To figure our the role of CeA CRF neurons in anxiety-promote wakefulness

失眠症是一种流行性疾病，而焦虑被认为是失眠的一个主要原因.然而，涉及的大脑区域尚不清楚。为了解决这个问题，我使用hM 3Dq蛋白激活杏仁核中产生促肾上腺皮质激素释放因子（CRF）的神经元，这种神经元被认为可以调节焦虑，这些小鼠的清醒时间增加了。这表明杏仁核CRF神经元在焦虑诱导的觉醒中起作用。然而，杏仁核CRF神经元的神经元活动与我们从杏仁核CRF神经元激活促进唤醒的预测相反。因此，我对hM 3Dq蛋白表达区域进行了分类。结果发现，不仅杏仁核有表达，位于杏仁核前部的前连合外侧部后肢间质核（IPACL）也有表达。为了确定IPACL和杏仁核的差异，我用原位杂交技术对CRF神经元表达的神经递质进行了RNA染色。强啡肽在IPACL和杏仁核CRF神经元均呈低表达。神经降压素在IPACL中不表达，与杏仁核CRF神经元几乎100%重叠。IPACL和杏仁核CRF神经元都是GABA能神经元。在此基础上，我敲除CRF神经元中的强啡肽（神经降压素，或CRF），以进一步了解肽在觉醒中的功能。我的研究结果表明，IPACL/杏仁核CRF神经元中强啡肽的敲除与对照相比增加了觉醒。以上结果表明，强啡肽可能参与了IPACL/杏仁核CRF神经元的觉醒调节。

The insomnia is a pandemic disease now, and anxiety is though as a main reason. However, the brain areas involved in are unclear. To address it, I used hM3Dq protein to activate corticotropin-releasing factor (CRF) producing neurons in amygdala, which is known to regulate anxiety, and the time in wake increased in these mice. This indicates the amygdala CRF neurons play a role in anxiety-induced wakefulness. However, the neuronal activity of amygdala CRF neurons is opposite to our prediction from wake-promoted by activation of amygdala CRF neurons. Therefore, I categorized the hM3Dq-protein expressed regions. I found out that not only amygdala, but also interstitial nucleus of the posterior limb of the anterior commissure, lateral part (IPACL), which is located in anterior part of amygdala, expressed. To identify the different between IPACL and amygdala, I used in situ hybridization to stain RNA of neurotransmitters expressing in CRF neurons. Dynorphin had low expression in both IPACL and amygdala CRF neurons. Neurotensin had no expression in IPACL and nearly 100% overlapping with amygdala CRF neurons. Both IPACL and amygdala CRF neurons are GABAergic. Based on these, I knockout dynorphin (neurotensin, or CRF) in CRF neurons to further understand the peptide function in wakefulness. My results indicated that knockout of dynorphin in IPACL/amygdala CRF neurons increased the wakefulness compared to control. All my results showed that the regulation of wakefulness in IPACL/amygdala CRF neurons might be caused by dynorphin peptide.