In vivo GCaMP imaging reveals distinct subsets of dorsal root ganglion neurons driving spontaneous pain after nerve injury in mice

Abstract Spontaneous pain is a hallmark and debilitating feature of neuropathic pain, yet the mechanisms driving spontaneous pain remain poorly understood. Although abnormal spontaneous activity (SA) in primary sensory neurons has been observed after nerve injury, it remains unclear how such activity is organized across neuronal populations within the dorsal root ganglion (DRG) and how it relates to different pain components. Here, we used large-scale in vivo calcium imaging to examine spontaneous and stimulus-evoked activity in lumbar DRG neurons after spared-nerve injury (SNI). Spared-nerve injury significantly increased the incidence of single-neuron SA in DRG neurons. By combining functional imaging with retrograde labeling, we found that this activity arose not only from injured afferents (51.8%) but also from a substantial population of spared neurons (48.2%) within the same ganglion. Among spared neurons exhibiting spontaneous activity, nearly half (48.4%) showed no detectable responses to a broad panel of mechanical and thermal stimuli under our recording conditions, whereas most responsive neurons retained modality selectivity. Importantly, the incidence of single-neuron SA was positively associated with spontaneous pain-like behavior, but not with mechanical hypersensitivity, after SNI, supporting a direct link between SA of DRG neurons and spontaneous neuropathic pain. Together, these findings show that SA in the DRG is organized across both anatomical and functional dimensions, providing a framework for understanding how abnormal peripheral neuronal activity relates to spontaneous neuropathic pain.