Mechanisms of anesthetic-induced synaptic plasticity

项目摘要 遗传多态性最近成为神经认知障碍（NCD）的危险因素， 麻醉和手术，但在我们的理解是否或如何突触功能障碍发挥了作用， 在这些认知缺陷中的作用。这些知识对于评估必要医疗程序的风险至关重要 需要麻醉的疾病，特别是对脆弱人群该提案的长期目标是确定 持续全身麻醉剂对突触可塑性作用的细胞和分子机制。整体 本申请的目的是鉴定参与持久性结构树突棘的信号通路。 麻醉剂暴露后的变化。树突棘是成熟突触上的突触后结构， 对学习和记忆至关重要，并与多种认知和发育功能障碍有关。 神经系统疾病中心假设是全身麻醉剂减少脑源性神经营养因子 因子（BDNF）释放，导致突触前功能受损，树突棘结构改变， 神经元活动和空间学习和记忆的缺陷。这一假设是基于 申请人实验室获得的初步数据。这项研究的基本原理是，一旦 已知麻醉剂如何诱导永久性脊柱丧失，这些分子靶点的药理学操作 在麻醉期间或设计避免这些影响的新麻醉剂将是可能的。指导 令人信服的初步数据，这一假设将通过三个具体的目标进行检验：1）确定BDNF的作用， 减少挥发性麻醉剂引起的突触囊泡（SV）胞吐作用; 2）明确BDNF在挥发性麻醉剂- 诱导的树突棘密度和形态的变化;和3）阐明BDNF在异氟醚- 诱导对突触可塑性的影响。对于目标1，已证实的光遗传学生物传感器（BDNF-1） pH、vGlut 1-pH和Ca 2+指示剂GCaMP 6），申请人已经确定为可行， 用于测试麻醉剂对BDNF释放的影响以及这些变化对抑制Ca 2+进入的影响， SV胞吐作用。对于目标2，将在分离的小鼠中研究由于BDNF减少而引起的结构脊柱变化。 使用延时荧光显微镜和高尔基体染色的海马培养物和完整脑切片， 分别对于目标3，异氟烷降低BDNF信号传导对实时神经元的功能结果， 与学习和记忆障碍相对应的活动将用纤维光度测定法进行评估， 行为测试这些功能和结构的研究将进行与或使用神经元或切片从 最近，由于人类常见的多态性（Val 66 Met），转基因小鼠的BDNF分泌减少， 被认为是NCD的危险因素。这个模型提供了一个创新的方法来阐明机制 潜在的麻醉诱导的突触前和突触后功能障碍与学习障碍相关， 记忆在高危人群中预防麻醉后的长期突触缺陷具有广泛的意义。 对术前风险评估和麻醉恢复的重要性。

Project Summary Genetic polymorphisms have recently emerged as risk factors for neurocognitive disorder (NCD) following anesthesia and surgery, but there is a gap in our understanding whether or how synaptic dysfunction plays a role in these cognitive deficits. Such knowledge is critical to assessing the risk of necessary medical procedures that require anesthesia, particularly in vulnerable populations. The long-term goal of this proposal is to define the cellular and molecular mechanisms of sustained general anesthetic actions on synaptic plasticity. The overall objective in this application is to identify signaling pathways involved in persistent structural dendritic spine changes following anesthetic exposure. Dendritic spines are postsynaptic structures on mature synapses that are critical for learning and memory and are associated with cognitive and developmental dysfunction in multiple neurological disorders. The central hypothesis is that general anesthetics reduce brain-derived neurotrophic factor (BDNF) release, resulting in impaired presynaptic function, alterations in dendritic spine structure, and deficits in neuronal activity and spatial learning and memory. This hypothesis has been formulated on the basis of preliminary data obtained in the applicant’s laboratory. The rationale for the proposed research is that, once it is known how anesthetics induce permanent spine loss, pharmacological manipulation of these molecular targets during anesthesia or design of new anesthetic agents that avoid these effects will be possible. Guided by compelling preliminary data, this hypothesis will be tested by three specific aims: 1) Identify the role of BDNF in reducing synaptic vesicle (SV) exocytosis by volatile anesthetics; 2) Define the role of BDNF in volatile anesthetic- induced changes on dendritic spine density and morphology; and 3) Elucidate the role of BDNF in isoflurane- induced effects on hippocampal-dependent synaptic plasticity. For Aim 1, proven optogenetic biosensors (BDNF- pH, vGlut1-pH and the Ca2+ indicator GCaMP6), already established as feasible in the applicant’s hands, will be used to test anesthetic effects on BDNF release and the impact of these changes on inhibition of Ca2+ entry and SV exocytosis. For Aim 2, structural spine changes due to reduced BDNF will be investigated in dissociated hippocampal cultures and intact brain slices using time-lapse fluorescence microscopy and Golgi staining, respectively. For Aim 3, the functional outcome of reduced BDNF signaling by isoflurane on real-time neuronal activity corresponding to impairments in learning and memory will be assessed with fiber photometry and behavioral testing. These functional and structural studies will be conducted with or using neurons or slices from transgenic mice with reduced BDNF secretion due to a common human polymorphism (Val66Met) recently identified as a risk factor for NCD. This model provides an innovative approach to elucidate mechanisms underlying anesthetic-induced pre- and post-synaptic dysfunction associated with impairments in learning and memory. Prevention of long-term synaptic deficits following anesthesia in at-risk individuals have broad translational importance to preoperative risk assessment and anesthesia recovery.