AAV1-CAG-GCaMP6f

In humans, deletion of SCN9A, encoding the voltage-gated sodium channel Na_V1.7, results in severe pain insensitivity and anosmia. Conditional deletion of Na_V1.7 in mouse sensory neurons also abolished pain, suggesting that the site of analgesia is the nociceptor. Here, researchers demonstrate through in vivo calcium imaging and extracellular recordings that Na_V1.7 knockout mice have essentially normal nociceptor activity. However, glutamate and substance P release from central terminals of spinal nociceptors is greatly reduced by an opioid-dependent mechanism. The analgesic effect was also significantly reversed by central rather than peripheral application of opioid antagonists. In contrast, the lack of neurotransmitter release from olfactory sensory neurons was not associated with opioids. Male and female humans with Na_V1.7 null mutations exhibit reversible analgesia with naloxone. Thus, opioid-dependent inhibition of neurotransmitter release is the primary mechanism of Na_V1.7 null analgesia in mice and humans.

Here, despite altered distributions of cold and mechanical responses, the researchers used calcium imaging to find little evidence of reduced nociceptor excitability in animals lacking Na_V1.7, with no changes in peak response amplitude to any stimulus, the prevalence of multimodality, or the number of noxious heat responses. Broadly similar results were obtained by calcium imaging in WT and KO^Adv mice that virally expressed GCaMP6f (Figure 1A-D).

Figure 1. In vivo calcium imaging of Na_V1.7-deficient sensory neurons virally expressing GCaMP6f. (A) Confocal z-stacks of DRGs in vivo from Na_V1.7 KO^Adv mice that virally expressed GCaMP6f. AAV1-CAG-GCaMP6f was injected intraperitoneally into P2 mouse pups. (B) Bar graphs summarizing the distribution of all sensory neurons that responded to different noxious stimuli in WT and KO^Adv animals. These animals have significantly fewer cells that respond to cold compared to Pirt-GCaMP3 mice, likely due to biased expression of virally delivered GCaMP6f. (D) Bar graph showing similar prevalence of multimodal nociceptors in WT and KO^Adv mice. (E-F) Scatter plots showing similar peak calcium responses (ΔF/F0) elicited by different noxious stimuli in WT and KO^Adv. (MacDonald D I, et al., 2020)