Quantifying the contribution of somatosensory afferent types and changes therein to pain sensitivity using transcutaneous optogenetic stimulation in behaving mice

Abstract

Optogenetics provides an unprecedented opportunity to delineate how different somatosensory afferents contribute to sensation, including pain. By expressing channelrhodopsin-2 (ChR2) in certain afferents, those afferents can be selectively activated by transcutaneous photostimuli applied to behaving mice. Despite the great care taken to precisely target expression of ChR2, imprecise photostimulation has hindered quantitative behavioral testing. Here, using a robot to reproducibly photostimulate behaving mice and precisely measure their paw withdrawal, we show that selectively activating nociceptors with ramped photostimuli evokes faster withdrawal than co-activating nociceptive and non-nociceptive afferents, consistent with gate control. We also show that inflammation-induced hyperexcitability in nociceptors is sufficient to increase pain sensitivity. Electrophysiological testing confirmed that inflammation increases nociceptor excitability without affecting phototransduction. Data further suggest that withdrawal latency depends on the number of nociceptors activated rather than how strongly each nociceptor is activated. Consistent with changes described in nociceptor somata, the behavioral consequences of peripherally blocking different voltage-gated sodium (NaV) channels showed that nociceptor axons normally rely on NaV1.8 but upregulate NaV1.7 after inflammation, with important clinical implications for drug efficacy. Collectively, these results demonstrate the utility of optogenetic pain testing when reproducibly delivered and strategically designed photostimuli are used.SIGNIFICANCE STATEMENTTranscutaneous optogenetic stimulation was first applied to behaving mice to explore the neural basis for pain over a decade ago. Despite great care taken to control which afferents express optogenetic actuators, the sensitivity of such testing has been hindered by crude photostimulation methods and imprecise response measurement. Here, we demonstrate highly quantitative optogenetic pain testing using robotic stimulation and withdrawal detection. By comparing paw withdrawal to equivalent nociceptor activation with and without activation of non-nociceptive afferents, we demonstrate the antinociceptive effect of the latter input. We also demonstrate increased pain sensitivity due to inflammation-induced hyperexcitability in nociceptors and the associated change in NaVisoform expression. We also show that withdrawal from ramped optogenetic stimulation reflects how many nociceptors are recruited.