Activity-dependent silencing reveals functionally distinct itch-generating sensory neurons

Abstract

The peripheral terminals of primary sensory neurons detect histamine and non-histamine itch-provoking ligands through molecularly distinct transduction mechanisms. It remains unclear, however, whether these distinct pruritogens activate the same or different afferent fibers. Using a strategy of reversibly silencing specific subsets of murine pruritogen-sensitive sensory axons by targeted delivery of a charged sodium-channel blocker, we found that functional blockade of histamine itch did not affect the itch evoked by chloroquine or SLIGRL-NH2, and vice versa. Notably, blocking itch-generating fibers did not reduce pain-associated behavior. However, silencing TRPV1(+) or TRPA1(+) neurons allowed allyl isothiocyanate or capsaicin, respectively, to evoke itch, implying that certain peripheral afferents may normally indirectly inhibit algogens from eliciting itch. These findings support the presence of functionally distinct sets of itch-generating neurons and suggest that targeted silencing of activated sensory fibers may represent a clinically useful anti-pruritic therapeutic approach for histaminergic and non-histaminergic pruritus.

Figure 1. Application of pruritogens leads to a QX-314-mediated blockade of sodium currents selectively in pruritogen-sensitive trigeminal ganglion (TG) neurons

(a) Photomicrography (left) and representative traces (right) of changes in intracellular calcium concentration recorded from cultured trigeminal ganglia neurons at left. Red arrow indicates small trigeminal neuron that responded (red trace) to histamine (60 seconds, 100 μM). Blue arrow indicates cell that did not respond (blue trace) to application of histamine. Dotted line indicates the time point of the photomicrography frame. (b) Representative, normalized traces of sodium currents recorded from histamine-positive (left) and histamine-negative (right) neurons before (black) and 10 minutes after (green) application of 100 μM histamine together with 5 mM QX-314. (c) Time course of the changes in amplitude of peak sodium current. The specific TRPV1 antagonist capsazepine (20 μM) abolished the histamine/QX-314-mediated decrease in sodium current. Results are mean ± SEM of peak sodium current relative to control (n = 3 for each group. For statistical analysis see Supplementary Table 2). (d) Photomicrography (left) and representative traces (right, dotted line indicates the time point of the photomicrography frame) of changes in intracellular calcium concentration following application of chloroquine (60 seconds, 100 μM). (e) Representative, normalized traces of sodium current recorded from chloroquine-positive (left) and chloroquine-negative (right) neurons before (black) and 10 minutes after (green) application of 100 μM chloroquine together with 5 mM of QX-314. (f). Time course of changes in amplitude of peak sodium current. The specific TRPA1 antagonist HC-030031 (100 μM) abolished chloroquine/QX-314-mediated decrease in sodium current. Results are mean ± SEM of peak sodium current relative to control (n = 3 for each group. For statistical analysis see Supplementary Table 4).

Figure 2. Co-administration of QX-314 and pruritogens inhibits subsequent pruritogen-evoked scratching

(a, c, e) Sequential pruritogen cheek injections at 30-minute inter-stimulus intervals (ISI) evoke similar levels of cheek scratching (itch) bouts. (b, d, f) Pruritogen-evoked scratching is inhibited 30-minutes after conditioning injection of pruritogen together with 1% QX-314. (a) Conditioning injection: histamine (100 μg/20 μl), total scratching bouts in 30 minutes (TSB/30 minutes) = 64.3 ± 7.5 bouts. Test injection: histamine (100 μg/10 μl), TSB/30 minutes = 49.2 ± 9.7, P > 0.05, n = 6, degrees of freedom (d.f.) = 10. (b) Conditioning injection: histamine (100 μg/20 μl) + 1% QX-314, TSB/30 minutes = 56.0 ± 8.3. Test injection: histamine (100 μg/10 μl), TSB/30 minutes = 7.5 ± 3.8, P < 0.001, n = 6, d.f. = 10. (c) Conditioning injection: chloroquine (CQ, 50 μg/20 μl), TSB/30 minutes = 103.0 ± 19.1. Test injection: chloroquine (50 μg/10 μl), TSB/30 minutes = 109.0 ± 20.8, P > 0.05, n = 7, d.f. = 12. (d) Conditioning injection: chloroquine (50 μg/20 μl) + 1% QX-314, TSB/30 minutes = 110.0 ± 13.7. Test injection: chloroquine (50 μg/10 μl), TSB/30 minutes = 31.8 ± 13.3, P < 0.01, n = 6, d.f. = 10. (e) Conditioning injection: SLIGRL (50 μg/20 μl), TSB/30 minutes = 80.8 ± 9.6. Test injection: SLIGRL (50 μg/10 μl), TSB/30 minutes = 71.4 ± 12.0, P > 0.05, n=5, d.f. = 8. (f) Conditioning injection: SLIGRL (50 μg/20 μl) + 1% QX-314, TSB/30 minutes = 71.2 ± 16.1. Test injection: SLIGRL (50 μg/10 μl), TSB/30 minutes = 17.7 ± 8.0, P < 0.05, n = 6, d.f. = 10. Figure results are mean ± SEM of total scratching bouts per minute for 30 minutes after conditioning injection and test injection. Total scratching bouts in 30 minutes (TSB/30 minutes) are mean ± SEM of total scratching bouts in 30 minutes following injection. P-values represent comparison of value of mean total scratching bouts in 30 minutes evoked by test injection to those evoked by conditioning injection.

