Anesthesia impairs developmental axon pruning and functional neuronal networks

 描述（由申请人提供）：全身麻醉（GA）允许护理患病和早产儿，但在大脑发育的关键阶段（即突触发生）使用全身麻醉可诱导未成熟神经元的凋亡性死亡以及长期神经认知和行为障碍。脑发育过程中功能性神经元网络的形成依赖于及时和适当的神经元轴突选择和修剪，这一过程高度依赖于神经元活性和神经营养因子，特别是脑源性神经营养因子（BDNF）的稳态。由于GA是一种有效的神经元活性抑制剂和BDNF稳态的有效调节剂，我们的一般假设是，短暂暴露于GA在大脑发育的关键阶段，导致长期损害的适当轴突选择和修剪的神经元，生存的最初的凋亡“攻击”。这反过来又导致在剩余的（“正常”）神经元中形成功能性神经元网络的持久损害。我们的长期目标是了解GA如何调节负责长期记忆储存和巩固的功能性神经元网络的形成，并建立GA诱导的BDNF调节的轴突修剪和突触神经传递之间的联系。基本原理是，这种理解将使药理学靶向的关键步骤， 轴突修剪和神经元回路的形成以防止GA诱导的突触发生损伤和记忆/学习缺陷。使用GA诱导的发育神经毒性的体内大鼠模型，我们将专注于海马中苔藓纤维投射的锥体下束（IPB）（从齿状回中的颗粒神经元到CA 3区域中的锥体神经元），这是一种在学习和记忆中起关键作用的神经元回路，并且被很好地确立为轴突修剪的模型。我们将讨论GA损害BDNF-p75 NTR信号传导的特定假设，从而阻碍轴突的正确选择并最终损害突触神经传递。具体目标1：将检查两种静脉麻醉剂，通常用于儿童，并具有不同的作用机制：NMDA拮抗剂氯胺酮和GABAA激动剂丙泊酚通过形态分析的IPB和其突触接触，使用光学和电子显微镜。我们的初步数据表明，氯胺酮损害发育IPB修剪，这与BDNF和pro-BDNF的显著下调相一致。我们将使用BDNF+/-和Ngfr-/-（p75 NTR-/-）突变小鼠研究BDNF和p75 NTR对GA诱导的IPB修剪损伤的重要性。将使用p75 NTR药理学拮抗剂TAT-Pep 5阻断p75 NTR或使用病毒载体LV-syn-p75 NTR在p75 NTR-/-小鼠的齿状回中敲入（“恢复”）p75 NTR来检查BDNF-p75 NTR级联的重要性。具体目标#2：将确定GA诱导的轴突修剪和选择损伤与IPB神经元回路中突触可塑性损伤之间的功能联系。我们将使用离体膜片钳切片记录海马CA 3区和齿状回的突触神经传递，研究GA对p75 NTR-/-和BDNF+/-突变小鼠及其野生型（WT）小鼠的影响。我们将尝试使用p75 NTR拮抗剂TAT-Pep 5来增强GA在WT小鼠中对突触传递的作用，并将尝试通过在p75 NTR-/-小鼠的齿状回中敲入p75 NTR来阻断GA的作用。

 DESCRIPTION (provided by applicant): General anesthesia (GA) allows for care of sick and premature children, but its use during critical stages of brain development (i.e. synaptogenesis) induces apoptotic death of immature neurons and long-term neurocognitive and behavioral impairments. The formation of functional neuronal networks during brain development depends on timely and proper neuronal axon selection and pruning, a process highly dependent on neuronal activity and homeostasis of neurotrophic factors, especially brain-derived neurotrophic factor (BDNF). Since GA is a potent inhibitor of neuronal activity and a potent modulator of BDNF homeostasis, our general hypothesis is that transient exposure to GA during critical stages of brain development causes long-term impairments of proper axon selection and pruning in neurons that survive the initial apoptotic `attack'. This, in turn, causes lasting impairments in formation of functional neuronal networks among the remaining (`normal') neurons. Our long-term goals are to understand how GA modulates the formation of functional neuronal networks responsible for long-term memory storage and consolidation and to establish the links between GA- induced BDNF-modulated axon pruning and synaptic neurotransmission. The rationale is that this understanding will enable pharmacological targeting of critical steps in axon pruning and formation of neuronal circuitries to prevent GA- induced impairment of synaptogenesis and memory/learning deficits. Using in vivo rat models of GA-induced developmental neurotoxicity we will focus on the infrapyramidal bundle (IPB) of mossy fiber projections in the hippocampus (from granular neurons in the dentate gyrus to pyramidal neurons in the CA3 region), a neuronal circuitry that plays a crucial role in learning and memory and is well established as a model of axon pruning. We will address specific hypothesis that GA impairs BDNF-p75NTR signaling, thereby hampering proper selection of axons and ultimately impairing synaptic neurotransmission. Specific Aim #1: will examine two intravenous anesthetics commonly used in children and having distinct mechanisms of action: the NMDA antagonist ketamine and the GABAA agonist propofol via morphometric analyses of the IPB and its synaptic contacts using light and electron microscopy. Our preliminary data suggest that ketamine impairs developmental IPB pruning that coincides with a significant down-regulation of BDNF and pro-BDNF. We will examine the importance of BDNF and p75NTR on GA-induced impairment in IPB pruning using BDNF+/- and Ngfr-/- (p75NTR-/-) mutant mice. The importance of the BDNF-p75NTR cascade will be examined using the p75NTR pharmacologic antagonist TAT-Pep5 to block p75NTR or the viral vector LV-syn-p75NTR to knock-in (`restore') p75NTR in dentate gyrus of p75NTR-/- mice. Specific Aim #2: will determine the functional link between GA-induced impairments in axon pruning and selection and the impairment of synaptic plasticity in IPB neuronal circuits. We will examine GA effects in p75NTR-/- and BDNF+/- mutant mice and their wild type (WT) counterparts using ex-vivo patch clamp slice recordings of synaptic neurotransmission in CA3 and dentate gyrus of the hippocampus. We will attempt to potentiate the effect of GA in WT mice on synaptic transmission using the p75NTR antagonist TAT-Pep5 and will attempt to block the effects of GA by knocking-in p75NTR in the dentate gyrus of p75NTR-/- mice.