5-HT和多巴胺对运动中枢模式发生器神经元调节的新靶点及作用机制研究

5-HT and dopamine play a significant role in both the generation of stable rhythmic locomotion and recovery of locomotion after spinal cord injury (SCI). The cellular and molecular mechanisms behind this influence are, however, still not very clear. Results we have so far obtained show that Cav1.3 calcium channels, located in distal dendrites of locomotor generator neurons in the lamprey spinal cord, not only play a key role during rhythmic oscillations, but also contribute to the postinhibitory rebound (PIR) depolarization following a period of inhibition/hyperpolarization, - one of the intrinsic membrane properties related to the left - right alternation and the stability of locomotion. Furthermore, 5-HT, acting via 5-HT1A receptors, reduce the PIR by targeting Cav1.3 calcium channels, as does dopamine, acting via D2 receptors. The current goal is to simultaneously observe the influence of 5-HT and dopamine on NMDA-induced membrane potential oscillations and on calcium fluctuations, using real-time multiphoton confocal calcium imaging in combination with intracellular recordings of membrane potential. With this approach, we hope to unravel the targets of action and the subtypes of receptors which are responsible for the modulatory influence of 5-HT and dopamine; and in particular the mechanisms related to calcium and Cav1.3 calcium channels. Such new and detailed knowledge of the neuronal and molecular mechanisms related to the generation and modulation of locomotion will add importantly to the theoretical platform that is necessary for advancing strategies for the recovery of locomotion after SCI, including designing new drugs for treatment.

5-HT和多巴胺在正常运动的产生及调节和脊髓损伤运动功能恢复中起重要作用。但其机制尚未阐明。我们已有的研究发现:七鳃鳗脊髓运动中枢模式发生器神经元远端树突上的Cav1.3钙通道不仅在与运动节律产生相关的膜电位振荡的形成中起着关键作用，而且也与运动行为左右交替和稳定性密切相关的后抑制反弹特性的形成中起关键作用；同时5-HT和多巴胺分别作用于5-HT1A和D2受体，并通过影响Cav1.3钙通道而降低后抑制反弹，进而促进运动的产生。本课题拟在此结果基础上将实时多光子激光共聚焦显微镜和电生理技术相结合，研究5-HT和多巴胺影响膜电位振荡和与之同时发生的钙离子振荡；发现5-HT和多巴胺对膜电位振荡具有影响及所结合的受体亚型，明确Cav1.3钙通道是5-HT和多巴胺影响膜电位振荡和后抑制反弹进而调节运动的双重靶点，完善运动产生及调节的细胞分子机制，为脊髓损伤后运动功能的恢复提供设计治疗药物的理论依据。

我们对5-HT和多巴胺对运动中枢模式发生器神经元调节的新靶点及细胞分子水平作用机制开展了研究，发现“在七鳃鳗运动网络神经元存在平台电位时程长和短两种类型的膜电位振荡： 具有明显去极化平台时相的“平台样”振荡和没有明显去极化平台时相的“尖峰样”振荡；随着NMDA浓度的增加，振荡频率增加，但不是无限制地增加，NMDA达到一定浓度时，频率达到最大。这种情况均发生在上述膜电位振荡的两种模式中。我们还发现：膜电位振荡的频率与膜电位水平密切相关：去极化会使频率增加，此现象发生在不同浓度NMDA诱导膜电位振荡发生的情况下；它们依赖于脊髓中5-HT内源性释放的水平的高低来调整转换。高水平的5-HT活性将呈现平台样振荡，这与缓慢而有力的运动相关，主要存在于七鳃鳗在水下抵抗水流压力增加或在沙子中挖洞时；相反低水平5-HT活性将呈现尖峰样振荡，主要发生在快速运动时。我们在神经细胞水平观察5-HT和其5-HT1A受体阻断剂spiperone对NMDA诱导的膜电位振荡的影响，发现5-HT作用阻断电压依赖性钙通道，从而调节钙依赖钾通道活性。5-HT延长NMDA诱导的膜电位振荡平台电位时程，而5-HT1A受体阻断剂spiperone缩短此平台电位时程，说明5-HT通过作用在5-HT1A受体亚型对NMDA诱导的膜电位振荡产生影响, 5-HT是通过5-HT1A受体实现对运动的调节的。我们进一步观察了多巴胺对NMDA诱导的膜电位振荡平台电位时程的影响,发现多巴胺降低平台电位时程, 而延长平台之间的周期, 振荡幅度和整个周期改变不显著。我们将实时多光子激光共聚焦显微镜和电生理技术相结合，探讨L，N和 P/Q亚型钙离子通道在七鳃鳗脊髓运动网络神经元上分布部位，进一步证实了与5-HT和多巴胺的调节作用位点相关的L亚型钙离子通道存在部位在脊髓神经元远端树突，而与之调节作用不相关的N, P/Q亚型主要存在于脊髓神经元胞体和近端树突。这些结果不仅仅提供脊髓神经网络正常运动时的信息并且也将为明确脊髓损伤等一些运动神经系统疾病发病机制提供依据，为脊髓损伤后运动功能的恢复提供设计治疗药物的理论依据。