BLR&D Research Career Scientist Award Application

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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.