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.

Conflict of interest statement

The authors have no conflicts of interest to declare.

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

Figures

Figure 1.
Figure 1.
Assessments of the excitability of nociceptors and non-nociceptors based on veratridine-response profiles. Dorsal root ganglia (DRG) contain a heterogeneous population of sensory neurons. Nociceptors are typically small in size, express the Nav1.8 channel, and respond to nociceptive compounds (eg, ATP and capsaicin). Most nociceptors responded to veratridine with an oscillatory (OS), rapid decay (RD), or intermediate decay (ID) profiles. Non-nociceptors are typically large in size, do not express the Nav1.8 channel, and do not respond to nociceptive compounds. Most non-nociceptors respond to veratridine with a slow-decay (SD) profile. The percentages of the OS and SD veratridine profiles plus the percentage of veratridine-irresponsive (VTD-) neurons can be used for an efficient assay of the excitability DRG neurons. Changes in the SD population reflect changes in non-nociceptors (blue, typically 15%-20% of all neurons). Changes in the OS population (red, typically 30%-40% of all neurons) reflect changes in nociceptors. Changes in the veratridine-irresponsive population (gray, typically 30%-40% of all neurons) can reflect sensitisation of high-threshold and normally “silent” neurons. The 2 minor profiles, ID and RD, are mostly nociceptors but can be excluded from an assay for simplicity as both account for less than 5% to 10% of all neurons.
Figure 2.
Figure 2.
The OS population is reduced in mice lacking most nociceptors. (A) Representative images from control and 1.8-DTA cultures loaded with fura-2AM for imaging (contrast enhanced for both). Ablation of Nav1.8-expressing neurons in 1.8-DTA mouse leaves behind mostly non-nociceptive large neurons. Scale bar is 50 μm. (B) Example traces from our imaging protocol. Dashed lines indicate the periods of agonist application in recordings typically 25 to 35 minutes long. The 4 agonists and KCL are applied in the same order for all coverslips. The first row shows examples of neurons irresponsive to veratridine but respond to capsaicin and AITC, the second row shows neurons responding to veratridine and 2 nociceptive agonists, whereas the third row shows neurons responding to all 4 agonists: veratridine (VTD), capsaicin (CAP), α, β-methylene ATP (ATP), and allyl isothiocyanate (AITC). (C) In control DRG, 75% of neurons respond to one or more of the 3 nociceptive compounds and are classified as nociceptors (top) while only 8.3% do so in the 1.8-DTA confirming the loss of 89% of nociceptors. (D) Ablation of Nav1.8-expressing neurons decreases the percentage of veratridine-irresponsive neurons, decreases the percentage of OS neurons, and increases the percentage of SD neurons. VTD? CTR = 38.6 ± 3.1 vs VTD? DTA = 22.5 ± 2.6; SDCTR = 17.11 ± 2.8 vs SDDTA = 66.4 ± 1.4; OSCTR = 33.7 ± 2.1 vs OSDTA = 6.9 ± 1.9; IDCTR = 5.9 ± 1.1 vs IDDTA = 3.8 ± 1.5; RDCTR = 2.6 ± 0.9 vs RDDTA = 0.3 ± 0.2%. One-way ANOVA with Sidak''s test. Pie charts represent mean percentages in the histogram. (E) In control DRG, veratridine-irresponsive neurons can be nociceptors (yellow section, 26.4% of all neurons) or non-nociceptors (orange section, 11.6% of all neurons). Veratridine-irresponsive or “silent” nociceptors are almost completely lost in 1.8-DTA (become 0.8% of all neurons). Overall, there are less veratridine-irresponsive neurons in 1.8-DTA. Data for C–E are from 940 neurons from 3 control mice and 360 neurons from 4 1.8-DTA. ANOVA, analysis of variance; DRG, dorsal root ganglia.
