An unbiased and efficient assessment of excitability of sensory neurons for analgesic drug discovery

Abstract

Alleviating chronic pain is challenging, due to lack of drugs that effectively inhibit nociceptors without off-target effects on motor or central neurons. Dorsal root ganglia (DRG) contain nociceptive and non-nociceptive neurons. Drug screening on cultured DRG neurons, rather than cell lines, allows for the identification of drugs most potent on nociceptors with no effects on non-nociceptors (as a proxy for unwanted side effects on central nervous system and motor neurons). However, screening using DRG neurons is currently a low-throughput process, and there is a need for assays to speed this process for analgesic drug discovery. We previously showed that veratridine elicits distinct response profiles in sensory neurons. Here, we show evidence that a veratridine-based calcium assay allows for an unbiased and efficient assessment of a drug effect on nociceptors (targeted neurons) and non-nociceptors (nontargeted neurons). We confirmed the link between the oscillatory profile and nociceptors, and the slow-decay profile and non-nociceptors using 3 transgenic mouse lines of known pain phenotypes. We used the assay to show that blockers for Nav1.7 and Nav1.8 channels, which are validated targets for analgesics, affect non-nociceptors at concentrations needed to effectively inhibit nociceptors. However, a combination of low doses of both blockers had an additive effect on nociceptors without a significant effect on non-nociceptors, indicating that the assay can also be used to screen for combinations of existing or novel drugs for the greatest selective inhibition of nociceptors.

Painful and painless mutations of SCN9A and SCN11A voltage-gated sodium channels

“…Equally important, for the evaluation of any drug combination, is the potential effects on non-nociceptors as well as nociceptors. We recently described a relevant assay [88] and provided proof-of-concept data that showed a combination of Na v 1.7 and Na v 1.8 blockers produced a reduction in the excitability of DRG neurons close to that measured in Na v 1.7 KO [89]. Changing the constituents and doses in VGSC blocker combinations may enable clinicians to manage chronic pain safely over the long term.…”

“…Deletion of Na v 1.7 causes an increase in action potential threshold in small-diameter DRG neurons [107]. Deletion of Na v 1.7 causes a major decrease in DRG neuron responsiveness to the VGSC opener veratridine [89]. Mechanically evoked spiking of C-fibres in the skin-nerve preparation was reduced in Na v 1.7 KO mice [49].…”

“…Na V 1.8 was cloned in 1996 and its strong expression in medium and small sensory neurons (the sizes of most nociceptors) made it the best and most obvious target for analgesic drug development [2]. The Na V 1.8 knockout mouse was reported 3 years later and although it showed a pain deficit, its phenotype was compromised by a compensatory upregulation of Na v 1.7 [3] with clear functional consequences [3,89]. However, knockdown of Na V 1.8 by antisense oligonucleotides in adult rats inhibited neuropathic pain [75,129].…”

Reversal of Peripheral Neuropathic Pain by the Small-Molecule Natural Product Narirutin via Block of Nav1.7 Voltage-Gated Sodium Channel

“…As VTD specifically functions on the Na V channel, these data imply that Narirutin affects Na V rather than Ca V channels. Given that nociceptors and non-nociceptors respond to VTD with distinct response profiles [ 26 ], a VTD-based neuron classification assay helps us to better investigate the potential analgesic mechanism of Narirutin. Generally, VTD elicits four distinct response profiles in sensory neurons: oscillatory (OS), slow decay (SD), rapid decay (RD), and intermediate decay (ID) ( Figure 2 b).…”

Myocardial infarction reduces cardiac nociceptive neurotransmission through the vagal ganglia

“…In animals after MI, however, the same application of nociceptive chemicals affected the activity of a greater proportion (48%) of all cardiac neurons, and the predominant responses observed were inhibitory. It has also been previously shown that acute application of nociceptive chemicals, such as capsaicin, bradykinin ( 36 , 37 ), and veratridine ( 38 ), causes neuronal depolarization and predominantly increases in activity and firing rates of the majority of nociceptive neurons in the peripheral autonomic ganglia when evaluated either in vivo or using whole-cell voltage-clamp recordings ( 39 , 40 ). In vivo nodose recordings from guinea pigs with healthy hearts, which were consistent with the healthy porcine animal data in this study, showed that epicardial application of bradykinin activated the majority (74%) of identified cardiac nociceptive neurons, with a decrease in firing rate observed in only 26% of recorded neurons ( 22 ).…”