Distinct Nav1.7-dependent pain sensations require different sets of sensory and sympathetic neurons

Abstract

Human acute and inflammatory pain requires the expression of voltage-gated sodium channel Nav1.7 but its significance for neuropathic pain is unknown. Here we show that Nav1.7 expression in different sets of mouse sensory and sympathetic neurons underlies distinct types of pain sensation. Ablating Nav1.7 gene (SCN9A) expression in all sensory neurons using Advillin-Cre abolishes mechanical pain, inflammatory pain and reflex withdrawal responses to heat. In contrast, heat-evoked pain is retained when SCN9A is deleted only in Nav1.8-positive nociceptors. Surprisingly, responses to the hotplate test, as well as neuropathic pain, are unaffected when SCN9A is deleted in all sensory neurons. However, deleting SCN9A in both sensory and sympathetic neurons abolishes these pain sensations and recapitulates the pain-free phenotype seen in humans with SCN9A loss-of-function mutations. These observations demonstrate an important role for Nav1.7 in sympathetic neurons in neuropathic pain, and provide possible insights into the mechanisms that underlie gain-of-function Nav1.7-dependent pain conditions.

Figure 1. Advillin and Wnt1 Cre expression pattern and pain behaviour of Advillin-Cre mice.

(a,b) Expression pattern of Cre activity. Arrow: trigeminal, arrowhead: DRG (scale bar 1 mm). X-gal staining of Advillin-Cre positive (c) DRG and (d) SCG sections, and Wnt1-Cre positive (e) DRG and (f) SCG sections (scale bar 100 μm). Acute nociceptive responses of heterozygous Advillin-Cre (orange columns) and littermate (white columns) mice (N shown as littermate/AdCre^+/−). (g) Motor coordination: Rotarod test (N=15/16). (h) Light touch: von Frey (N=15/16). (i) Mechanical pain: Randall–Selitto test (N=13/16). (j) Thermal spinal reflex: Hargreaves' test (N=15/16). (k) Noxious cooling: acetone test (N=10/11). (l) Supraspinal thermal: hotplate test at 50 and 55 °C (N=6/6). All behavioural data analysed by t-test. Results are presented as mean±s.e.m.

Figure 2. Advillin-Cre does not affect inflammatory or neuropathic pain behaviour.

Advillin-Cre^+/− (orange boxes/columns), littermates (white boxes/columns). (a,b) Behavioural responses of Advillin-Cre^+/− (N=12) wild-type littermate (N=8) mice following intraplantar injection of 20 μl of 5% formalin. (c) von Frey 50% threshold response of Advillin-Cre^+/− (N=9) and wild-type littermate (N=8) mice following intraplantar injection of 20 μl of complete Freund's adjuvant. (d) Hargreaves' test responses of Advillin-Cre^+/− mice (N=9) and wild-type littermate (N=8) mice following intraplantar injection of 20 μl of complete Freund's adjuvant. (e) Hargreaves' test response of Advillin-Cre^+/− mice (N=12) and wild-type littermates (N=10) mice following intraplantar injection of 20 μl of carrageenan. (f) von Frey 50% threshold response of Advillin-Cre^+/− mice (N=12) and wild-type littermates (N=10) mice following L5 SNT. All data analysed by two-way analysis of variance followed by the Bonferroni post hoc test. Results are presented as mean±s.e.m.

Figure 3. Nociceptive responses of different tissue-specific Nav17 KO mice.

Littermate (white columns), Nav1.7^Nav1.8 (blue columns), Nav1.7^Advill (red columns) and Nav1.7^Wnt1 (green columns), N shown as littermate/Nav1.7^Nav1.8/Nav1.7^Advill/Nav1.7^Wnt1. (a) Randall–Selitto test (noxious mechanical stimulus; N=23/6/13/5). (b) Acetone cooling test (noxious cooling stimulus; N=29/6/7/5). (c) Hargreaves' test (spinal noxious heat stimulus; N=22/6/7/7). (d) Hotplate test (supraspinal noxious heat stimulus; N=37/8/19/10): in vivo electrophysiological spinal cord recording from Nav1.7^Advill (red boxes) and littermate (white boxes) mice. (e) Thermally evoked WDR responses (N=6 per group). (f) Mechanically evoked WDR responses (N=6 per group). (g) Hotplate test following 6-OHDA treated (purple column, N=10) and untreated (white column, N=12) C57/Black6 mice. (h) Hotplate test of Nav1.7^Advill (red column, N=7) and littermate mice (white column, N=12) following chemical 6-OHDA treatment. (a–d) Data analysed by t-test, (e–h) data analysed by two-way analysis of variance followed by the Bonferroni post hoc test. Results are presented as mean±s.e.m. ***P<0.01 and ***P<0.001 (individual points).