Figure 3.
Figure 3.
Reference veratridine-response patterns for “safe” and “unsafe” analgesic drugs. (A) Deletion of Nav1.7 causes a major loss of pain without adverse CNS or motor effects. The veratridine-response pattern of the Nav1.7KO represents that of a safe and potent analgesic. Nav1.7 deletion leads to a decrease in responsiveness to veratridine (increase VTD? population) due to a decrease in the OS population but not the SD population. VTD? CTR = 51.0 ± 3.51 vs VTD? KO = 80.4 ± 2.8; SDCTR = 9.1 ± 1.9 vs SDKO = 7.5 ± 1; OSCTR = 28.2 ± 2.2 vs OSKO = 6.7 ± 1.3; IDCTR = 7.4 ± 0.4 vs IDKO = 3.2 ± 0.6; RDCTR = 4.0 ± 0.6 vs RDKO = 1.9 ± 0.5%. One-way ANOVA with Sidak''s test. Pie charts represent mean values in the histogram. Data from 1448 neurons from 6 floxed-control mice and 1630 neurons from 6 Nav1.7KO. (B) 300 nM of the Nav1.6 blocker 4,9TTX reduces responsiveness to veratridine (increases VTD?) through decreases in both the SD and OS populations. The decrease in SD is greater than that in OS. VTD? CTR = 41.6 ± 3.1 vs VTD? 4,9TTX = 68.0 ± 1.7; SDCTR = 20.68 ± 2.6 vs SD4,9TTX = 10.0 ± 1.5; OSCTR = 28.7 ± 1.8 vs OS4,9TTX = 17.6 ± 2.2; IDCTR = 5.4 ± 0.9 vs ID4,9TTX = 3.1 ± 0.5; RDCTR = 3.5 ± 1.1 vs RD4,9TTX = 1.4 ± 0.3%. One-way ANOVA with Sidak''s test. Pie charts represent mean values in the histogram. Data from 635 untreated and 927 treated neurons from 6 C57Bl6 mice. (C) The compensatory increase in Nav1.7 channels in nociceptors of the Nav1.8KO increases responsiveness to veratridine (decreases VTD?) due to an increase in the OS population. VTD? CTR = 55.4 ± 3 vs VTD? KO = 37.1 ± 3.2; SDCTR = 6.9 ± 1.7 vs SDKO = 9.1 ± 1.1, OSCTR = 26.7 ± 2.0 vs OSKO = 40.3 ± 2.7, IDCTR = 7.0 ± 0.5 vs IDKO = 7.65 ± 1.1, RDCTR = 3.6 ± 0.6 vs RDKO = 5.7 ± 0.5%. One-way ANOVA with Sidak''s test. Pie charts represent mean values in the histogram. Data from 1493 neurons from 5 littermate-control mice and 1028 neurons from 4 Nav1.8KO. ANOVA, analysis of variance.
Figure 4.
Figure 4.
Evaluating the effect of PF-04856264 and A-803467 on nociceptors and non-nociceptors. (A) 1 μM PF-048 increases the percentage of veratridine-irresponsive neurons through a reduction of the OS but not the other 3 populations. VTD? CTR = 45.9 ± 3 vs VTD? 1 PF = 58.1 ± 4; SDCTR = 16.9 ± 2 vs SD1PF = 13.6 ± 1.7; OSCTR = 26.2 ± 1.7 vs OS1PF = 16.8 ± 3; IDCTR = 7.5 ± 1.3 vs ID1PF = 7.0 ± 1.4; RDCTR = 4.1 ± 1.6 vs RD1PF = 3.6 ± 0.9%. One-way ANOVA with Sidak''s test. Pie charts represent mean values in the histogram. Data from 813 untreated and 681 treated neurons from 7 C57Bl6 mice. (B) 5 μM PF-048 increases the percentage of veratridine-irresponsive neurons through a reduction of both the OS and SD populations. 