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Synapse formation, development and plasticity of corticspinal synapse : An in vitro slice culture study

皮质脊髓突触的突触形成、发育和可塑性：体外切片培养研究

基本信息

批准号：
15300137
负责人：
SAKURAI Masaki
金额：
$ 4.03万
依托单位：
Teikyo University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

We developed corticospinal projection system with functional synapses in vitro, using slices of the rat sensorimotor cortex and spinal cord (Takuma et al., 2002). In the present study, we found that corticospinal synapses are formed widely in the entire spinal cord until 7 DIV, but those in the ventral side are eliminated and the synapse distribution is restricted to the dorsal side by 14 DIV, as seen in the in vivo adult. This synapse elimination is blocked by an NMDA receptor blocker APV, indicating that this process is activity dependent. Retrograde double labeling of the ventral and dorsal nerve terminals and axon reflex studies showed that the synapse elimination is due to axon branch elimination in the spinal cord rather than cell death of the parent cortical neurons. There is a critical period for the blocking effect of APV : APV has no effect before 6 DIV and after 11 DIV and the effect is irreversible when APV is removed from the medium after 11DIV. This is the first in vitro model for critical period of the developmental plasticity.We also showed above-mentioned synapse elimination occurred in vivo in a quite similar time course: synapses formed in the ventral spinal cord at P7 are eliminated at P8 -P11. Field EPSPs produced by corticospinal synapses in vitro are largely positive, while those in vivo are negative at active synaptic sites. To confirm the positive field EPSPs in vitro indicate active synaptic sites, we recorded corticospinal synaptic activity with fluorescent voltage sensitive dye We found amplitudes of optical signals and field EPSPs are well correlated.To study competitive relationship between the ventral and dorsal sides and clarify the site of action of APV, we devised compartmented chamber for slice co-culture system.

我们利用大鼠感觉运动皮层和脊髓的切片，在体外开发了具有功能突触的皮质脊髓投影系统（Takuma et al., 2002）。在本研究中，我们发现整个脊髓在7 DIV之前广泛形成皮质脊髓突触，但腹侧的突触被消除，突触分布在14 DIV时仅限于背侧，这在体内成人中是可见的。这种突触消除被NMDA受体阻断剂APV阻断，表明这一过程是活性依赖的。逆行双标记腹背神经末梢和轴突反射的研究表明，突触的消除是由于脊髓内轴突分支的消除，而不是由于亲本皮质神经元的细胞死亡。APV的阻断作用存在一个关键时期，在6 DIV之前APV没有作用，在11DIV之后APV没有作用，11DIV之后将APV从介质中移除效果不可逆。这是第一个发育可塑性关键期的体外模型。我们还发现，上述突触消除在体内发生的时间过程非常相似：在P7形成的脊髓腹侧突触在P8 -P11被消除。在体外，皮质脊髓突触产生的场epsp大部分是阳性的，而在体内，活跃的突触部位产生的场epsp是阴性的。为了证实体外正场EPSPs指示活跃的突触位点，我们用荧光电压敏感染料记录了皮质脊髓突触的活动。我们发现光信号的振幅与场EPSPs有很好的相关性。为了研究腹、背侧的竞争关系，明确APV的作用部位，我们设计了隔室切片共培养系统。