Figure 6. P-CTX-1 induces spontaneous action potential firing in peripheral sensory afferents.

(a) Effect of P-CTX-1 (0.5 nM) on single C-fibres recorded from a skin-saphenous nerve preparation in the mouse. Selective inhibitors of Na_V1.7 (Pn3a, 1 μM); Na_V1.6 (GIIIA, 10 μM), TTX-s Na_V isoforms (TTX, 1 μM) and Na_V1.8 (A803467, 10 μM) were sequentially perfused at the receptive field to assess cumulative effects of Na_V inhibition on action potential firing. Each data point represents the number of action potentials/5 min from a single C-fibre receptive field. (b) Representative recording of a C-fibre silenced by the selective Na_V1.7 inhibitor Pn3a (1 μM). (c) Representative recording of a C-fibre unaffected by Pn3a (1 μM), GIIIA (10 μM) and TTX (1 μM) and silenced by the Na_V1.8 inhibitor A803467 (10 μM). (d) Effect of P-CTX-1 (0.5 nM) on single A-fibres recorded from a skin-saphenous nerve preparation in the mouse. Selective inhibitors of either Na_V1.7 (Pn3a, 1 μM) or Na_V1.6 (GIIIA, 10 μM) almost completely silenced P-CTX-1-induced action potential firing in A-fibres. Each data point represents the number of action potentials/5 min from a single A-fibre receptive field. (e) Representative recording of an A-fibre silenced by Pn3a (1 μM) and (f) GIIIA (10 μM). (b,c and e,f) Each data point represents a single action potential and is plotted as a function of instantaneous frequency (1/s) to represent the time elapsed since the previous action potential. Arrows indicate time points of superfusion with compounds. (g) P-CTX-1 (1 nM) is a potent activator of colonic nociceptors (activating 14/19 afferents) and also recruits silent afferents (9/19 afferents). (h) Pre-incubation with the Na_V1.8 blocker A803467 (10 μM for 15 min) prevents P-CTX-1-induced firing of colonic nociceptors (7/7 afferents tested). Data are presented as mean ± SEM. Statistical significance was determined using (a) One-way ANOVA with Friedman post-hoc test or (d) student’s matched observations t-test and defined as p < 0.05 (*).