Selective optogenetic activation of Na(V)1.7–expressing afferents in Na(V)1.7-ChR2 mice induces nocifensive behavior without affecting responses to mechanical and thermal stimuli

In small and large spinal dorsal root ganglion neurons, subtypes of voltage-gated sodium channels, such as Na(V)1.7, Na(V)1.8, and Na(V)1.9 are expressed with characteristically localized and may play different roles in pain transmission and intractable pain development. Selective stimulation of each specific subtype in vivo may elucidate its role of each subtype in pain. So far, this has been difficult with current technology. However, Optogenetics, a recently developed technique, has enabled selective activation or inhibition of specific neural circulation in vivo. Moreover, optogenetics had even been used to selectively excite Na(V)1.8-expressing dorsal root ganglion neurons to induce nocifensive behavior. In recent years, genetic modification technologies such as CRISPR/Cas9 have advanced, and various knock-in mice can be easily generated using such technology. We aimed to investigate the effects of selective optogenetic activation of Na(V)1.7-expressing afferents on mouse behavior. We used CRISPR/Cas9-mediated homologous recombination to generate bicistronic Na(V)1.7–iCre knock-in mice, which express iCre recombinase under the endogenous Na(V)1.7 gene promoter without disrupting Na(V)1.7. The Cre-driver mice were crossed with channelrhodopsin-2 (ChR2) Cre-reporter Ai32 mice to obtain Na(V)1.7(iCre/+);Ai32/+, Na(V)1.7(iCre/iCre);Ai32/+, Na(V)1.7(iCre/+);Ai32/Ai32, and Na(V)1.7(iCre/iCre);Ai32/Ai32 mice. Compared with wild–type mice behavior, no differences were observed in the behaviors associated with mechanical and thermal stimuli exhibited by mice of the aforementioned genotypes, indicating that the endogenous Na(V)1.7 gene was not affected by the targeted insertion of iCre. Blue light irradiation to the hind paw induced paw withdrawal by mice of all genotypes in a light power-dependent manner. The threshold and incidence of paw withdrawal and aversive behavior in a blue-lit room were dependent on ChR2 expression level; the strongest response was observed in Na(V)1.7(iCre/iCre);Ai32/Ai32 mice. Thus, we developed a non-invasive pain model in which peripheral nociceptors were optically activated in free-moving transgenic Na(V)1.7–ChR2 mice.