兴奋性与抑制性神经网络整合在全身麻醉诱发发育早期中枢神经毒性中的作用及机制

Both spontaneous synchronized neural electrical activity ( excitatory calcium waves) and genes play a very important instructive role in the refinement of synapses and the formation of neural circuits during early brain development. Our previous work has shown that retinal calcium waves are generated by a transient and specific network of cell-autonomous oscillators(starburst amacrine cells)synchronized by reciprocally excitatory synapse inputs; GABA、NMDA etc excitatory and inhibitory synaptic inputs manipulate the spontaneous synchronized neural electrical activity patterns of and the activities of individual neurons through their coordinated and sequential tranisition. Therefore, we speculate that general anesthetics working through GABA and /or NMDA receptors might first affect the intergration of excitatory and inhibitory neuroanl networks, then interfere with the precise and sequential transistions of the spatiotemporal properties of calcium waves, result in the abnormal expressions of puma,Fas,Bax etc pro_apoptosis genes and Bcl_2,Bcl_xL etc anti_apoptosis genes, and finally result in the structural and functional disturbance of neural synapses and circuits. To verify this speculation, we will use the mutal excitatory and inhibitory neural nedtwork formed by retinal starburst amacrine cells as a simple whole brain experimental model, the spatiotemporal patterns of excitatory calcium waves, the compond action potential composed of action potential, excitatory postsynaptic potential, oscillatory potential and calcium activated cAMP sensitive long lasting hyperpolarizing potential, the excitatory and inhibitory conduct ratio, etc as the indexes of network integration, to study the effects of general anesthtics on the integration of excitory and inhibitory networks,the patterns of spontaneous synchronized neural electrical activity and the calcium signaling pathway in neuclear, to dissect the roles and mechanisms of integration between excitatory and inhibitory neuronal networks in anesthesia induced neuronal apostosis during the early development, and provide a new thought and possible targets for the treatment of anesthesia induced neurotoxicities in developing central nervous system and early development_related diseases.

在大脑发育早期，自发性协同神经电活动（兴奋性钙波）与基因编码一起对突触的再精细化和神经环路的形成起着重要的指导作用。我们前期研究发现：视网膜兴奋性钙波由具有自发性节律的星爆细胞整合相互兴奋的突触传入产生；GABA、NMDA等兴奋性与抑制性神经突触传入通过协同有序转变调控自发性协同神经电活动模式。由此推测，通过GABA或/和NMDA受体起作用的全身麻醉药可能通过影响兴奋性和抑制性神经网络整合，干预自发性协同神经电活动模式的精确有序转变，导致puma 、Fas、Bax等促凋亡基因及Bcl-2、Bcl-xL等抑制凋亡基因表达异常。为证实以上假设，本项目采用视网膜星爆细胞间形成的相互兴奋与抑制的神经网络这一简化的整体脑实验模型，研究兴奋性与抑制性神经网络整合在全身麻醉诱发的发育早期神经毒性中的作用与机制，为全身麻醉引起的中枢神经系统毒性及发育早期相关疾病的干预提供新的思路和可能的靶点。

在大脑发育早期，尤其是突触形成高峰期接受全身麻醉，可以引起动物中枢神经树突和丝状伪足形态与数量的改变，线粒体形态与功能的明显损伤，神经元大量凋。这一现象引起人们对婴幼儿麻醉安全的极大关注。既往的研究表明，自发性节律性神经电活动与基因一起对突触的再精细化和神经环路的形成起着非常重要的调节作用。然而，二者是否参与全身麻醉药引发的发育神经毒性尚不清楚。本研究应用电生理、免疫组化学及TUNEL等技术探讨大鼠视网膜自发性节律性神经电活动在全身麻醉诱发发育早期神经元凋亡中的作用、机制及可能的治疗措施。同时, 还探讨了circ RNAs的作用。全身麻醉药氯胺酮诱发的视网膜神经元凋亡与生理性凋亡仅存在程度上的差异，凋亡高峰期均发生在出生后7-9天，且具有剂量和时间依赖性；氯胺酮诱发神经元凋亡的同时也抑制了自发性节律性电活动，并呈发育阶段依赖性；类似腺苷酸环化酶激活剂Forskolin，多巴胺明显增强神经元自发性节律性电活动，减轻生理性及氯胺酮诱发的神经元凋亡；多巴胺D1，D2受体、腺苷A2A受体、cAMP/PKA信号通路及G蛋白耦联受体激酶2参与多巴胺减轻氯胺酮诱发的神经元凋亡；多巴胺也同样能够减轻乙醇诱发的神经元凋亡；氯胺酮不仅通过影响谷氨酸能介导的神经网络电活动引起凋亡，而且还通过影响胆碱能介导的神经网络电活动引起凋亡；适度的应激反应可能通过cAMP/PKA、PLC、PLD以及ERK1/2信号通路减轻氯胺酮所致视网膜节细胞层神经元凋亡。另外，伴随着生理性凋亡高峰，一系列与凋亡相关基因来源的circ RNAs （Thoc1, Akt3, Rere, Rbm5, Ranbp9, Pias1, Optn, Naa35, Melk, Epha7, Ddx19a, Braf, Bnip2, Birc6, and Arhgap10）出现明显的升高或降低，提示这些circ RNAs可能在基因层面上参与了神经元凋亡的调控。.总之，在突触形成高峰期，胆碱能神经系统介导的自发性节律性神经网络电活动向谷氨酸能神经系统有序转换紊乱及相互作用参与了全身麻醉药诱发的发育神经毒性。circ RNAs可能在基因调控层面上参与神经元凋亡的调控。本研究为全身麻醉引起的中枢神经系统毒性及发育早期相关疾病的干预提供了新的思路和治疗途径。