Optogenetic Long-Term Manipulation of Behavior and Animal Development

Channelrhodopsin-2 (ChR2) is widely used for rapid photodepolarization of neurons, yet, as it requires high-intensity blue light for activation, it is not suited for long-term in vivo applications, e.g. for manipulations of behavior, or photoactivation of neurons during development. We used “slow” ChR2 variants with mutations in the C128 residue, that exhibit delayed off-kinetics and increased light sensitivity in Caenorhabditis elegans. Following a 1 s light pulse, we could photodepolarize neurons and muscles for minutes (and with repeated brief stimulation, up to days) with low-intensity light. Photoactivation of ChR2(C128S) in command interneurons elicited long-lasting alterations in locomotion. Finally, we could optically induce profound changes in animal development: Long-term photoactivation of ASJ neurons, which regulate larval growth, bypassed the constitutive entry into the “dauer” larval state in daf-11 mutants. These lack a guanylyl cyclase, which possibly renders ASJ neurons hyperpolarized. Furthermore, photostimulated ASJ neurons could acutely trigger dauer-exit. Thus, slow ChR2s can be employed to long-term photoactivate behavior and to trigger alternative animal development.通道视紫红质-2（Channelrhodopsin-2，ChR2）被广泛应用于神经元的快速光诱导去极化，但因其激活需要高强度蓝光，故不适用于长期体内研究场景，例如行为操控或发育过程中的神经元光激活。我们针对C128位点残基进行突变，构建了“慢型”ChR2变体，这类变体在秀丽隐杆线虫（Caenorhabditis elegans）中展现出延迟的关闭动力学特性与更高的光敏感性。在施加1秒光脉冲后，我们可通过低强度光对神经元与肌肉细胞进行长达数分钟的光诱导去极化；若辅以重复短时间刺激，该效应甚至可维持长达数天。对命令性中间神经元中的ChR2(C128S)进行光激活，可引发运动行为的长期改变。最后，我们可通过光学手段诱导动物发育产生显著改变：对调控幼虫生长的ASJ神经元进行长期光激活，可绕过daf-11突变体自发进入的dauer幼虫态。该类突变体缺乏鸟苷酸环化酶，这可能导致ASJ神经元处于超极化状态。经光刺激的ASJ神经元还可快速触发dauer幼虫态退出。综上，慢型ChR2变体可用于长期光操控动物行为，并诱导动物发育发生替代性改变。