The effects of anesthetics on brain development

描述(由申请人提供)：流行病学研究表明，儿童时期接触麻醉剂与随后的学习和行为障碍的增加之间存在关联。这些数据得到了动物研究的补充，动物研究表明，暴露在麻醉剂中的大鼠幼鼠表现出持久的学习障碍。以前的研究人员已经证明，麻醉剂可以增加发育中的大脑中的细胞凋亡，但很少有人评估麻醉剂对剩余神经元发育的影响。我们的初步数据引起了人们的担忧，即一些临床上相关剂量的常用麻醉药可能会干扰轴突生长锥对引导它们到达正确树突靶点的提示的反应，从而潜在地扰乱对学习等更高功能至关重要的脑回路的形成。在这项提案中，我们将研究主要种类的麻醉剂对轴突引导和神经回路形成的影响，以期发现这种有害影响的机制，并确定如何设计麻醉方案来避免或最大限度地减少这种影响。具体目的是：(1)测试全身麻醉剂对轴突生长锥体对引导线索反应的影响。需要检验的假设是，具有GABAA受体和/或NMDA受体活性的麻醉剂可以实质上干扰生长锥功能，包括对指导提示的感知和反应。利用时间推移显微镜和轴突形态的定量分析，我们将在缺乏指导线索的情况下确定主要麻醉剂对轴突生长锥体运动的影响。然后，使用塌陷试验和梯度转向试验，我们将测试麻醉剂是否干扰轴突生长锥体对排斥和吸引的引导线索的反应。最后，我们将确定其作用机制是否是麻醉药对GABAA受体和/或NMDA受体的影响，从而导致生长激素钙的水平和分布发生变化，生长锥钙是引导反应的主要细胞内调节因子。(2)确定全身麻醉药对轴突引导的影响是否会导致回路形成的错误。需要检验的假设是麻醉剂可以扰乱轴突靶向，从而导致回路形成错误。在暴露于麻醉剂的轴突引导的器官型切片培养模型中，轴突的轨迹将被评估发育中的错误，以确定哪些药物可能具有有害影响。随后，苔藓纤维和Schaffer侧支的发育，这两个参与关键学习回路的海马区，将被 在麻醉暴露后进行评估，以确定哪些药剂、什么剂量和什么暴露时间可以扰乱电路的形成。这些研究将在器官型脑片培养中进行，使用定量显微镜和电生理学技术。结果将通过对暴露于切片培养中确定干扰轴突引导和束形成的麻醉剂的完整小鼠的这些束的显微分析来证实。

DESCRIPTION (provided by applicant): Epidemiologic studies demonstrate an association between childhood exposure to anesthesia and an increase in subsequent learning and behavioral disorders. These data are complemented by animal studies showing that rat pups exposed to anesthetics exhibit lasting deficits in learning. Previous investigators have demonstrated that anesthetic agents can increase apoptosis in the developing brain, but little has been done to assess the effects of anesthetics on the development of the neurons that remain. Our preliminary data raise a concern that some commonly used anesthetics presented at clinically relevant doses may interfere with the response of axonal growth cones to the cues that guide them to the correct dendritic targets, thus potentially disrupting the formation of brai circuits critical for higher functions, such as learning. In this proposal we will examine the effets of the major classes of anesthetic agents on axon guidance and circuit formation, with a view towards discovering mechanisms of this deleterious effect and determining how anesthetic plans may be designed to avoid or minimize it. The Specific Aims are: (1) Test the effects of general anesthetic agents on the response of axonal growth cones to guidance cues. The hypothesis to be tested is that anesthetic agents with activity at GABAA receptors and/or NMDA receptors can substantially interfere with growth cone function, including sensing of and response to guidance cues. Using time-lapse microscopy and quantitative analysis of axon morphology we will determine the effects of the major classes of anesthetic agents on axonal growth cone motility in the absence of guidance cues. Then, using a collapse assay and gradient turning assay, we will test whether anesthetics interfere with the response of the axonal growth cone to repulsive and attractive guidance cues. Finally, we will determine whether the mechanism of action is an effect of anesthetics on GABAA receptors and/or NMDA receptors that leads to alterations in the levels and distribution of growth cone Ca2+, a principal intracellular regulator of the guidance response. (2) Determine whether effects of general anesthetics on axon guidance can lead to errors in circuit formation. The hypothesis to be tested is that anesthetic agents can disrupt axon targeting, thus leading to errors in circuit formation. The trajectory of axons in an organotypic slice culture model of axon guidance that is exposed to anesthetics will be assessed for errors in development to determine which agents may have deleterious effects. Subsequently, the development of the mossy fibers and the Schaffer collaterals, two tracts of the hippocampus that participate in a critical learning circuit, will be assessed after anesthetic exposure to determine which agents, what doses, and what exposure times can disrupt circuit formation. These studies will take place in organotypic brain slice culture using quantitative microscopy and electrophysiologic techniques. Results will be confirmed by microscopic analysis of these tracts in intact mice exposed to the anesthetic agents determined in slice culture to interfere with axon guidance and tract formation.