Pan-neuronal functional imaging and anesthesia

挥发性麻醉剂，如异氟烷，产生全身麻醉的所有阶段，包括无意识， 健忘症、镇痛和肌肉松弛。尽管它们无处不在，但它们的基本作用机制 这些药物仍然未知。阐明临床麻醉产生的机制是 这是麻醉学领域尚未解决的基础性研究问题。volatile的日常使用 麻醉剂并非没有临床风险。婴儿期多次接触麻醉剂可能导致 老年人在麻醉后会出现持续的术后认知功能障碍。 从历史上看，该领域的研究沿着两条轨道进行：要么是寻找特异性的分子分析， 挥发性麻醉剂的受体（一种方法，由于与麻醉剂的扩散相互作用， 许多受体的作用不能适当地联合收割机），或神经元活动的粗略测量， 使用脑电图和功能磁共振成像（从根本上说，这是有限的分辨率）的大脑的整个区域。很明显， 突触尺度和EEG尺度之间的长度分辨率存在巨大差距。我们假设 麻醉，因此失去意识，是由于中断之间的沟通， 在小神经元网络水平上的神经元，远低于EEG和fMRI的分辨率极限。我们 研究麻醉剂对完整的、活的神经网络内的相互通信的影响， 神经元分辨率我们使用C。线虫是世界上神经结构最简单、最易处理的生物， 这种麻醉是可以诱导的使用光片显微镜，结合固定和钙- 在神经元启动子下表达的敏感荧光团，以及我们定制的用于 通过图像分析和信号提取，我们跟踪并捕获了基本上整个神经系统的活动 并检查其在不同麻醉暴露水平下的行为， 人体手术使用双光子成像，我们能够在小鼠中进行类似的实验， 在清醒和麻醉状态下提取躯体感觉皮层的选定区域的活动。 我们将使用一个系统的差异表达的固定神经元荧光团在C。优雅，让精确的 在光片成像下识别单个神经元。结合C.线虫连接体， 我们将确定麻醉与意识状态下的神经通路，麻醉剂如何改变 化学和电突触连接来诱导这些状态，神经元连接的变化， 允许麻醉状态恢复到意识状态，从而描绘出 老年人的临床术后认知功能障碍和年轻人的神经发育障碍。我们 将把我们的成像和分析技术扩展到脊椎动物斑马鱼，在斑马鱼中建立一个系统发育的桥梁。 麻醉剂的作用从简单的生物到人类。我们将证明，这些变化在神经元 活动与在人类EEG中看到的麻醉的总体统计标志一致。

Volatile anesthetics, such as isoflurane, produce all stages of general anesthesia including unconsciousness, amnesia, analgesia and muscle relaxation. Despite their ubiquity, the fundamental mechanisms of action of these drugs remains unknown. Elucidating the mechanisms by which clinical anesthesia is produced is the foundational unanswered research question in the specialty of anesthesiology. The routine use of volatile anesthetics is not without clinical risk. Multiple exposures to anesthetics in infancy leads to possible behavioral problems in later life, and persistent post-operative cognitive dysfunction is seen in the elderly after anesthesia. Historically, research in this field has proceeded along two tracks: either molecular analysis looking for specific receptors for the volatile anesthetics (an approach that has largely foundered due to diffuse interactions with many receptors whose effects do not combine appropriately), or the gross measurement of neuronal activity in entire regions of the brain using EEG and fMRI (which are fundamentally limited by resolution). Clearly, there is an enormous gap in length resolution between synaptic-scale and EEG-scale. We hypothesized that the onset of anesthesia, and hence the loss of consciousness, is due to disruption in the communication between neurons at the level of small neuronal networks that lie well below the resolution limit of EEG and fMRI. We study the effect of anesthetic agents on intercommunication within intact, living neural networks, with single neuron resolution. We use C. elegans, the creature with the simplest, most tractable neuronal architecture in which anesthesia is known to be inducible. Using light-sheet microscopy, a combination of fixed and calcium- sensitive fluorophores expressed under neuronal promoters, and our customized supercomputing toolchain for image analysis and signal extraction, we track and capture the activity of essentially the entire nervous system and examine its behavior under varying levels of anesthetic exposure normalized to comparable levels used in human surgery. Using two-photon imaging, we are able to perform similar experiments in the mouse and extract activity from selected regions of the somatosensory cortex in both awake and anesthetized states. We will use a system of differential expression of fixed neuronal fluorophores in C. elegans to allow the precise identification of individual neurons under light-sheet imaging. In combination with the C. elegans connectome, we will determine the neuronal pathways that underlie anesthetized vs conscious states, how anesthetics alter chemical and electrical synaptic connections to induce these states, the changes in neuronal connectivity that permit the anesthetized state to resolve back into consciousness, and hence delineate the mechanisms of clinical post-operative cognitive dysfunction in the old and neurodevelopmental impairment in the young. We will extend our imaging and analysis techniques into vertebrate zebrafish, building a phylogenetic bridge in the action of anesthetics from simple creatures to humans. We will demonstrate that these changes in neuronal activity are consistent with the gross statistical hallmarks of anesthesia seen in the EEG in humans.