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

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. Abstract 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.

Co-targeting myelin inhibitors and CSPGs markedly enhances regeneration of GDNF-stimulated, but not conditioning-lesioned, sensory axons into the spinal cord

“…Various AAV vectors, including AAV2, demonstrate neuronal tropism ( Mason et al, 2010 ; Fischer et al, 2011 ). Several AAV serotypes exhibit differential tropism for specific subpopulations of DRG neurons: AAV6 preferentially transduces small-sized neurons; AAV5, 8, and 9, large neurons ( Jacques et al, 2012 ; Yu et al, 2013 ; Wu et al, 2016 ; Kubota et al, 2019 ). We found that AAV2 preferentially transduces NF+ neurons.…”

Specific gene expression in unmyelinated dorsal root ganglion neurons in nonhuman primates by intra-nerve injection of AAV 6 vector

“… 18 , 19 , 20 , 21 However, other serotypes of AAV vectors exhibit different target specificity to DRG neurons. For example, AAV8 and AAV9 vectors have been reported to be effective in delivering genes into large-diameter DRG neurons 22 , 23 that signal deep muscle and tactile sensations, but not nociception. Furthermore, by taking advantage of the target specificity of AAV vectors, the use of optogenetics to selectively modulate nociceptive, 20 , 21 , 24 , 25 , 26 as well as non-nociceptive, 23 , 26 DRG neuron activity has also been reported.…”

“…For example, AAV8 and AAV9 vectors have been reported to be effective in delivering genes into large-diameter DRG neurons 22 , 23 that signal deep muscle and tactile sensations, but not nociception. Furthermore, by taking advantage of the target specificity of AAV vectors, the use of optogenetics to selectively modulate nociceptive, 20 , 21 , 24 , 25 , 26 as well as non-nociceptive, 23 , 26 DRG neuron activity has also been reported. Similarly, sustained relief of chronic pain has been achieved in a rodent model, by target-selective delivery of calcium ion (Ca 2+ ) channel-related peptides, together with the AAV6 vector.…”

“…The aim of our study was to examine whether the target specificity of AAV6 and AAV9 vectors to DRG neurons that relay nociceptive or other somatosensory signals, respectively (as established in rodent models 18 , 19 , 20 , 21 , 22 , 23 ), can be reproduced in nonhuman primates. We injected each of the vectors into the sciatic nerve in both the rat and common marmoset (a New World monkey) and compared their target specificity within DRG neurons.…”

Insights Into Spinal Dorsal Horn Circuit Function and Dysfunction Using Optical Approaches

“…However, even with dorsal roots removed, optogenetic excitation of central terminals of primary afferent neurons is sufficient to produce postsynaptic responses in spinal cord neurons (Wang and Zylka, 2009;Foster et al, 2015). For an investigation of primary afferent-evoked responses within the spinal cord, optogenetic stimulation provides the benefit of selectively stimulating a genetically distinct subset of primary afferents, as compared to electrical stimulation (Tashima et al, 2018;Kubota et al, 2019). This provides unparalleled specificity to define circuitry within the spinal cord.…”