In vivo optogenetic activation of Nav1.8+ cutaneous nociceptors and their responses to natural stimuli

Abstract

Optogenetic methods that utilize expression of the light-sensitive protein channelrhodopsin-2 (ChR2) in neurons have enabled selective activation of specific subtypes or groups of neurons to determine their functions. Using a transgenic mouse model in which neurons natively expressing Na_v1.8 (a tetrodotoxin-resistant voltage-gated sodium channel) also express the light-gated channel ChR2, we have been able to determine the functional properties of Na_v1.8-expressing cutaneous nociceptors of the glabrous skin in vivo. Most (44 of 53) of the C-fiber nociceptors isolated from Na_v1.8-ChR2⁺ mice were found to be responsive to blue (470 nm) light. Response characteristics, including conduction velocity and responses to mechanical stimuli, were comparable between nociceptors isolated from Na_v1.8-ChR2⁺ and control mice. Interestingly, while none of the non-light-responsive C-fibers were sensitive to heat or cold, nearly all (77%) light-sensitive fibers were excited by mechanical and thermal stimuli, suggesting that Na_v1.8 is predominantly expressed by C-fiber nociceptors that are responsive to multiple stimulus modalities. The ability to activate peripheral nociceptors with light provides a method of stimulation that is noninvasive, does not require mechanical interruption of the skin, and accesses receptive fields that might be difficult or impossible to stimulate with standard stimuli while allowing repeated stimulation without injuring the skin.NEW & NOTEWORTHY Transgenic mice that express the blue light-sensitive protein channelrhodopsin2 (ChR2) in nociceptive nerve fibers that contain voltage-gated sodium channel Na_v1.8 were used to determine functional properties of these afferent fibers. Electrophysiological recordings in vivo revealed that most nociceptive fibers that possess Na_v1.8 are C-fiber nociceptors that respond to multiple stimulus modalities. Furthermore, responses evoked by blue light stimulation were comparable to those elicited by noxious mechanical, heat, and cold stimuli.

Keywords: C-fiber; channelrhodopsin2; electrophysiology; peripheral neuron; tibial nerve.

Fig. 1.

Light-responsive nociceptors respond with higher discharge rates as light intensity increases. A: mean responses of light-responsive C-fibers (n = 43) to increasing intensity-of-light stimuli. B: representative example of a light-responsive C-fiber nociceptor. 1: conduction latency: multiple traces were overlapped to show consistency of conduction latency (CV = 0.38 m/s). The left arrow indicates the onset of the stimulus and the right arrow indicates the action potential of the fiber of interest. 2: response evoked by 147 mN for 5 s. 3: responses evoked by 5 s stimulation with light stimuli of increasing intensity.

Fig. 2.

Responses to thermal stimuli did not differ between control and Na_v1.8-ChR2⁺ mice. A: means ± SE response thresholds of C-fibers responsive to heat and/or cold did not differ between control and Na_v1.8-ChR2⁺ mice. B: means ± SE cumulative responses of C-fibers responsive to heat and/or cold did not differ between control and Na_v1.8-ChR2⁺ mice. C: responses to a range of temperatures did not differ for cold-responsive C-fibers in control (n = 15) and Na_v1.8-ChR2⁺ (n = 10) mice. D: responses to a range of temperatures did not differ for heat-responsive C-fibers in control (n = 11) and Na_v1.8-ChR2⁺ (n = 14) mice. E: representative examples of evoked responses of a light-responsive C-mechanoheatcold nociceptor fiber. Top: responses to 5-s heat stimuli of increasing temperature; middle: responses to 10-s cold stimuli of decreasing temperature; bottom: responses to 5-s light stimuli of increasing intensity. F: evoked responses from a single light-responsive CH fiber. Top: responses to 5-s heat stimuli of increasing temperature; bottom: responses to 5-s light stimuli of increasing intensity.