Vidal-Ortiz Aurelio, Blanco-Centurion Carlos, Shiromani Priyattam J
Laboratory of Sleep Medicine and Chronobiology, Ralph H. Johnson Veterans Healthcare System, Charleston, SC, USA.
Department of Psychiatry and Behavioral Sciences, Medical University of South Carolina, Charleston, SC, USA.
Sleep. 2024 Mar 11;47(3). doi: 10.1093/sleep/zsad217.
To determine how a waking brain falls asleep researchers have monitored and manipulated activity of neurons and glia in various brain regions. While imaging Gamma-Aminobutyric Acid (GABA) neurons in the zona incerta (ZI) we found a subgroup that anticipates onset of NREM sleep (Blanco-Centurion C, Luo S, Vidal-Ortiz A, Swank C, Shiromani PJ. Activity of a subset of vesicular GABA-transporter neurons in the ventral ZI anticipates sleep onset. Sleep. 2021;44(6):zsaa268. doi:10.1093/sleep/zsaa268.). To differentiate the GABA subtype we now image and optogenetically manipulate the ZI neurons containing the transcription factor, Lhx6. In the first study, Lhx6-cre mice (n = 5; female = 4) were given rAAV-DJ-EF1a-DIO-GCaMP6M into the ZI (isofluorane anesthesia), a GRIN lens implanted, and 21days later sleep and fluorescence in individual Lhx6 neurons were recorded for 4 hours. Calcium fluorescence was detected in 132 neurons. 45.5% of the Lhx6 neurons were REM-max; 30.3% were wake-max; 11.4% were wake + REM max; 9% were NREM-max; and 3.8% had no change. The NREM-max group of neurons fluoresced 30 seconds ahead of sleep onset. The second study tested the effects of unilateral optogenetic stimulation of the ZI Lhx6 neurons (n = 14 mice) (AAV5-Syn-FLEX-rc[ChrimsonR-tdTomato]. Stimulation at 1 and 5 Hz (1 minute on- 4 minutes off) significantly increased percent REM sleep during the 4 hours stimulation period (last half of day cycle). The typical experimental approach is to stimulate neurons in both hemispheres, but here we found that low-frequency stimulation of ZI Lhx6 neurons in one hemisphere is sufficient to shift states of consciousness. Detailed mapping combined with mechanistic testing is necessary to identify local nodes that can shift the brain between wake-sleep states.
为了确定清醒的大脑是如何进入睡眠状态的，研究人员监测并操控了大脑各个区域的神经元和神经胶质细胞的活动。在对未定带（ZI）中的γ-氨基丁酸（GABA）神经元进行成像时，我们发现了一个亚群，它能预测非快速眼动睡眠（NREM）的开始（布兰科-森图里昂C、罗S、维达尔-奥尔蒂斯A、斯旺克C、希罗曼尼PJ。腹侧ZI中一部分囊泡GABA转运体神经元的活动可预测睡眠开始。《睡眠》。2021年；44(6):zsaa268。doi:10.1093/sleep/zsaa268。）。为了区分GABA亚型，我们现在对含有转录因子Lhx6的ZI神经元进行成像并通过光遗传学方法进行操控。在第一项研究中，将rAAV-DJ-EF1a-DIO-GCaMP6M注入Lhx6-cre小鼠（n = 5；雌性 = 4）的ZI（异氟烷麻醉），植入一个梯度折射率透镜，21天后记录单个Lhx6神经元4小时的睡眠和荧光情况。在132个神经元中检测到钙荧光。45.5%的Lhx6神经元在快速眼动（REM）睡眠时荧光最强；30.3%在清醒时荧光最强；11.4%在清醒 + REM睡眠时荧光最强；9%在NREM睡眠时荧光最强；3.8%没有变化。NREM睡眠时荧光最强的神经元亚群在睡眠开始前30秒发出荧光。第二项研究测试了对ZI Lhx6神经元（n = 14只小鼠）进行单侧光遗传学刺激的效果（AAV5-Syn-FLEX-rc[ChrimsonR-tdTomato]）。在1赫兹和5赫兹（开1分钟 - 关4分钟）下进行刺激，在4小时刺激期（日周期的后半段）显著增加了REM睡眠的比例。典型的实验方法是刺激双侧半球的神经元，但在这里我们发现，对一侧半球的ZI Lhx6神经元进行低频刺激就足以改变意识状态。要确定能够在清醒 - 睡眠状态之间转换大脑的局部节点，需要进行详细的图谱绘制并结合机制测试。
