破坏会导致其他几种行为异常。他们有哺乳问题，在新奇偏好和被动回避测试中有较高的恐惧反应分数，在明暗选择测试中表现出更强的光厌恶，对恐惧诱导的环境和声学启动的听源性癫痫具有高反应。在突变体中还观察到了海马结构神经细胞层和嗅球肾小球的解剖缺陷。因此，t…更多的HE FYN信号通路应该为哺乳动物神经系统的形态、电生理和行为事件提供深入的了解。其次，我们重点研究了Fyn在脑形成过程中的分子功能以及几种行为的形成。我们检测了不同的惊厥药物，包括戊四氮、印防己毒素、荷包牡丹碱、红藻氨酸、N-甲基-D-天冬氨酸(NMDA)和士的宁诱发癫痫的敏感性。当注射戊四氮、印防己毒素、荷包牡丹碱、红藻氨酸或NMDA时，FYN缺陷小鼠更容易出现肌阵挛发作。另一方面，当给予士的宁时，在Fyn缺陷的小鼠中没有发现癫痫敏感性的差异。这些结果提供了抗GABA能药物和中枢兴奋性氨基酸激动剂对Fyn缺陷小鼠癫痫异常易感性的证据。并利用双杂交酵母系统从新生小鼠脑内分离到5个与Fyn基因直接相关的基因克隆。序列分析表明，其中三个是已报道的分子SON、tctex-1和hnRNP K，两个克隆编码新的序列。据报道，hnRNP K已与Fyn关联。获得了新克隆82的全长cDNA，其推导的氨基酸序列与RNA结合蛋白同源。许多Fyn结合分子的分离表明，在小鼠的大脑中，Fyn通过与多个分子结合来调节多个信号通路，其中一些通路在决定某种类型的行为方面发挥着关键作用。第三，我们新培育了NMDA受体亚型的基因敲除小鼠：谷氨酸受体epsilon1、epsilon2、epsilon4、delta2和GAD65。这些基因缺陷的小鼠分别存在空间学习障碍、哺乳行为缺陷、自发活动减少、突触畸形和对致痫药物的不同敏感性。我们认为用更少的时间就能取得成功

项目成果

Yagi,T.: "Gene targeting of fyn tyrosine kinase in murine nervous system.Genes for development,Cell growh and infectious diseases" Genes for development,Cell growth and infectious diseases,John Libbey Eurotext in Paris. 20. 17-27 (1995)

Yagi,T.：“小鼠神经系统中 fyn 酪氨酸激酶的基因靶向。发育、细胞生长和传染病的基因”发育、细胞生长和传染病的基因，John Libbey Eurotext，巴黎。

Miyakawa,T.,Yagi,T.,Kagiyama,A.and Niki,H.: "Radial maze performance,open-field and elevated plus-maze behaviors in Fyn-kinase deficient mice ; Further evidence for increased fearfulness" Molecular Brain Research. 37. 145-150 (1996)

Miyakawa,T.、Yagi,T.、Kagiyama,A. 和 Niki,H.：“Fyn 激酶缺陷小鼠的径向迷宫表现、开放场和升高的十字迷宫行为；恐惧增加的进一步证据”分子脑研究

Asada,H.,et al: "Mice lacking the 65 kDa isoform of glutamic acid decarboxylase (GAD65) maintain normal levels of GAD67 and GABA in their brains but are susceptible to seizures" Biochemical and Biophysical Research Communications. 229. 891-895 (1996)

Asada, H. 等人：“缺乏 65 kDa 谷氨酸脱羧酶 (GAD65) 亚型的小鼠大脑中 GAD67 和 GABA 保持正常水平，但容易癫痫发作”《生物化学和生物物理研究通讯》。

Umemori,H.,et al: "Initial events of myelination involve Fyn tyrosine kinase signalling" Nature. 367. 572-576 (1994)

Umemori, H., et al：“髓鞘形成的初始事件涉及 Fyn 酪氨酸激酶信号传导”Nature。

八木健 等: "遺伝子欠損マウスにおける行動異常解析" 実験医学. 12. 2456-2460 (1994)

Ken Yagi 等人：“基因缺陷小鼠的行为异常分析”《实验医学》12. 2456-2460 (1994)。