5 μM PF-048 is equally potent on the SD and OS profiles reducing both to about 50% of control values. VTD? CTR = 44.1 ± 2.7 vs VTD? 5 PF = 71.8 ± 1.5; SDCTR = 13.2 ± 1.6 vs SD1PF = 6.3 ± 1.0; OSCTR = 28.1 ± 2.4 vs OS5PF = 12.4 ± 1.6; IDCTR = 7.9 ± 1.6 vs ID5PF = 5.9 ± 1.1; RDCTR = 6.2 ± 1.8 vs RDPF = 3.8 ± 0.7%. One-way ANOVA with Sidak''s test. Pie charts represent mean values in the histogram. Data from 605 untreated and 338 treated neurons from 5 C57Bl6 mice. (C) 100 nM A-80 does not change the percentage of veratridine-responsive neurons but reduces the OS population by about 50%. VTD? CTR = 51 ± 4.9 vs VTD? 100A80 = 54.8 ± 4.9; SDCTR = 12.9 ± 3.2 vs SD100A80 = 14.4 ± 2; OSCTR = 25.1 ± 2.6 vs OS100A80 = 11.61 ± 2.6; IDCTR = 5.2 ± 1.3 vs ID100A80 = 10.7 ± 2.5; RDCTR = 6.6 ± 0.8 vs RD100A80 = 7.3 ± 1.6%. One-way ANOVA with Sidak''s test. Pie charts represent mean values in the histogram. Data from 607 untreated and 594 treated neurons from 6 C57Bl6 mice. (D) 300 nM A-80 increases the percentage of veratridine-irresponsive neurons through a reduction of both the OS and SD populations. The reduction in the OS population is slightly greater than that in the SD profile. VTD? CTR = 40.8 ± 2.3 vs VTD? 300A80 = 66.2 ± 2.4; SDCTR = 19.1 ± 1.7 vs SD300A80 = 11.5 ± 2; OSCTR = 28.0 ± 1.3 vs OS300A80 = 12.2 ± 1.2; IDCTR = 8.5 ± 1.5 vs ID300A80 = 6.8 ± 1.5; RDCTR = 4.8 ± 0.9 vs RD300A80 = 3.3 ± 0.7%. One-way ANOVA with Sidak''s test. Pie charts represent mean values in the histogram. Data from 1197 untreated and 1294 treated neurons from 7 C57Bl6 mice. ANOVA, analysis of variance.
Figure 5.
Figure 5.
The additive effects of a combination of PF-04856264 and A-803467 on nociceptors. (A) A combination of 1 μM PF-048 and 100 nM A-80 increases the percentage of veratridine-irresponsive neurons through a decrease of the OS population only. VTD? CTR = 40.8 ± 3.5 vs VTD? A80PF = 71.5 ± 3.7; SDCTR = 19.2 ± 2.5 vs SDA80PF = 12.2 ± 1.5; OSCTR = 30.4 ± 2.4 vs OSA80PF = 11.5 ± 2; IDCTR = 5.7 ± 0.8 vs IDA80PF = 2.4 ± 0.8; RDCTR = 3.7 ± 0.4 vs RDA80PF = 2.1 ± 1.1%. One-way ANOVA with Sidak''s test. Pie charts represent mean values in the histogram. Data from 980 untreated and 1114 treated neurons from 7 C57Bl6 mice. (B) Comparison of the changes in the OS and SD populations caused by VGSC blockers to those of the Nav1.7KO. The higher doses of PF and A80 and 4,9TTX caused a significant reduction in the SD population. Notice that the combined action of the lower doses of the PF and A80 produced the closest reduction of the OS population to Nav1.7 deletion. ANOVA, analysis of variance; VGSC, voltage-gated sodium channel.
Figure 6.
Figure 6.
Applications of the veratridine-based calcium assay. The assay is a very efficient method to characterise changes in a heterogeneous population of neurons and therefore has several applications. The assay can be used to identify lead analgesic drugs either by validating hits from cell line–based screens on all types of DRG neurons or identification of hits by a direct screen on DRG neurons. The assay can be used to assess how stem cell–derived neurons compare to primary neurons of the same type more efficiently than by patch clamping. The assay can be used to compare neurons derived from patients'' IPSC with known or unknown genetic mutations. The assay is suited to characterise pathologies that develop over time as in diabetes, aging, or cancer. Finally, the assay can be used to efficiently characterise changes in DRG from the large number of transgenic strains generated by phenotyping consortia. DRG, dorsal root ganglia.
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