Department of Neurophysiology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan.
Research Fellow of the Japan Society for the Promotion of Science, Chiyoda-ku, Tokyo, Japan.
J Physiol. 2019 Oct;597(19):5025-5040. doi: 10.1113/JP278292. Epub 2019 Aug 28.
We demonstrated optical activation of primary somatosensory afferents with high selectivity to fast-conducting fibres by means of adeno-associated virus 9 (AAV9)-mediated gene transduction in dorsal root ganglion (DRG) neurons. AVV9 expressing green fluorescent protein showed high selectivity and transduction efficiency for fast-conducting, large-sized DRG neurons. Compared with conventional electrical stimulation, optically elicited volleys in primary afferents had higher sensitivity with stimulus amplitude, but lower sensitivity with stimulus frequency. Optically elicited dorsal root volleys activated postsynaptic neurons in the segmental spinal pathway. This proposed technique will help establish the causal relationships between somatosensory afferent inputs and neural responses in the CNS as well as behavioural outcomes in higher mammals where transgenic animals are not available.
Previously, fundamental structures and their mode of action in the spinal reflex circuit were determined by confirming their input-output relationship using electrophysiological techniques. In those experiments, the electrical stimulation of afferent fibres was used as a core element to identify different types of reflex pathways; however, a major disadvantage of this technique is its non-selectivity. In this study, we investigated the selective activation of large-diameter afferents by optogenetics combined with a virus vector transduction technique (injection via the sciatic nerve) in non-transgenic male Jcl:Wistar rats. We found that green fluorescent protein gene transduction of rat dorsal root ganglion (DRG) neurons with a preference for medium-to-large-sized cells was achieved using the adeno-associated virus 9 (AAV9) vector compared with the AAV6 vector (P = 0.021). Furthermore, the optical stimulation of Channelrhodopsin 2 (ChR2)-expressing DRG neurons (transduced by AAV9) produced compound action potentials in afferent nerves originating from fast-conducting nerve fibres. We also confirmed that physiological responses to different stimulus amplitudes were comparable between optogenetic and electrophysiological activation. However, compared with electrically elicited responses, the optically elicited responses had lower sensitivity with stimulus frequency. Finally, we showed that afferent volleys evoked by optical stimulation were sufficient to activate postsynaptic neurons in the spinal reflex arc. These results provide new ways for understanding the role of sensory afferent input to the central nervous system regarding behavioural control, especially when genetically manipulated animals are not available, such as higher mammals including non-human primates.
我们通过腺相关病毒 9（AAV9）介导的背根神经节（DRG）神经元基因转导，展示了对快速传导纤维具有高选择性的初级体感传入的光激活。表达绿色荧光蛋白的 AAV9 对快速传导的大尺寸 DRG 神经元具有高选择性和转导效率。与传统的电刺激相比，光激发的初级传入冲动在刺激幅度上具有更高的敏感性，但在刺激频率上的敏感性较低。光激发的背根冲动激活了脊髓节段性通路中的突触后神经元。这项拟议的技术将有助于在高级哺乳动物中建立体感传入输入与中枢神经系统神经反应以及行为结果之间的因果关系，而这些动物中没有转基因动物。
以前，通过使用电生理技术来确认其输入-输出关系，确定了脊髓反射回路中的基本结构及其作用方式。在这些实验中，传入纤维的电刺激被用作识别不同类型反射通路的核心要素；然而，该技术的一个主要缺点是其非选择性。在这项研究中，我们通过病毒载体转导技术（通过坐骨神经注射），结合光遗传学，在非转基因雄性 Jcl：Wistar 大鼠中研究了大直径传入纤维的选择性激活。我们发现，与 AAV6 载体相比，腺相关病毒 9（AAV9）载体更有利于大鼠背根神经节（DRG）神经元的绿色荧光蛋白基因转导，偏爱中到大细胞（P=0.021）。此外，光刺激 ChR2 表达的 DRG 神经元（由 AAV9 转导）产生源自快速传导神经纤维的传入神经的复合动作电位。我们还证实，不同刺激幅度的生理反应在光遗传学和电生理学激活之间是可比的。然而，与电激发反应相比，光激发反应在刺激频率上的敏感性较低。最后，我们表明，光学刺激引起的传入冲动足以激活脊髓反射弧中的突触后神经元。这些结果为理解感觉传入输入对中枢神经系统在行为控制方面的作用提供了新途径，特别是在无法获得基因操作动物时，例如包括非人类灵长类动物在内的高级哺乳动物。
