BACKGROUND Optogenetic silencing techniques have expanded the causal understanding of the functions of diverse neuronal cell types in both the healthy and diseased brain. A widely used inhibitory optogenetic actuator is eNpHR3.0, an improved version of the light-driven chloride pump halorhodopsin derived from Natronomonas pharaonis. A major drawback of eNpHR3.0 is related to its pronounced inactivation on a time-scale of seconds, which renders it unsuited for applications that require long-lasting silencing. RESULTS Using transgenic mice and Xenopus laevis oocytes expressing an eNpHR3.0-EYFP fusion protein, we here report optimized photo-stimulation techniques that profoundly increase the stability of eNpHR3.0-mediated currents during long-term photo-stimulation. We demonstrate that optimized photo-stimulation enables prolonged hyperpolarization and suppression of action potential discharge on a time-scale of minutes. CONCLUSIONS Collectively, our findings extend the utility of eNpHR3.0 to the long-lasting inhibition of excitable cells, thus facilitating the optogenetic dissection of neural circuits.

中文翻译：

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优化了光刺激视紫红质对神经元的长期抑制作用。

背景技术光遗传沉默技术已经扩大了对健康和患病大脑中多种神经元细胞类型功能的因果理解。广泛使用的抑制性光遗传学致动器是eNpHR3.0，它是源自法老王藻（Natronomonas pharaonis）的光驱动氯化物泵卤代视紫红质的改良版。eNpHR3.0的主要缺点与在几秒钟的时间尺度上的明显失活有关，这使其不适用于需要长时间沉默的应用。结果使用表达eNpHR3.0-EYFP融合蛋白的转基因小鼠和非洲爪蟾卵母细胞，我们在此报告了优化的光刺激技术，该技术可在长期光刺激期间显着增加eNpHR3.0介导的电流的稳定性。我们证明优化的光刺激能够在几分钟的时间范围内延长超极化和抑制动作电位放电。结论总的来说，我们的发现将eNpHR3.0的应用扩展到了对可兴奋细胞的长期抑制，从而促进了神经回路的光遗传学解剖。