Identification and analyses of molecules and neural circuits regulating REM sleep

2018 Fiscal Year Annual Research Report

• Project/Area Number: 18J11827
• Research Institution: University of Tsukuba
• Principal Investigator: Liu ChihーYao 筑波大学, グローバル教育院(HBP), 特別研究員(DC2)
• Project Period (FY): 2018-04-25 – 2020-03-31
• Keywords: Rapid-eye-movement sleep / Sublaterodorsal nucleus

Outline of Annual Research Achievements

In order to investigate the genetic identity of neuronal populations in sublaterodorsal nucleus (SLD), a putative generator of rapid-eye-movement (REM) sleep in mammals, two genes were identified in our laboratory as potential markers of SLD neurons. In my project, I planned to search for the roles of SLD neurons expressing each of the two genes in the regulation of REM sleep. Therefore, I have generated two Cre-KI mice in which the promoters of each of both genes drive the expression of Cre recombinase, designated as riDMT-Cre and rpDMT-Cre mice. Due to the heterogeneity of pontine tegmental areas including SLD, it has been difficult to identify the neurons regulating REM sleep and manipulate their activities. In this study, I harnessed Cre-loxP system to genetically label two novel neuronal populations in SLD so that I can manipulate its neuronal activity by DREADD and ablate them by selective expression of DTA. As a result, I successfully found that two functionally distinct neuronal populations in the SLD, riDMT and rpDMT neurons, exert a bidirectional control of REM sleep. The function of riDMT neurons is involved in the switch between NREM sleep and REM sleep. While the first gene is impaired, it may disturb the balance between NREM sleep and REM sleep. In contrary, rpDMT neurons are involved in generating REM sleep and contribute to muscle atonia during REM sleep. Thus, the discovery of a bidirectional control in SLD may help us further understand the biology of REM sleep.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

This study is aiming to investigate the genetic identity of neuronal populations in sublaterodorsal nucleus (SLD), a putative generator of rapid-eye-movement (REM) sleep in mammals. When using the DREADD to activate the target neurons, I found that different doses of CNO can induce different sleep patterns so that finding out the most appropriate dose is one of the critical point for activating the desired behavioral change. Moreover, in some cases, the desired behavioral change activated by DREADD necessitates the precise injection of AAV in bilateral areas. Therefore, the post-analysis of the targeting sites in the stereotactic surgery is crucial for the proper interpretation of experimental results. Although trying different doses of CNO and more injection for correct targeting have taken time during the progress of this study, it makes the experimental results more solid and reliable. In the meantime, it also prompted me to take more details into account when designing other similar experiments. So far, I successfully found that two neuronal population in SLD are either REM sleep-inhibiting or REM sleep-promoting, suggesting a bidirectional control of REM sleep occurred in the SLD. Surprisingly, I found that the gene used to label riDMT-Cre neurons may also be involved in the regulation of REM sleep in an unfamiliar environment, contributing to the physiology of REM sleep from a cellular level to circuit level. Therefore, I think the progress of this study is good.

Strategy for Future Research Activity

In this study, it suggests that riDMT neurons in SLD participate in a putative NREM/REM sleep switch, functionally reminiscent of Atoh1-E10.5-medial neurons located nearby SLD (Hayashi et al, Science, 2015). A further investigation into a possible relation between riDMT neurons and Atoh1-E10.5-medial neurons and NALCN is of interest. Besides, a sodium leak channel, NALCN, has been found to regulate the excitability of REM sleep-inhibiting neurons (Funato et al, Nature, 2016). It is also intriguing to study the function of NALCN in riDMT neurons. My results also showed that the genetic manipulation of REM sleep is feasible. In the past, when studying the function of sleep, sleep deprivation is a general strategy to see the impact of sleep loss on the trait of interest. If a trait is altered after sleep deprivation, one may consider the sleep is responsible for the altered trait. However, classical sleep deprivation is performed by applying mechanistic stimuli that may cause some non-specific disturbance, confounding the interpretation. Recently, by genetically manipulate the neural circuit of wake/sleep control, genetic sleep deprivation has been realized for investigating the function of sleep (Holth et al, Science, 2019). The mouse models generated in this study allow us to manipulate the ratio of NREM/REM sleep, even further expanding our ability to explore the mystery of sleep.