CRISPR/Cas9-mediated in vivo gene editing reveals that neuronal 5-HT1A receptors in the dorsal raphe nucleus contribute to body temperature regulation in mice
CRISPR/Cas9-mediated in vivo gene editing reveals that neuronal 5-HT1A receptors in the dorsal raphe nucleus contribute to body temperature regulation in mice
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DOI：
10.1016/j.brainres.2019.06.009
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发表时间：
2019-09
期刊：
影响因子：
2.9
通讯作者：
N. Nishitani;Yu Ohmura;K. Nagayasu;Norihiro Shibui;S. Kaneko;Akiko Ohashi;Takayuki Yoshida;A. Yamanaka;M. Yoshioka
中科院分区：
文献类型：
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作者：
N. Nishitani;Yu Ohmura;K. Nagayasu;Norihiro Shibui;S. Kaneko;Akiko Ohashi;Takayuki Yoshida;A. Yamanaka;M. Yoshioka
中枢神经系统中的5-羟色胺(5-HT)通过作用于14种已知的受体亚型，调节从基本的体内平衡控制到高级大脑功能的各种生物学功能。然而，由于大多数受体缺乏高度选择性的配体，通常仍不清楚哪种受体亚型负责特定的功能。虽然5-羟色胺受体基因敲除小鼠是有用的，但各种受体在脑内的广泛分布使得很难剖析特定脑区和细胞类型的受体功能。CRISPR/CAS9介导的病毒基因组编辑技术的最新进展可能会克服这一问题。在本研究中，我们构建了在神经元特异性启动子的控制下，针对Htr1a(SgHtr1a)和Cre重组酶的单引导(Sg)RNA的病毒载体。将病毒载体注射到Cre依赖Cas9基因敲除小鼠的中缝背核(DRN)，诱导Cre依赖的Cas9主要在DRN、5-羟色胺和GABA神经元中表达。错配裂解分析和Sanger测序显示在靶点形成插入或缺失。通过将表达sgHtr1a的病毒载体微量注射到Cre依赖的Cas9敲入小鼠的DRN中，可以减弱5-HT1a受体激动剂诱导的低温，并增强选择性5-羟色胺再摄取抑制剂(SSRI)的抗抑郁作用。这些结果表明，活体CRISPR/Cas9介导的5-羟色胺受体基因敲除策略为阐明特定细胞类型和脑区域的5-羟色胺受体功能提供了一种可靠且低成本的方法。此外，我们还证明DRN中的神经元5-HT1A受体调节体温和SSRI的抗抑郁作用。
Serotonin (5-HT) in the central nervous system regulates a variety of biological functions, from the basic homeostatic control to higher brain functions, by acting on fourteen known receptor subtypes. However, it is still usually unclear which receptor subtype is responsible for a specific function due to the lack of highly selective ligands for most of these receptors. Although 5-HT receptor knockout mice are useful, the brain-wide distribution of various receptors makes it difficult to dissect receptor functions in specific and brain regions and cell types. Recent advances in CRISPR/Cas9-mediatedin vivogenome editing technology may overcome this problem. In this study, we constructed a viral vector expressing a single guide (sg)RNA targetingHtr1a(sgHtr1a) and Cre recombinase under the control of a neuron-specific promoter. Injection of the viral vector into the dorsal raphe nucleus (DRN) of Cre-dependent Cas9 knock-in mice induced Cre-dependent Cas9 expression mainly in DRN serotonin and GABA neurons. Mismatch cleavage assay and Sanger sequencing showed insertion or deletion formation at the target site. 5-HT1Areceptor agonist-induced hypothermia was attenuated and antidepressant effect of a selective serotonin reuptake inhibitor (SSRI) was enhanced by microinjection of the viral vector expressing sgHtr1a into the DRN of Cre-dependent Cas9 knock-in mice. These results suggest that thisin vivoCRISPR/Cas9-mediated 5-HT receptor gene knockout strategy provides a reliable and low-cost method for elucidating 5-HT receptor functions in specific cell types and brain regions. Further, we demonstrate that the neuronal 5-HT1Areceptor in the DRN regulates body temperature and antidepressant effect of SSRI.