Figure 3. Silencing pruritogen-responsive neurons does not alter behavioral responses to non-itch stimuli

Forelimb wipes (pain-related behavior) evoked by (a) capsaicin (0.1%, 10 μl) and (b) AITC (0.15%, 10 μl) 30 minutes after cheek conditioning injections of vehicle or 1% QX-314 together with pruritogens (histamine, chloroquine (CQ)) or algogens (capsaicin, AITC). Hindpaw withdrawal to (c) punctate mechanical (von Frey) stimulus, (d) a radiant heat (52° C) stimulus, or (e) focally applied cold stimulus (see online methods) 30 minutes after intraplantar conditioning injections of vehicle or 1% QX-314 together with pruritogens or algogens. (f) Latency to bite, lick or attempt removal of an adhesive dot on the plantar surface of the hindpaw 30 minutes after intraplantar conditioning injections of vehicle, 1% QX-314 together with pruritogens or algogens was compared with a group receiving saline or intraplantar injection of 5% lidocaine (after 5 minutes). P-values represent comparison to vehicle (white column) value (not significant, P > 0.05; *, P < 0.05; ** P < 0.01). Error bars, SEM. n = 5–7 for all groups.

Figure 4. Distinct primary afferents mediate histaminergic itch and non-histaminergic itch

Pruritogen-evoked cheek scratching (itch) bouts 30 minutes after different conditioning injections indicated at the x-axes. (a) Intradermal test injection of histamine (100 μg/10 μl) alone, (b) chloroquine (CQ, 50 μg/10 μl) alone or (c) SLIGRL (50 μg/10 μl) alone 30 minutes after conditioning injection of vehicle or pruritogens together with 1% QX-314 P-values represent comparison to vehicle (white column) value (not significant, P > 0.05; *, P < 0.05; ** P < 0.01). Error bars, SEM. n = 5–7 for all groups.

Figure 5. Proportional representation of coincident trigeminal cell responses to low dose chloroquine and histamine, and their overlapping responsiveness with capsaicin and AITC

Venn diagram of calcium responses of 564 cultured trigeminal (TG) neurons to 10 μM histamine, 10 μM chloroquine (CQ), 1 μM capsaicin, and 100 μM AITC. (a) Histamine (10 μM) activated 23 of 564 TG neurons, while 17 responded to 10 μM chloroquine. Among histamine-activated cells 19 of 23 responded to histamine but not chloroquine, and 4 of 23 responded to both histamine and chloroquine. For TG cells responding to chloroquine 13 of 17 responded to chloroquine but not histamine. (b) Capsaicin activated 235 of 564 cells. Among histamine responsive cells, 16 of 23 also responded to capsaicin. (c) AITC activated 161 of 564 TG neurons, and more than half of chloroquine-responding cells (9 of 17) responded to AITC. (d) AITC activated 9 of 23 of histamine-responding cells. (e) Capsaicin activated 5 of 17 of chloroquine-responsive TG neurons.

Figure 6. Selective silencing of nociceptor populations differentially inhibits histamine itch and non-histamine Itch

Cheek scratching (itch) following intradermal test injection of (a) histamine (100 μg/10 μl) alone (n = 6), (b) chloroquine (CQ, 50 μg/10 μl) alone (n = 6–7), and (c) SLIGRL (50 μg/10 μl) (n = 5–6) 30 minutes after a conditioning injection of vehicle (0.9% NaCl, 20 μl) or 1% QX-314 together with capsaicin or AITC. P-values represent comparison to vehicle (white column) value (not significant, P > 0.05; *, P < 0.05; **, P < 0.01; *** P < 0.001). Error bars, SEM.

Figure 7. TRPV1⁺/TRPA1⁺ fibers suppress itch

(a) Hindlimb scratching (itch) evoked by capsaicin (0.1%, 10 μl) thirty-minutes after intradermal injection of vehicle (0.9% NaCl, 20 μl), capsaicin (0.1%) together with 1% QX-314 (20 μl) or AITC (0.15%) together with 1% QX-314 (20 μl). Note the significant increase in capsaicin-mediated scratching following application of AITC with QX-314. (b) Thirty-minutes after injection of vehicle (0.9% NaCl, 20 μl) intradermal cheek injection of AITC (0.15%, 10 μl) generated little hindlimb scratching over the next 30 minutes and this was not changed following a conditioning injection of AITC together with 1% QX-314. However, the AITC evoked scratching increased following intradermal cheek injection of capsaicin (0.1%) together with 1% QX-314 (20 μl). P-values represent comparison to vehicle value (not significant, P > 0.05; *, P < 0.05). Error bars, SEM. n = 6 for all groups.