权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

In vitro reconstruction of corticospinal tract

皮质脊髓束的体外重建

基本信息

批准号：
11670611
负责人：
SAKURAI Masaki
金额：
$ 1.92万
依托单位：
Tekyo University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670611/
关键词：
Slice culture Coculture Corticospinal tract Cerebral cortex Spinal cord Synapse formation

项目摘要

In order to study biological properties of the corticospinal tract, we have reconstructed this system in an in vitro slice culture preparation. Motor cortex and spinal cord slices, prepared from newborn rats, were co-cultured on pored membranes for 16-24 days. Anterograde labeling with biocytin showed that substantial neural connections had formed between the cortex and spinal cord slices. Retrograde labeling with HRP or DiI demonstrated that the parent cells were located primarily in the deeper layer of the cortex, as is found in vivo. Stimulation of the deep layer of the cortex elicited extracellular postsynaptic responses and intracellular excitatory postsynaptic potentials (EPSPs) in the co-cultured spinal cord that were mediated by the AMPA/KA type glutamate receptor. The intracellular injection of biocytin after EPSPs were recorded showed that one-third of these cells were large stellate cells, which are thought to be motoneurons, while a large portion of the remaining labeled cells were bipolar cells of smaller sizes Using this reconstructed in vitro preparation, we recorded field EPSPs (fEPSPs) along a 100 μm-interval lattice in the spinal gray matter, which allowed the quantitative evaluation of synapse formation. The fEPSP amplitudes were more than two-fold larger when the forelimb cortex was co-cultured with cervical cord rather than lumbar cord. However, hindlimb cortex did not show this preference. The fEPSP amplitudes were more than twice as large when the dorsal side of the spinal cord was adjacent to the cortex than the ventral side. In summary, we have reconstructed the corticospinal projection and synapses in vitro using cortical and spinal explants. This system allows for an efficient quantitative evaluation of synapse formation and for studies of postsynaptic cells. Our results suggest that synapse formation shows preferences along and perpendicular to the neuraxis of the spinal cord.

为了研究皮质脊髓束的生物学特性，我们在体外切片培养制备中重建了这个系统。将从新生大鼠制备的运动皮质和脊髓切片在多孔膜上共培养16 - 24天。生物胞素顺行标记显示皮质和脊髓切片之间形成了大量的神经连接。HRP或DiI逆行标记表明，母细胞主要位于皮层的深层，如在体内发现的。刺激皮层深层可引起共培养脊髓的细胞外突触后反应和细胞内兴奋性突触后电位（EPSP），这些反应均由AMPA/KA型谷氨酸受体介导。记录EPSP后细胞内注射生物胞素显示，这些细胞中有三分之一是大型星状细胞，被认为是运动神经元，而剩余的标记细胞中有很大一部分是较小尺寸的双极细胞。使用这种重建的体外制备，我们记录了脊髓灰质中沿100 μ m间隔晶格的场EPSP（fEPSP），这允许对突触形成进行定量评估。当前肢皮质与颈髓而不是腰髓共培养时，fEPSP振幅是2倍以上。然而，后肢皮层没有表现出这种偏好。当脊髓背侧邻近皮质时，fEPSP振幅是腹侧的两倍多。总之，我们已经重建了皮质脊髓投射和突触在体外使用皮质和脊髓外植体。该系统允许一个有效的定量评价突触的形成和突触后细胞的研究。我们的研究结果表明，突触的形成显示出偏好沿着和垂直于脊髓的神经轴。