Figure 4. Behavioural responses of different tissue-specific Nav1.7 and Nav1.8 KO mice to cooling and extreme cold.

N shown as littermate/Nav1.7^Wnt1. (a) Nav1.7^Wnt1 (green columns) and littermate (white columns) core (N=12/13) and skin temperature (N=12/9). (b) Behavioural response of Nav1.8 KO (turquoise column, N=5) and littermate (white column, N=7) mice to the acetone cooling test. (c) Behavioural response of Nav1.8 KO (turquoise columns, N=8) and littermate (white columns, N=6) thermal place preference test. (d–f) Littermate (white columns), Nav1.7^Nav1.8 (blue columns), Nav1.7^Advill (red columns), Nav1.7^Wnt1 (green columns) N shown as littermate/Nav1.7^Nav1.8/Nav1.7^Advill/Nav1.7^Wnt1. (d) Behavioural response of all three Nav1.7 tissue-specific knockout mice to thermal place preference test (N=15/8/8/8). (e) Behavioural response of all three Nav1.7 tissue-specific knockout mice to the von Frey test. (f) Behavioural response of all three Nav1.7 tissue-specific knockout mice to the Rotarod test (N=23/8/13/9). (a,b) Data analysed by t-test, (c–f) data analysed by two-way analysis of variance followed by the Bonferroni post hoc test. Results are presented as mean±s.e.m. **P<0.01 (individual points).

Figure 5. Reduced electrically evoked wind-up and substance P release in the spinal cord of Nav1.7^Advill mice.

Littermate (white columns/boxes), Nav1.7^Advill (red columns/boxes) N shown as littermate/Nav1.7^Advill. (a) TTX-sensitive and TTX-resistant inward sodium currents recorded from Nav1.7^Advill DRG neurons (N=11/6) Current–voltage relationships for sodium currents in littermate control (N=11) and Nav1.7^Advill DRG neurons (N=6) in the presence (b) or absence (c) of 500 nM TTX. (d) WDR spinal recording from Nav1.7^Advill and littermate mice in response to electrical stimulation of the sciatic nerve (N=6 per group). (e) Electrically evoked substance P release into the dorsal horn measured by radioimmunoassay in Nav1.7^Advill and littermate mice (N=7/8). (f) First and second phase responses observed in Nav1.7^Advill mice after intraplantar injection of 20 μl of 5% formalin (N=6/5). All data analysed by two-way analysis of variance followed by the Bonferroni post hoc test. Results are presented as mean±s.e.m. *P<0.05, **P<0.01 and ***P<0.001 (individual points).

Figure 6. Behavioural responses of Nav1.7^Advill and Nav1.7^Wnt1 mice following L5 SNT.

(a) Mechanical sensitization following L5 SNT of Nav1.7^Advill (red boxes, N=6) and littermate (white boxes, N=9) mice. (b) Mechanical sensitization following L5 SNT of Nav1.7^Wnt1 (green boxes, N=9) and littermate (white boxes, N=12) mice. (c) Mechanical sensitization in littermate mice treated with 6-OHDA (purple column, N=6) or vehicle (white column, N=6) 28 days after L5 SNT surgery. All analysed by two-way analysis of variance followed by the Bonferroni post hoc test. Results are presented as mean±s.e.m. *P<0.05, **P<0.01 and ***P<0.001 (individual points).

Figure 7. The neuron-specific role of Nav1.7 in different pain states.

Nav1.7 expressed in Nav1.8-positive sensory neurons (blue) is required for mechanical pain as well as inflammatory thermal and mechanical hyperalgesia. Nav1.7 expressed in Nav1.8-negative neurons (red) is required for thermal acute pain sensing (but not extreme cold, which is dependent on Nav1.8421). A contribution of both sensory and sympathetic (grey) Nav1.7-mediated signalling is required for neuropathic pain and responses to the hotplate test.