Department of Anesthesiology, Faculty of Medicine, University of Miyazaki, Miyazaki, Miyazaki, Japan.
Department of Pharmacology, Faculty of Medicine, University of Miyazaki, Miyazaki, Miyazaki, Japan.
PLoS One. 2022 Oct 6;17(10):e0275751. doi: 10.1371/journal.pone.0275751. eCollection 2022.
In small and large spinal dorsal root ganglion neurons, subtypes of voltage-gated sodium channels, such as NaV1.7, NaV1.8, and NaV1.9 are expressed with characteristically localized and may play different roles in pain transmission and intractable pain development. Selective stimulation of each specific subtype in vivo may elucidate its role of each subtype in pain. So far, this has been difficult with current technology. However, Optogenetics, a recently developed technique, has enabled selective activation or inhibition of specific neural circulation in vivo. Moreover, optogenetics had even been used to selectively excite NaV1.8-expressing dorsal root ganglion neurons to induce nocifensive behavior. In recent years, genetic modification technologies such as CRISPR/Cas9 have advanced, and various knock-in mice can be easily generated using such technology. We aimed to investigate the effects of selective optogenetic activation of NaV1.7-expressing afferents on mouse behavior. We used CRISPR/Cas9-mediated homologous recombination to generate bicistronic NaV1.7-iCre knock-in mice, which express iCre recombinase under the endogenous NaV1.7 gene promoter without disrupting NaV1.7. The Cre-driver mice were crossed with channelrhodopsin-2 (ChR2) Cre-reporter Ai32 mice to obtain NaV1.7iCre/+;Ai32/+, NaV1.7iCre/iCre;Ai32/+, NaV1.7iCre/+;Ai32/Ai32, and NaV1.7iCre/iCre;Ai32/Ai32 mice. Compared with wild-type mice behavior, no differences were observed in the behaviors associated with mechanical and thermal stimuli exhibited by mice of the aforementioned genotypes, indicating that the endogenous NaV1.7 gene was not affected by the targeted insertion of iCre. Blue light irradiation to the hind paw induced paw withdrawal by mice of all genotypes in a light power-dependent manner. The threshold and incidence of paw withdrawal and aversive behavior in a blue-lit room were dependent on ChR2 expression level; the strongest response was observed in NaV1.7iCre/iCre;Ai32/Ai32 mice. Thus, we developed a non-invasive pain model in which peripheral nociceptors were optically activated in free-moving transgenic NaV1.7-ChR2 mice.
在小型和大型脊髓背根神经节神经元中，电压门控钠离子通道的亚型，如 NaV1.7、NaV1.8 和 NaV1.9，表达具有特征性的局部定位，并可能在疼痛传递和难治性疼痛发展中发挥不同的作用。在体内选择性刺激每种特定亚型可能阐明其在疼痛中的作用。到目前为止，这在当前技术下是困难的。然而，光遗传学是一种最近开发的技术，它使在体内选择性激活或抑制特定的神经循环成为可能。此外，光遗传学甚至被用于选择性地兴奋表达 NaV1.8 的背根神经节神经元以诱导伤害感受行为。近年来，基因修饰技术如 CRISPR/Cas9 的发展，使得使用这种技术很容易生成各种基因敲入小鼠。我们旨在研究选择性光遗传学激活表达 NaV1.7 的传入神经对小鼠行为的影响。我们使用 CRISPR/Cas9 介导的同源重组生成双顺反子 NaV1.7-iCre 基因敲入小鼠，该小鼠在内源性 NaV1.7 基因启动子下表达 iCre 重组酶，而不破坏 NaV1.7。Cre 驱动小鼠与通道视紫红质-2 (ChR2) Cre 报告 Ai32 小鼠交配，以获得 NaV1.7iCre/+;Ai32/+、NaV1.7iCre/iCre;Ai32/+、NaV1.7iCre/+;Ai32/Ai32 和 NaV1.7iCre/iCre;Ai32/Ai32 小鼠。与野生型小鼠行为相比，上述基因型小鼠的机械和热刺激相关行为没有差异，表明内源性 NaV1.7 基因不受靶向插入 iCre 的影响。蓝光照亮后爪以光功率依赖的方式诱导所有基因型小鼠的后爪退缩。后爪退缩和在蓝光照射房间中的回避行为的阈值和发生率取决于 ChR2 表达水平；在 NaV1.7iCre/iCre;Ai32/Ai32 小鼠中观察到最强的反应。因此，我们开发了一种非侵入性疼痛模型，其中在自由移动的转基因 NaV1.7-ChR2 小鼠中光学激活外周伤害感受器。
