BLR&D Research Career Scientist Award Application

7000万美国人患有某种睡眠障碍。行为、情绪和记忆恶化 睡眠不足会导致更严重的问题睡眠障碍是一种常见的 这是我们退伍军人的共同抱怨。由于过度觉醒导致的睡眠不足是创伤后应激障碍的一种症状，但它是 不知道为什么患有PTSD的退伍军人无法入睡。我的研究重点是识别和绘制 大脑神经元诱导睡眠。我的研究的总体影响是，它将提供第一个直接的 神经元的特殊表型和它们负责诱导睡眠的回路之间的联系。这将使 在唤醒驱动非常强烈的情况下，例如在失眠症中， 创伤后应激障碍，或在过度嗜睡时保持清醒，如阻塞性肺病患者。 睡眠呼吸暂停或非典型抑郁症。 一系列的实验使用光遗传学和药物遗传学来识别大脑中的功能回路。 个脑袋大脑包含许多不同类型的细胞，通过光遗传学和药物遗传学， 现在有可能分解大脑，以确定负责复杂行为的罪魁祸首神经元， 就像睡觉一样。我的实验室是睡眠神经生物学领域第一个使用光遗传学诱导睡眠的实验室 （Konadhode等人，2013年）。我们激活了下丘脑中一种特殊的神经元表型， 发现它在一天中老鼠应该醒着的时候诱导睡眠。我们现在已经 在大鼠身上也显示了同样的效果，这表明激活这些神经元可以驱动哺乳动物的睡眠。我们现在 他们想测试在高唤醒状态下的睡眠诱导效果，如恐惧条件反射（PTSD）或焦虑。 结合光遗传学和药物遗传学，我正在使用脑深部成像方法， 对大脑中表现型特异性神经元的活动进行成像。该方法测量 荧光的遗传编码的钙指标在个别神经元。荧光信号 通过连接到微型显微镜（2g）的显微内窥镜捕获。显微内窥镜可以放置在 提供了前所未有的神经元活动记录。我用它来获取 睡眠和清醒时特定神经回路活动的视觉证据。 另一系列的研究使用的是PICTURITY方法来绘制由光遗传学激活的电路。 透明化是一种新的神经解剖学方法，可以使死后组织（如大脑）透明化。 PI与RHJVAMC研究人员合作，获得了蔡司Lightsheet显微镜，并使用它来 产生大脑神经元回路的3D重建（见Shiromani和Peever，2017年）。我的意图是 使用PERIITY可视化TBI啮齿动物模型中的死后大脑，并对透明心脏进行成像， 肝脏和肾脏。其目的是提供病变组织中回路中断的视觉记录。 第四个系列的实验使用基因转移的方法来修复有缺陷的电路和恢复 行为我已经用它成功地纠正了小鼠模型的行为症状， 神经退行性睡眠障碍嗜睡症我们现在用基因转移的方法来阻断触发 异常行为，例如恐惧条件反射。总的来说，这些神经科学方法和工具有助于 RHJ VAMC的集体研究成果。

70 million Americans suffer from some sort of sleep disorder. Behavior, mood and memory deteriorate with sleep loss and it gets worse with continuing sleep deprivation. Sleep disturbance is a frequent and common complaint among our Veterans. Lack of sleep due to hyperarousal is one symptom of PTSD, but it is not known why Veterans with PTSD cannot fall asleep. My research focuses on identifying and mapping the brain neurons that induce sleep. The overall impact of my research is that it will provide the first direct evidence linking specific phenotypes of neurons and their circuits responsible for inducing sleep. This will make it possible to induce sleep in conditions where the arousal drive is very strong, such as in the insomnia of PTSD, or to maintain wakefulness when there is excessive sleepiness, such as in patients with obstructive sleep apnea or atypical depression. One series of experiments uses optogenetics and pharmacogenetics to identify functional circuits in the brain. The brain contains many different types of cells and through optogenetics and pharmacogenetics it is now possible to disassemble the brain to identify the culprit neurons responsible for complex behaviors, such as sleep. My lab was the first in the area of sleep neurobiology to use optogenetics to induce sleep (Konadhode et al., 2013, attached). We activated a specific phenotype of neurons in the hypothalamus and discovered that it induced sleep at a time of day when the mouse should have been awake. We have now shown the same effect in rats, indicating that activating these neurons drives sleep across mammals. We now want to test the sleep-inducing effect in conditions of high arousal, such as fear-conditioning (PTSD) or anxiety. In conjunction with optogenetics and pharmacogenetics, I am using the deep-brain imaging method to image the activity of phenotype-specific neurons in the brains. This method measures changes in fluorescence of a genetically encoded calcium indicator in individual neurons. The fluorescence signal is captured via a microendoscope attached to a miniature microscope (2g). The microendoscope can be placed anywhere in the brains of mice providing unprecedented record of neuronal activity. I am using it to obtain visual evidence of the activity of specific neuronal circuits during sleep and waking. Another series of studies uses the CLARITY method to map the circuit activated by optogenetics. CLARITY is a new neuroanatomical method that makes postmortem tissue, such as the brain, transparent. The PI collaborated with RHJVAMC researchers to acquire a Zeiss Lightsheet microscope and used it to produce a 3D reconstruction of brain neuronal circuits (see Shiromani and Peever, 2017 attached). My intent is to use CLARITY to visualize postmortem brains in rodent models of TBI, and also image a transparent heart, liver and kidney. The goal is to provide a visual record of the break in a circuit in diseased tissue. The fourth series of experiments use the gene transfer approach to fix defective circuits and restore behavior. I have used it to successfully to correct behavioral symptoms in a mouse model of the neurodegenerative sleep disorder, narcolepsy. We are now using the gene transfer method to block triggering of abnormal behavior, for example in fear-conditioning. Overall, these neuroscience methods and tools aid the collective research effort at RHJ VAMC.