Department of Physiology and Pharmacology, Tohoku University Graduate school of Medicine, Sendai, Japan.
Research Fellow of the Japan Society for the Promotion of Science (JSPS Research Fellow), Tokyo, Japan.
Sci Rep. 2018 Apr 3;8(1):5435. doi: 10.1038/s41598-018-23810-8.
Rats are excellent animal models for experimental neuroscience. However, the application of optogenetics in rats has been hindered because of the limited number of established transgenic rat strains. To accomplish cell-type specific targeting of an optimized optogenetic molecular tool, we generated ROSA26/CAG-floxed STOP-ChRFR(C167A)-Venus BAC rats that conditionally express the step-function mutant channelrhodopsin ChRFR(C167A) under the control of extrinsic Cre recombinase. In primary cultured cortical neurons derived from this reporter rat, only Cre-positive cells expressing ChRFR(C167A) became bi-stable, that is, their excitability was enhanced by blue light and returned to the baseline by yellow~red light. In bigenic pups carrying the Phox2B-Cre driver, ChRFR(C167A) was specifically expressed in the rostral parafacial respiratory group (pFRG) in the medulla, where endogenous Phox2b immunoreactivity was detected. These neurons were sensitive to blue light with an increase in the firing frequency. Thus, this transgenic rat actuator/reporter system should facilitate optogenetic studies involving the effective in vivo manipulation of the activities of specific cell fractions using light of minimal intensity.
大鼠是实验神经科学的优秀动物模型。然而，由于已建立的转基因大鼠品系数量有限，光遗传学在大鼠中的应用受到了阻碍。为了实现优化的光遗传学分子工具的细胞类型特异性靶向，我们生成了 ROSA26/CAG-floxed STOP-ChRFR(C167A)-Venus BAC 大鼠，该大鼠在外部 Cre 重组酶的控制下条件性表达步骤功能突变体通道视紫红质 ChRFR(C167A)。在这种报告大鼠衍生的原代皮质神经元中，只有表达 ChRFR(C167A)的 Cre 阳性细胞变得双稳态，即它们的兴奋性通过蓝光增强，并通过黄/红光恢复到基线。在携带 Phox2B-Cre 驱动器的双基因幼仔中，ChRFR(C167A)特异性表达在延髓中的头侧旁面神经呼吸组 (pFRG) 中，在那里检测到内源性 Phox2b 免疫反应性。这些神经元对蓝光敏感，其放电频率增加。因此，这种转基因大鼠执行器/报告基因系统应该有助于光遗传学研究，使用最小强度的光有效地对特定细胞亚群的活性进行体内操作。
