Neuronal Basis Underlying Volatile Anesthetic Induced Hypnosis

项目摘要 尽管麻醉剂对单个离子通道的影响已经取得了很大进展， 麻醉剂产生其行为效应的神经基质的鉴定仍然是 挑战性在表征麻醉状态的关键成分中，我们关注挥发性麻醉剂- 诱导性催眠，定义为对非伤害性刺激缺乏意识。了解麻醉剂 产生催眠已经成为神经科学的一个中心问题，正如过去的多份报告所述， 十年的研究表明，现有的全身麻醉药每年可能会伤害4000万美国患者中的一部分 需要麻醉的人一个假设的替代麻醉诱导的无意识是产生一个 昏迷一种可逆的生理无意识状态，如睡眠，在这种状态下，病人被锁在门外，无法进入 清醒的状态还能恢复自然睡眠的恢复作用。在我们最初的获奖期间，我们 发现挥发性麻醉剂确实可以直接去除促进睡眠的视前核的一个子集， 下丘脑（POAH）神经元。我们的实验室和其他实验室还表明，每种经过测试的全身麻醉剂 （除了氯胺酮）使腹外侧视前区（VLPO）神经元去极化。然而，准确的重叠程度 在触发内源性睡眠的神经元和那些被麻醉暴露激活的神经元之间， 麻醉剂“劫持”内源性睡眠回路的后果仍然未知。核心假设 这种更新的一个重要方面是，挥发性麻醉药将通过增强活性来赋予其部分催眠特性 但神经元激活关键差异将区分内源性POAH神经元 麻醉催眠的非快速眼动睡眠。在目标1中，我们将使用一种新的基因编码钙检测器， 永久标记体内睡眠活跃的POAH神经元，随后确定 也被挥发性麻醉剂去极化。同样，我们将确定麻醉去极化的分数 POAH神经元在NREM和REM睡眠期间活跃。在目标2中，我们将采用开创性的体内 内窥镜显微镜观察并确定POAH神经元在小鼠周期中的真实的时间神经元活动 自然地跨越觉醒，NREM和REM睡眠，以及在这些相同的POAH神经元中， 仔细滴定挥发性麻醉剂的暴露量最后，在目标3中，我们将调节睡眠活性蛋白的体内活性。 POAH神经元通过神经元回路标记和最近发现的组织实现精确控制 区分睡眠活跃神经元与状态无关或觉醒活跃神经元的特异性表达标记物 邻居病毒驱动的“设计者受体"的条件表达 药物”（DREADDs）将允许我们去兴奋或过度兴奋POAH神经元，以确定随后的 激活或抑制睡眠活跃的POAH神经元对麻醉状态稳定性的影响。 累积起来，这些目标将使我们能够评估催眠引起的神经元会聚/发散， 挥发性麻醉剂和睡眠

PROJECT SUMMARY Although much progress has been made deciphering the effects of anesthetics upon individual ion channels, identification of the neural substrates upon which anesthetics act to produce their behavioral effects remains challenging. Of the key components that characterize the anesthetized state, we focus on volatile anesthetic- induced hypnosis, defined as a lack of awareness to non-noxious stimuli. Understanding how anesthetics produce hypnosis has become more than a central question for neuroscience, as multiple reports over the past decade suggest that existing general anesthetics may annually harm a subset of the 40 million US patients who require anesthesia. One hypothetical alternative to anesthetic-induced unconsciousness is to generate a state of reversible physiological unconsciousness, such as sleep, in which the patient is locked out of access to the state of wakefulness yet recoups restorative benefits of natural sleep. In our original award period, we discovered that volatile anesthetics do directly depolarize a subset of sleep-promoting, preoptic anterior hypothalamic (POAH) neurons. Our lab and others have also shown that every tested general anesthetic (except for ketamine) depolarizes ventrolateral preoptic (VLPO) neurons. However, the exact degree of overlap among neurons triggering endogenous sleep and those activated by anesthetic exposure as well as precise consequences of anesthetic “hijacking” of endogenous sleep circuitry remain unknown. The central hypothesis of this renewal is that volatile anesthetics will impart a portion of their hypnotic properties by enhancing activity in endogenous POAH neurons, but that key differences in neuronal activation will distinguish endogenous NREM sleep from anesthetic hypnosis. In Aim 1, we will use a novel genetically encoded calcium detector to permanently mark sleep-active POAH neurons in vivo and subsequently determine the exact fraction that is also depolarized by volatile anesthetics. Similarly, we will determine the fraction of anesthetic-depolarized POAH neurons that active during NREM and REM sleep. In Aim 2, we will employ groundbreaking in vivo endoscopic microscopy to visualize and determine real time neuronal activity of POAH neurons as mice cycle naturally across wakefulness, NREM, and REM sleep as well as in these same POAH neurons during a carefully titrated volatile anesthetic exposure. Finally, in Aim 3 we will modulate in vivo activity of sleep-active POAH neurons achieving precise control through neuronal circuit labeling and by recently discovered tissue specific expression markers that distinguish sleep-active neurons from state-indifferent or wake-active neighbors. Conditional expression of virally driven “designer receptors exclusively activated by designer drugs” (DREADDs) will permit us to depolarize or hyperpolarize POAH neurons to determine the ensuing effects of activating or inhibiting sleep-active POAH neurons upon the stability of the anesthetic state. Cumulatively, these aims will enable us to assess the neuronal convergence/divergence of hypnosis caused by volatile anesthetics and sleep.