Direct voltage imaging of peripheral axons in vivo reveals ongoing nociceptor firing in healthy mice

Abstract

The electrical output of a neuron is generated and conducted along its axon, yet the axon has remained largely inaccessible to direct measurement of membrane potential in intact tissue. Genetically encoded voltage indicators have enabled optical recording of membrane potential dynamics from neuronal cell bodies and dendrites in vivo, but direct voltage imaging of identified axons in the living animal has remained largely unexplored. This gap is especially consequential for nociceptive neurons, whose signals are generated and propagated at peripheral terminals far from the soma. Here, we expressed genetically encoded voltage indicators in trigeminal neurons and, using high-speed widefield imaging with holographic targeted illumination, recorded membrane-voltage dynamics directly from corneal nociceptive terminal arbors and their parent axons in vivo, in intact tissue. Individual terminals responded to capsaicin and menthol with depolarization accompanied by trains of spiking activity. Unexpectedly, a substantial fraction generated ongoing action-potential-like firing in the absence of an applied stimulus. This firing was sodium-channel-dependent, synchronized across sister terminal branches, and shared its dynamics with the parent axon. Ongoing terminal firing persisted during lidocaine delivery to the trigeminal ganglion, supporting a peripheral origin distal to the ganglion, and was abolished by inhibition of NaV1.8 or HCN/Ih conductances. Thus, direct voltage imaging reveals ongoing and spatially coordinated electrical activity within intact peripheral sensory arbors. More broadly, this approach provides access to the generation and integration of neuronal output within fine axonal compartments in vivo.