Abstract
OBJECTIVES: Sensitization of C-fiber nociceptors plays a critical role in spontaneous and ongoing pain in patients with chronic pain. We recently demonstrated that C-fiber afferents can be reversibly blocked through activity-dependent conduction slowing, suggesting that selective activation of C-fiber afferents may represent a novel strategy for pain relief. We hypothesized that electrical peripheral nerve stimulation (ePNS) and dorsal root ganglion (DRG) stimulation exhibit distinct activation thresholds for unmyelinated C-fiber afferents. MATERIALS AND METHODS: We characterized the activation thresholds of A- and C-fiber afferents during ePNS and DRG stimulation using single-fiber electrophysiological recordings from split nerve filaments and optical GCaMP6f imaging of intact DRGs. We further quantified the distribution of the sodium channel subtype NaV1.6 in afferent axons and somata and evaluated its contribution to neuronal excitability using NEURON-based computational modeling. RESULTS: Single-fiber recordings showed that activation of C-fiber axons required approximately tenfold higher stimulus amplitudes than Aα/β-fiber axons during ePNS. In contrast, DRG stimulation within a narrow amplitude range robustly activated both small- and large-diameter DRG neurons, with putative C-fiber afferents comprising 57% of the activated population, indicating markedly reduced differential activation thresholds compared with ePNS. Analysis of published single-cell RNA-sequencing datasets revealed high NaV1.6 expression in TRPV1-positive C-fiber nociceptors. Immunohistochemical staining demonstrated prominent clustering of NaV1.6 in the stem axons of most DRG neurons, including small-diameter C-fiber afferents, whereas NaV1.6 was absent from C-fiber axons in the sciatic nerve. NEURON simulations further showed that NaV1.6 clustering at the stem axon is a key determinant of activation thresholds during DRG stimulation. CONCLUSIONS: These findings identify a structural and molecular mechanism underlying the efficient activation of C-fiber afferents by DRG stimulation and provide mechanistic insight into the superior therapeutic efficacy of DRG stimulation for the treatment of C-fiber-mediated chronic pain.