前边缘皮层自噬降低参与慢性神经病理痛维持的机制研究

新近研究发现，前额叶的前边缘皮层（PL）亚区在慢性痛中起到重要的作用。自噬参与神经元内突触相关蛋白的降解，维持神经元正常功能和稳态。我们预实验发现，坐骨神经部分结扎损伤(SNI)的大鼠，到了神经病理痛28天的晚期，PL脑区自噬水平降低、自噬受损。本申请拟研究PL脑区神经元自噬参与慢性神经病理痛维持的机制。首先，观察模型大鼠PL脑区神经元自噬水平的变化，结合CaMKII-Cre/PV-Cre转基因大鼠和CRISPR/Cas9技术，研究上调及下调兴奋性锥体神经元和抑制性中间神经元自噬水平对痛行为与情绪的影响；其次，通过脑片膜片钳技术探讨自噬水平对神经元兴奋性及突触传递效能的影响；最后，通过流式细胞荧光分选和RNA-seq技术，探讨神经元自噬及其参与慢性痛的分子机制。本项目希望验证：PL脑区神经元自噬减弱，突触内蛋白降解受阻，兴奋性神经元功能和突触传递效能因而增强，从而维持慢性神经病理痛。

The morphology and function of neurons in pain matrix have undergone significant changes in chronic neuropathic pain, but the underlying mechanisms are not fully understood. We and others have shown that the prelimbic cortex (PL) of the prefrontal cortex plays an important role in the development of chronic pain..Autophagy is not only involved in the degradation of synaptic-related proteins, but also plays an important role in maintaining neuronal morphology and function, suggesting that autophagy is possibly vital for neuronal excitability and synaptic transmission..Preliminary data of the present grant application indicate that autophagy in the PL is significantly impaired in the late stage of neuropathic pain. Based on this, the present grant application aims to elucidate the mechanisms of autophagy in neuropathic pain at molecular and cellular levels..Firstly, we will examine the level of autophagy in the PL of rats with neuropathic pain after spinal nerve injury (SNI), and further observe the effects of up- or down-regulated autophagy on pain behaviors. Secondly, we will observe the effects of up- or down-regulated autophagy in different types of neuron on pain behaviors in SNI rats, respectively. Thirdly, we will investigate the effects of autophagy on the neuronal function and synaptic transmission in the PL by patch clamp recording on brain slice. Finally, we will explore the possible molecular mechanisms of autophagy in PL neurons involved in chronic pain by fluorescence activated cell sorting (FACS) and RNA-seq techniques. .Our present grant application will testify the following hypothesis: the decreased level of autophagy of neurons in the PL will impede the degradation of synaptic proteins, and thus leads to the increased neuronal excitability and enhanced AMPA-mediated synaptic transmission, thus contributes to neuropathic pain.