The Roles of Genetically Distinct Cortical Neuron Types in General-Anesthesia- and Sleep-Induced Slow Waves

项目概要/摘要。可逆性意识丧失是两个主要医学领域的关键部分： 全身麻醉和睡眠全身麻醉剂和非快速眼动（NREM）睡眠都诱导 皮层脑电图（EEG）中的慢波（0.1-4 Hz）。目前尚不清楚慢波是否会产生 在不同麻醉剂和NREM睡眠期间，产生相同的神经回路活动。博士 Melonakos的初步数据表明，具有不同分子靶点的麻醉剂具有不同的缓慢作用。 波机制（目标1假设）。此外，虽然右美托咪定麻醉共享神经回路， 对于NREM睡眠，它也可能具有明显的直接皮层效应，可能导致不同的慢波活动 (Aim 2假设）。本研究的目的是通过绘制皮层神经活动图来验证这些假设 关于麻醉和NREM睡眠的EEG慢波。为了做到这一点，Melonakos博士将 学习如何在行为自由的啮齿动物中进行钙成像实验。然后他将记录钙图像 从Ca 2 +/钙调蛋白依赖性蛋白激酶IIa阳性（CaMKIIa+），小清蛋白阳性（PV+）， 生长抑素阳性（SST+）和血管活性肠肽阳性（VIP+）皮质神经元 麻醉和睡眠引起的慢波。将测试丙泊酚、氯胺酮和右美托咪定麻醉。 博士然后，Melonakos将比较麻醉剂和全身麻醉之间的神经活动 睡觉最后，他将通过以下方式确定SST+神经元在慢波中的作用（目标3假设）：（1）观察 通过刺激臂旁核破坏慢波后皮层神经元的活动， 脑干中的唤醒区，和（2）在麻醉和睡眠诱导的慢波期间抑制SST+神经元。 在该项目的K99阶段，Melonakos博士将由Christa Nehs博士和Emery Brown博士指导， 马萨诸塞州哈佛医学院的麻醉和睡眠神经回路专家和教员 医院（MGH）和马萨诸塞州理工学院（MIT）。Melonakos博士还将与Dr. 迈克尔·哈塞尔莫（波士顿大学）、南希·科佩尔（波士顿大学）和丹尼尔·阿哈罗尼（波士顿大学） 加州、洛杉矶）。他将接受Hasselmo和Aharoni博士的钙成像培训， 布朗博士的分析科佩尔博士将指导梅洛纳科斯博士，因为他将他的发现定位在假设的缓慢 波动机制的研究。Melonakos博士还将学习光遗传学 Nehs博士和麻省理工学院的课程中的刺激技术。导师，合作者和其他成员 当他接近独立时，MGH社区还将为他提供专业指导，包括 在撰写拨款、同行评审、教学和教师求职方面的培训。科学和专业培训 博士Melonakos获得将使他能够开发一个独立的研究计划，以研究麻醉剂的直接 vs.间接影响。对慢波机制的理解有可能改进协议 用于全身麻醉的监护和睡眠障碍的治疗，有利于患者的安全和健康。

Project Summary/Abstract. Reversible loss of consciousness is a crucial part of two major medical fields: general anesthesia and sleep. General anesthetics and non-rapid-eye-movement (NREM) sleep both induce slow waves (0.1-4 Hz) in the cortical electroencephalogram (EEG). It is unknown whether slow waves generated with different anesthetic agents and during NREM sleep are generated with the same neural circuit activity. Dr. Melonakos’ preliminary data suggests that anesthetic agents with different molecular targets have distinct slow wave mechanisms (Aim 1 Hypothesis). In addition, although dexmedetomidine anesthesia shares neural circuits with NREM sleep, it may also have distinct direct cortical effects, possibly leading to different slow wave activity (Aim 2 Hypothesis). The purpose of this research is to test these hypotheses by mapping cortical neural activity with respect to the EEG slow waves of both anesthesia and NREM sleep. In order to do this, Dr. Melonakos will learn how to perform calcium imaging experiments in freely behaving rodents. He will then record calcium images from Ca2+/calmodulin-dependent protein kinase IIa-positive (CaMKIIa+), parvalbumin-positive (PV+), somatostatin-positive (SST+), and vasoactive intestinal peptide-positive (VIP+) cortical neurons during anesthesia- and sleep-induced slow waves. Propofol, ketamine, and dexmedetomidine anesthesia will be tested. Dr. Melonakos will then compare the neural activity between the anesthetics and between general anesthesia and sleep. Finally, he will identify the role of SST+ neurons in slow waves (Aim 3 Hypothesis) by (1) looking at the activity of cortical neurons following disruption of slow waves by stimulation of the parabrachial nucleus, an arousal area in the brainstem, and (2) inhibiting SST+ neurons during anesthesia- and sleep-induced slow waves. During the K99 phase of this project, Dr. Melonakos will be mentored by Drs. Christa Nehs and Emery Brown, experts in anesthesia and sleep neurocircuitry and faculty at Harvard Medical School, Massachusetts General Hospital (MGH), and Massachusetts Institute of Technology (MIT). Dr. Melonakos will also collaborate with Drs. Michael Hasselmo (Boston University), Nancy Kopell (Boston University), and Daniel Aharoni (University of California, Los Angeles). He will be trained in calcium imaging by Drs. Hasselmo and Aharoni, and statistical analysis by Dr. Brown. Dr. Kopell will guide Dr. Melonakos as he orients his findings within hypothesized slow wave mechanisms from the field of computational neuroscience. Dr. Melonakos will also learn optogenetics stimulation techniques from Dr. Nehs and in a course at MIT. The mentors, collaborators, and other members of the MGH community will also provide him with professional guidance as he nears independence, including training in grant writing, peer review, teaching, and the faculty job search. The scientific and professional training Dr. Melonakos receives will enable him to develop an independent research program to study anesthetics’ direct vs. indirect effects. The resulting understanding of slow wave mechanisms has potential to improve the protocols used to monitor general anesthesia and treat sleep disorders, thus benefiting patient safety and health.