Optogenetic recruitment of spinal reflex pathways from large-diameter primary afferents in non-transgenic rats transduced with AAV9/Channelrhodopsin 2.

Shinji Kubota,Moeko Kudo,Tatsuya Umeda,Kazuhiko Seki,Masahiko Takada,Wupuer Sidikejiang,Ken‐Ichi Inoue

doi:10.1113/jp278292

Abstract

Key points 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.

Highlights

Optogenetics describes the combination of genetic and optical methods (Zemelman et al 2002; Boyden et al 2005) to manipulate the activity of neuronal networks with the aim of determining their roles in the control of specific behaviours
Eight male rats were used to determine the cellular tropism of AAV6 and AAV9 vectors expressing green fluorescent protein (GFP) for DRG neurons
We observed GFP expression in the L4 and L5 DRG neurons following the injection of these AAV vectors into the left sciatic nerve

Summary

Introduction

Optogenetics describes the combination of genetic and optical methods (Zemelman et al 2002; Boyden et al 2005) to manipulate the activity of neuronal networks with the aim of determining their roles in the control of specific behaviours. This technique uses the gene expression of light-activated microbial proteins, opsins, in target neuronal (Boyden et al 2005) or non-neuronal cell populations (Gradinaru et al 2009). Neuroscience studies have taken advantage of this technology to determine the causal relationships of neuronal activity and their behavioural relevance by manipulating neuronal activity and monitoring consequential behavioural changes in awake, behaving animals (Adamantidis et al 2007; Gradinaru et al 2009). The application of optogenetics to the PNS has clear advantages for both therapeutic (Llewellyn et al 2010; Daou et al 2013; Liske et al 2013; Copits et al 2016; Iyer et al 2016; Srinivasan et al 2018) and physiological (Park et al 2016; Abe & Yawo, 2017; Arcourt et al 2017) perspectives, a number of technical difficulties have precluded the application (Montgomery et al 2016)

Objectives

Methods

Results

Conclusion