Ma Caifen, Zhou Ning, Ma Kang, Niu Jiandong, Mi Ting, He Zhenquan, Wen Yujun, Liu Chunhong, He Zhongyi, Niu Jianguo
Department of Human Anatomy, School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, China.
Key Laboratory of Craniocerebral Diseases of Ningxia Hui Autonomous Region, Ningxia Medical University, Yinchuan, China.
Front Neurosci. 2023 Mar 14;17:1122803. doi: 10.3389/fnins.2023.1122803. eCollection 2023.
Fear and sleep impairments common co-exist, but the underlying mechanisms remain unclear. Hypothalamic orexinergic neurons are involved in the regulation of sleep-wake and fear expression. The ventrolateral preoptic area (VLPO) is an essential brain region to promote sleep, and orexinergic axonal fibers projecting to the VLPO are involved in the maintenance of sleep-wake. Neural pathways from hypothalamic orexin neurons to the VLPO might mediate sleep impairments induced by conditioned fear.
To verify above hypothesis, electroencephalogram (EEG) and electromyogram (EMG) were recorded for analysis of sleep-wake states before and 24 h after conditioned fear training. The retrograde tracing technique and immunofluorescence staining was used to identify the projections from the hypothalamic orexin neurons to the VLPO and to observe their activation in mice with conditioned fear. Moreover, optogenetic activation or inhibition of hypothalamic orexin-VLPO pathways was performed to observe whether the sleep-wake can be regulated in mice with conditioned fear. Finally, orexin-A and orexin receptor antagonist was administered into the VLPO to certify the function of hypothalamic orexin-VLPO pathways on mediating sleep impairments induced by conditioned fear.
It was found that there was a significant decrease in the non-rapid eye movement (NREM) and rapid eye movement (REM) sleep time and a significant increase in the wakefulness time in mice with conditioned fear. The results of retrograde tracing technique and immunofluorescence staining showed that hypothalamic orexin neurons projected to the VLPO and observed the CTB labeled orexin neurons were significantly activated (c-Fos+) in the hypothalamus in mice with conditioned fear. Optogenetic activation of hypothalamic orexin to the VLPO neural pathways significantly decreased NREM and REM sleep time and increased wakefulness time in mice with conditioned fear. A significant decrease in NREM and REM sleep time and an increase in wakefulness time were observed after the injection of orexin-A into the VLPO, and the effects of orexin-A in the VLPO were blocked by a pre-administrated dual orexin antagonist (DORA).
These findings suggest that the neural pathways from hypothalamic orexinergic neurons to the VLPO mediate sleep impairments induced by conditioned fear.
恐惧与睡眠障碍常常并存，但其潜在机制仍不清楚。下丘脑促食欲素能神经元参与睡眠 - 觉醒调节以及恐惧表达。腹外侧视前区（VLPO）是促进睡眠的关键脑区，投射至VLPO的促食欲素能轴突纤维参与睡眠 - 觉醒维持。从下丘脑促食欲素神经元到VLPO的神经通路可能介导条件性恐惧诱导的睡眠障碍。
为验证上述假设，在条件性恐惧训练前及训练后24小时记录脑电图（EEG）和肌电图（EMG）以分析睡眠 - 觉醒状态。采用逆行追踪技术和免疫荧光染色来鉴定从下丘脑促食欲素神经元到VLPO的投射，并观察其在条件性恐惧小鼠中的激活情况。此外，对下丘脑促食欲素 - VLPO通路进行光遗传学激活或抑制，以观察条件性恐惧小鼠的睡眠 - 觉醒是否能被调节。最后，将促食欲素A和促食欲素受体拮抗剂注入VLPO，以证实下丘脑促食欲素 - VLPO通路在介导条件性恐惧诱导的睡眠障碍中的作用。
发现条件性恐惧小鼠的非快速眼动（NREM）和快速眼动（REM）睡眠时间显著减少，清醒时间显著增加。逆行追踪技术和免疫荧光染色结果显示，下丘脑促食欲素神经元投射至VLPO，且在条件性恐惧小鼠的下丘脑中观察到CTB标记的促食欲素神经元显著激活（c - Fos +）。对下丘脑促食欲素至VLPO神经通路进行光遗传学激活，显著减少了条件性恐惧小鼠的NREM和REM睡眠时间，并增加了清醒时间。向VLPO注射促食欲素A后，观察到NREM和REM睡眠时间显著减少，清醒时间增加，且预先给予的双重促食欲素拮抗剂（DORA）可阻断促食欲素A在VLPO中的作用。
这些发现表明，从下丘脑促食欲素能神经元到VLPO的神经通路介导了条件性恐惧诱导的睡眠障碍。
