Mechanisms of anesthetic-induced synaptic plasticity

项目摘要 基因多态最近被认为是神经认知障碍(NCD)的危险因素 麻醉和手术，但我们对突触功能障碍是否或如何发挥作用的理解存在差距 在这些认知缺陷中扮演的角色。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，特别是在脆弱人群中。这项提案的长期目标是定义 持续全身麻醉作用于突触可塑性的细胞和分子机制。整体而言 本应用的目的是确定与持续结构树突棘相关的信号通路。 麻醉暴露后的变化。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，并与多发性硬化症患者的认知和发育障碍有关 神经紊乱。中心假设是全身麻醉药减少了脑源性神经营养 因子(BDNF)释放，导致突触前功能受损，树突棘结构改变，以及 神经元活动、空间学习和记忆的缺陷。这一假说是在 申请人实验室获得的初步数据。提出这项研究的理由是，一旦它 已知麻醉剂如何导致永久性脊椎丧失，这些分子靶点的药理学操作 在麻醉期间或设计新的麻醉剂来避免这些影响是可能的。指导原则 令人信服的初步数据，这一假说将通过三个具体的目标进行检验：1)确定BDNF在 通过挥发性麻醉剂减少突触小泡(SV)的胞吐；2)确定BDNF在挥发性麻醉剂中的作用- 诱导树突棘密度和形态的改变；3)阐明BDNF在异氟醚中的作用。 诱导对海马区依赖突触可塑性的影响。对于目标1，经过验证的光遗传生物传感器(BDNF- PH，vGlut1-pH和钙指示剂GCaMP6)，已经在申请人手中确立为可行的，将 用来检测麻醉剂对脑源性神经营养因子释放的影响，以及这些变化对抑制钙内流和 SV胞吐。对于目标2，由于BDNF减少而引起的结构脊柱变化将在分离的情况下进行研究 使用延时荧光显微镜和高尔基体染色的海马区培养物和完整脑片， 分别进行了分析。对于目标3，异氟醚减少脑源性神经营养因子信号在实时神经元上的功能结果 与学习和记忆障碍相对应的活动将通过纤维光度法和 行为测试。这些功能和结构研究将使用或使用来自 最近人类一种常见多态(Val66Met)导致BDNF分泌减少的转基因小鼠 被确定为非传染性疾病的危险因素。该模型为阐明机理提供了一种创新的方法。 潜在的麻醉剂诱导的突触前和突触后功能障碍与学习和记忆障碍相关 记忆。高危个体麻醉后长期突触缺陷的预防具有广泛的意义 术前风险评估和麻醉恢复的翻译重要性。

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.