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Regressive and proliferative processes in the formation of cortico-spinal projection.

皮质脊髓投射形成中的退化和增殖过程。

基本信息

批准号：
20300138
负责人：
SAKURAI Masaki
金额：
$ 7.07万
依托单位：
Teikyo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2008
资助国家：
日本
起止时间：
2008 至 2010
项目状态：
已结题

项目摘要

We developed corticospinal projection system in vitro using slice co-cultures of rodent sensorimotor cortex and spinal cord, where we found thatcorticospinal (CS) synapses are formed diffusely in the spinal gray matter until 7 DIV butthose on the ventral side are eliminated later until 14 DIV in an NMDA receptor-dependent manner.In the present studies : (1) We labeled CS axons with EYFP transfected with electroporation and studied axonal regression from ventral side on long-term live imaging. There were three types of regressive events : retraction, amputation (autaxotomy), and degeneration. We analyzed their character in detail. (2) We co-cultured slices obtained from GluN2B or GluN2A KO mice and wild type animals. The synapse elimination was not seen when GluN2B KO spinal cord was cocultured with wild type cortex, indicating that postsynaptic (not presynaptic) GluN2B (rather than GluN2A) is essentially involved in the synapse elimination. Furthermore, differential effect of 2B and 2A seems to be downstream signaling rather than the difference in amount of Ca entry. (3) We studied developmentalchange in distribution of CS cells projecting to a single segment, C7, by injecting almost entire half gray matter of C7 at P7 and P56. At P7, projecting cells are widely distributed through motor-related cortex and somatosensory cortices, which was dramtically decreased in density at P56. This reduction was not due to cell death but axon elimination. (4) Wefound direct cortico-motoneuronal synapses at P7 rat by recording CS-EPSC from identified motoneuron retrogradely labeled with CTB. Only motoneurons innervate distal limb muscles received direct CS synapses.

本研究利用啮齿类动物感觉运动皮层和脊髓的切片共培养，在体外建立了皮质脊髓投射系统，发现皮质脊髓（CS）突触在脊髓灰质中弥漫性形成，直到7 DIV，而腹侧的CS突触在脊髓灰质中以NMDA受体依赖的方式消失，直到14 DIV。（1）我们用电穿孔转染的EYFP标记CS轴突，并在长期实时成像中从腹侧研究轴突消退。有三种类型的退行性事件：回缩、截肢（自体切开术）和退行性变。我们详细分析了他们的性格。(2)我们共培养从GluN 2B或GluN 2A KO小鼠和野生型动物获得的切片。当GluN 2B KO脊髓与野生型皮质共培养时，未观察到突触消除，表明突触后（非突触前）GluN 2B（而不是GluN 2A）基本上参与突触消除。此外，2B和2A的差异效应似乎是下游信号传导，而不是Ca内流量的差异。(3)我们通过在P7和P56注射几乎一半的C7灰质，研究了投射到单个节段C7的CS细胞分布的发育变化。在P7，投射细胞广泛分布于运动相关皮层和躯体感觉皮层，在P56其密度急剧下降。这种减少不是由于细胞死亡，而是由于轴突消除。(4)用CTB逆行标记运动神经元，在P7时记录皮质-运动神经元直接突触。只有支配远端肢体肌肉的运动神经元才能接受直接的CS突触。