脊髓损伤小鼠小脑皮层神经元活动及突触传递的重塑机制

Spinal cord injury (SCI) not only induces dysfunction of limbs under the damaged spinal levels, but also impairs the functional activity of higher level central nervous system. Cerebellum is a motor center. SCI could induce decreases in the number of granule cell (GC) axons. Our preliminary experiment showed that SCI induced a significant change in the Purkinje cell (PC) spontaneous spike firing activity, suggesting that SCI may induce structural disorder and functional remodeling in cerebellar cortical neuronal circuitry. However, the mechanism of SCI induced structural disorder and functional remodeling is unclear. Applicant will use electrophysiological and pharmacological methods to study the changed properties and pharmacological mechanisms of spontaneous activity in cerebellar cortical PC, molecular layer interneurons (MLI) and GC in anesthetized SCI mice at the middle and late period, and to explore the mechanism of SCI-induced changes in cerebellar cortical neuronal activity. We will also investigate the mechanism of changes in excitatory postsynaptic currents evoked at parallel fiber (PF)-PC, PF-MLI and mossy fiber (MF)-GC synapses in acute cerebellar slices of SCI mice, and to explore the signal mechanism and effect of SCI on long-term synaptic plasticity induced at PF-PC synapses. We aim to understand the mechanisms of SCI affect neuronal activity, synaptic transmission and PC-PC presynaptic long-term plasticity in cerebellar cortex, and to classify the mechanisms of remodeling cerebellar cortical neuronal activity and synaptic transmission after spinal cord injury.

脊髓损伤（SCI）导致损伤节段下肢体功能障碍，并影响上级中枢功能活动。小脑是重要的运动中枢，SCI会导致小脑皮层颗粒细胞（GC）轴突数量减少；预实验发现：SCI小鼠小脑皮层浦肯野细胞（PC）自发性放电活动发生显著变化，提示SCI可导致小脑皮层神经环路结构异常、功能重塑，但其机制尚不清楚。申请人拟应用电生理和药理学手段，研究SCI中晚期麻醉小鼠小脑皮层PC、分子层中间神经元（MLI）和GC自发性活动变化特点及药理机制，探讨SCI后小脑环路神经元活动变化机制；在急性小脑切片上，研究SCI小鼠小脑皮层平行纤维（PF）-PC、PF-MLI和苔藓纤维（MF）-GC兴奋性突触电流变化机制，探讨SCI对小脑皮层PF-PC突触前长时程可塑性的影响及其信号机制；明确SCI小鼠脑皮层神经元活动、突触传递及PF-PC长时程可塑性变化的受体、通道及信号通路机制，阐明SCI后小脑皮层神经元活动及突触传递的重塑机制。

脊髓损伤（SCI）导致损伤节段下肢体功能障碍，并影响上级中枢功能活动。小脑是重要的运动中枢，SCI会影响小脑功能。申请人应用电生理和药理学手段，研究了SCI小鼠小脑皮层PC、分子层中间神经元（MLI）和GC自发性活动变化特点及药理机制，探讨了SCI后小脑环路神经元活动变化机制。明确了SCI小鼠小脑皮层神经元活动、突触传递变化的受体、通道机制，阐明SCI后小脑皮层神经元活动及突触传递的重塑机制。具体结果如下：（1）SCI小鼠PC的自发性SS频率明显降低，而SS的峰峰间隔（ISI）和变异系数（CV）则明显增大，阻断AMPA受体后两组的SS变异系数均明显减小。（2）阻断GABAA受体后SCI小鼠PC的自发性SS频率明显增高而CV明显减小，假手术组则只有CV明显降低，SCI组的SS频率和CV变化值明显大于假手术组的变化值，阻断GABAA受体后SCI的PC的SS频率与假手术组正常值没有明显差异。（3）一氧化氮合酶（NOS）抑制剂能够降低SCI组MLI的放电频率，增加CV，给予NOS抑制剂后SCI组PC的SS自发性频率增高，CV明显降低，与假手术组没有差异。（4）SCI组小鼠使吹风刺激引起的抑制性P1的振幅明显升高，PC反应后简单峰电位（SS）暂停时间明显延长，而P1的半宽和衰减时间则减低。SCI组吹风反应引起的兴奋性成分 N1的振幅大小无明显变化，潜伏期相同， N1的半宽值和衰减时间却明显低。兴奋性AMPA受体阻断剂NBQX完全阻断了吹风刺激引起的小脑皮层PC的反应P1和 N1，两组结果相同。（5）小脑表面灌流促肾上腺皮质激素释放因子（CRF）受体阻断剂SCI组明显减小吹风反应引起的 P1的振幅和反应后 SS暂停时间，在假手术组无影响。SCI组CRF-R1阻断剂 BMS-763534能够明显减小P1的振幅和SS暂停时间，而给予CRF-R2阻断剂antisauvagine-30却无明显改变。（6）假手术组小脑表面给予CRF能够明显增大吹风反应引起的 P1振幅和反应后 SS暂停时间，两组比较CRF能够明显增加假手术组吹风反应P1振幅，但增大的 P1振幅与SCI组的大小无明显差异。实验证明了SCI后小脑皮层中NO系统和CRF系统发生了变化，结果为SCI 患者的诊治提供一定的实验数据。