Abstract Detection of somatosensory inputs requires conversion of external stimuli into electrical signals by activation of primary sensory neurons. The mechanisms by which heterogeneous primary sensory neurons encode different somatosensory inputs remains unclear. In vivo dorsal root ganglia (DRG) imaging using genetically-encoded Ca 2+ indicators (GECIs) is currently the best technique for this purpose by providing an unprecedented spatial and populational resolution. It permits the simultaneous imaging of &gt;1800 neurons/DRG in live mice. However, this approach is not ideal given that Ca 2+ is a second messenger and has inherently slow response kinetics. In contrast, genetically-encoded voltage indicators (GEVIs) have the potential to track voltage changes in multiple neurons in real time but often lack the brightness and dynamic range required for in vivo use. Here, we used soma-targeted ASAP4.4-Kv, a novel GEVI, to dissect the temporal dynamics of noxious and non-noxious neuronal signals during mechanical, thermal, or chemical stimulation in DRG of live mice. ASAP4.4-Kv is sufficiently bright and fast enough to optically characterize individual neuron coding dynamics. Notably, using ASAP4.4-Kv, we uncovered cell-to-cell electrical synchronization between adjacent DRG neurons and robust dynamic transformations in sensory coding following tissue injury. Finally, we found that a combination of GEVI and GECI imaging empowered in vivo optical studies of sensory signal processing and integration mechanisms with optimal spatiotemporal analysis. Highlights In vivo ultra fast and sensitive dynamic voltage imaging of peripheral primary sensory neurons by a newly generated genetically-encoded voltage indicator. Identification of mechanical, thermal, or chemical stimuli-evoked voltage signals with superior temporal resolution. Single-cell detection of changes in sub- and suprathreshold voltage dynamics across different disease conditions. Combination of voltage (by ASAP4.4-Kv) and Ca 2+ (by Pirt-GCaMP3) signals to facilitate the understanding of signal processing and integration of primary sensory neurons, especially for noxious versus non-noxious sensation.

Imaging sensory transmission and neuronal plasticity in primary sensory neurons with genetically-encoded voltage indicator, ASAP4.4-Kv