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

失眠现在是一种流行病，焦虑被认为是一个主要原因。然而，涉及的大脑区域尚不清楚。为了解决这个问题，我用hM3Dq蛋白激活了杏仁核中产生促肾上腺皮质激素释放因子（CRF）的神经元，这种神经元被认为是调节焦虑的，结果这些老鼠醒着的时间增加了。这表明杏仁核CRF神经元在焦虑引起的觉醒中起作用。然而，杏仁核CRF神经元的神经元活动与我们通过激活杏仁核CRF神经元来促进觉醒的预测相反。因此，我对hm3dq蛋白表达区进行了分类。我发现，不仅是杏仁核，位于杏仁核前部的前联合后肢的间质核，外侧部（IPACL）也表达。为了确定IPACL和杏仁核之间的区别，我使用原位杂交技术对CRF神经元中表达的神经递质RNA进行染色。Dynorphin在IPACL和杏仁核CRF神经元中均有低表达。神经紧张素在IPACL中无表达，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dynorphin (neurotensin，或CRF)，以进一步了解肽在清醒状态下的功能。我的研究结果表明，与对照组相比，IPACL/杏仁核CRF神经元中dynorphin的敲除增加了清醒度。这些结果表明，IPACL/杏仁核CRF神经元觉醒的调节可能是由肌啡肽引起的。

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.