Assessing the excitability changes of DRG neurons in models of diabetic neuropathy

Diabetic neuropathy (DN) is the most common type of neuropathy, with 25-50% of patients experiencing pain at any stage of its progression. However, the understanding of the pathophysiology of DN is still incomplete. No disease-modifying treatments have yet been developed and pain is only treated symptomatically with drugs targeting the central nervous system (CNS). These have limited efficacy and can produce serious side effects. Research into novel analgesics is turning away from the CNS and now focusing on the peripheral nervous system (PNS) and more specifically the pain-sensing neurons (nociceptors) to produce better-targeted and safer treatments. However, to this end, a suitable medium-to-high throughput screening tool is needed, able to discriminate nociceptors from non-nociceptors in a heterogeneous cell population, such as the dorsal root ganglia (DRG). Previously, our lab showed a link between the veratridine (VTD)-induced oscillatory (OS) and slow decay (SD) calcium response profiles with nociceptors and non-nociceptors, respectively. Here, we validated the VTD-response profiles as broad functional markers of these DRG subpopulations using mice with genetically ablated nociceptors (1.8-DTA). Then, by using voltage-gated sodium channel (VGSC) blockers with different specificities, we demonstrated that the VTD-Ca2+ imaging assay can be used as a drug screening platform for drugs, individually or in novel combinations. The VTD-Ca2+ imaging assay was then applied in combination with nociceptive agonists to investigate the excitability changes in distinct neuronal subpopulations during DN. In db/db mice, during the early metabolic phase, small- to medium-diameter nociceptors showed 1.4 -fold increased sensitivity to CAP and 1.2-fold increased VGSC excitability. In the late, NEU phase, small-diameter nociceptors showed increased sensitivity to CAP (1.6-fold) and increased VGSC excitability (1.4-fold), whereas medium nociceptors show decreased sensitivity to AITC (1.8-fold) . This is the first study to characterise phase- and subpopulation-specific excitability changes in the well-established db/db mouse model. Collectively, these results point to specific subpopulations of DRG neurons affected during the early and late stages of DN. These findings could aid in the better targeting of novel therapies for the treatment of DN and pain. Furthermore, we have demonstrated that the VTD-Ca2+ imaging assay can be applied as a tool for the characterisation of excitability changes in distinct DRG subpopulations during neuropathological conditions such as DN