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Research on mechanisms of development and collapse of the pial and glial limiting membranes, which comprise the cerebrospinal fluid-brain barrier

组成脑脊液-脑屏障的软脑膜和神经胶质限制膜的发育和塌陷机制研究

基本信息

批准号：
16500219
负责人：
KIKKAWA Satoshi
金额：
$ 1.92万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

We anterogradely labeled entorhinodentate axons in wild type and reeler mice at adult and neonatal stages. The results revealed that pioneer axons of the entrhinodentate tract develop similarly and pass through the hippocampal fissure at postnatal day 1(P1) in wild type and reeler mice. In the reeler mutant, however, follower axons can not pass through the fissure and detour around it, whereas those in wild type mice follow their pioneer and continue to pass through the fissure. Immunohistochemical analysis of postnatal development of the hippocampal area using anti-GFAP antibody demonstrated aberrant accumulation of GFAP-positive astroglia along the hippocampal fissure in the reeler but not wild type brain. This accumulation of astroglia seems to prevent the entorhinohippocampal axons from passing through the hippocampal fissure and forces them to detour along the fissure in the reeler mutant.Next, we double-labeled neocortical neurons in the fukutin-deficient chimeric mouse with antero- or retro-grade axonal tracers and anti-laminin antibody to investigate relationship between collapse of the glial limiting membranes due to fukutin-deficiency and malpositioning of neocortical neurons. The results showed that a population of neocortical neurons positioned ectopically in the area just beneath the disrupted limiting membranes which lack laminin immunoreactivity, suggesting that the glial limiting membranes have certain effects on cortical neuronal migration.Thirdly, we cloned and sequenced the complete coding region of zebrafish fukutin. Zebrafish fukutin showed a significant degree of sequence conservation. Fukutin mRNA was detected a wide variety of tissues including the brain and the spinal cord. We also tried knocking-down fukutin protein expression by antisense morpholino oligonucleotide microinjection into fertilized eggs. Further investigation of effects of fukutin knock-down on development of the central nervous system is currently being continued.

我们在成年和新生阶段的野生型和卷尾型小鼠中顺行标记了内鼻齿状轴突。结果表明，野生型和绞股型小鼠在出生后第1天(P1)，鼻齿状束的前驱轴突发育相似，并穿过海马裂。然而，在卷轴突变体中，跟随轴突不能穿过裂隙并绕过裂隙，而在野生型小鼠中，跟随轴突的轴突跟随它们的先驱继续穿过裂隙。用抗GFAP抗体对出生后海马区发育的免疫组织化学分析显示，在卷绕型脑中，GFAP阳性星形胶质细胞沿着海马裂异常聚集，而不是野生型脑。星形胶质细胞的这种积聚似乎阻止了内鼻海马轴突穿过海马裂，并迫使它们沿着海马裂绕道而行。接下来，我们用顺行或逆行轴突示踪剂和抗层粘连蛋白抗体对Fukutin缺陷嵌合小鼠的新皮质神经元进行双重标记，以探讨由于Fukutin缺乏导致的神经胶质限制膜崩溃与新皮质神经元错位之间的关系。结果表明，新皮质神经元群体位于破裂层界膜下缺乏层粘连蛋白免疫反应的区域，提示胶质细胞界膜对皮质神经元的迁移有一定的影响。斑马鱼Fukutin显示出显著的序列保守性。在包括大脑和脊髓在内的各种组织中都检测到了Fukutin mRNA。我们还尝试了通过向受精卵中注射反义吗啉寡核苷酸来下调Fukutin蛋白的表达。目前仍在继续进一步调查击倒河豚对中枢神经系统发育的影响。