Facial injections of pruritogens and algogens excite partly overlapping populations of primary and second-order trigeminal neurons in mice

Abstract

Intradermal cheek injection of pruitogens or algogens differentially elicits hindlimb scratching or forelimb wiping, suggesting that these behaviors distinguish between itch and pain. We studied whether pruritogens and algogens excite separate or overlapping populations of primary afferent and second-order trigeminal neurons in mice. Calcium imaging of primary sensory trigeminal ganglion (TG) cells showed that 15.4% responded to histamine, 5.8% to the protease-activated receptor (PAR)-2 agonist, 13.4% to allyl isothiocyanate (AITC), and 36.7% to capsaicin. AITC and/or capsaicin activated the vast majority of histamine- and PAR-2 agonist-sensitive TG cells. A chemical search strategy identified second-order neurons in trigeminal subnucleus caudalis (Vc) responsive to histamine, the PAR-2 agonist, or AITC. A minority of histamine or PAR-2 agonist-responsive Vc neurons responded to the other pruritogen, whereas a large majority of puritogen-responsive Vc neurons responded to capsaicin and/or AITC. A minority of AITC-responsive Vc neurons responded to pruritogens, whereas most responded to capsaicin. These data indicate that most primary and higher-order trigeminal sensory neurons are activated by both pruritic and algesic stimuli, although a minority exhibit selectivity. The results are discussed in terms of population codes for itch and pain that result in distinct behavioral responses of hindlimb scratching and forelimb wiping that are mediated at lumbar and cervical segmental levels, respectively.

Fig. 1.

Behavioral scratching and wiping elicited by cheek injection of pruritogens and algogens. Scatter plot shows mean numbers of scratch bouts and ipsilateral forelimb wipes (error bars: SE) elicited by each pruritic and/or algesic agent shown. Each chemical was injected intradermally in a volume of 10 μl. Doses (in nmol) were as follows: histamine, 272; protease-activated receptor (PAR)-2 agonist, 76; PAR-4 agonist, 73.4; chloroquine, 194; 5-HT, 47; capsaicin, 98.2; allyl isothiocyanate (AITC), 100.9; bradykinin, 1.0. The formalin dose was 16.7 μmol. For cowhage, a single spicule was inserted intradermally, left in for 5 s, and removed. Dashed line: equal numbers of wipes and scratch bouts. Data for histamine, PAR-2, and -4 agonists, 5-HT, capsaicin, AITC, bradykinin, and cowhage modified from Akiyama et al. 2010a.

Fig. 2.

Examples of 3 trigeminal ganglion (TG) cell responses to itch mediators. Graph plots 340/380 nm ratio as a function of time. Black bars indicate time of application of each indicated chemical.

Fig. 3.

Venn diagrams showing coincident responses of TG cells to pruritogens and algogens. A: TG cells selected based on their response(s) to AITC, histamine, and/or the PAR-2 agonist SLIGRL-NH2. Numbers within each sector indicate the percentage of cells that responded. B: as in A for cells responsive to capsaicin, histamine, and/or SLIGRL-NH2. C: as in B except that cells responsive to capsaicin and AITC are pooled.

Fig. 4.

Examples of trigeminal subnucleus caudalis (Vc) neurons identified by chemical search. A: unit identified by histamine search. Shown are peristimulus time histograms (PSTHs; bin width: 1 s) of unit responses to presentation (at arrow) of each indicates stimulus. This responded to intradermal injection of histamine in the ipsilateral cheek but not to any other stimulus except cheek pinch. Inset: recording site in superficial Vc on drawing of section through caudal medulla/C2. NPS, nucleus of solitary tract; PX, pyramidal decussation; Vc, trigeminal subnucleus caudalis. B: unit identified using SLIGRL-NH2 search. This unit was located in superficial Vc and responded to intradermal injection of SLIGRL-NH2, histamine, 5-HT, and AITC. C: unit identified by AITC search. This unit was located in superficial Vc and responded to AITC and briefly to histamine and capsaicin.

Fig. 5.

Averaged responses of Vc units isolated by chemical search strategy. A: histamine. Averaged PSTHs (bin width: 1 s) of 25 units isolated using histamine search strategy. Gray error bars: SE. Average firing following intradermal histamine (left PSTH) was significantly greater than prestimulus baseline out to 9 min after injection (P < 0.05, paired t-test). Right PSTHs show averaged responses of units that responded to indicated stimuli. Numbers in parentheses indicate the number that responded (>30% increase) of all units tested. The mean response of the 4 units to SLIGRL-NH2 1 min after injection did not reach statistically significant difference compared with preinjection baseline (P = 0.052). The same was true for the mean response of the 8 units to 5-HT (P = 0.053) and the 8 units to brush (P = 0.057). The mean response to pinch was significantly different compared with prestimulus baseline (P < 0.05). The mean response to capsaicin was significantly different from baseline at the 1st minute after injection (P < 0.05), whereas the mean response to AITC was significantly different out to the 6th minute after injection (P < 0.05 for minutes 1–6). The mean firing rate after saline injection was not significantly different from prestimulus baseline at any time (P > 0.5). Inset: histologically identified lesion sites (●) plotted on representative drawing of caudal medulla at the level of the pyramidal decussation. NTS, nucleus of the solitary tract; PX, pyramidal decussation. B: PAR-2 agonist SLIGRH-NH2 (format as in A). Mean response to SLIGRL-NH2 was significantly greater than prestimulus baseline out to minute 3 (P < 0.05). The mean response to histamine was significantly different for 1st minute after injection (P < 0.05). The mean response 1 min after 5-HT was not significantly different from baseline (P = 0.07). The mean brush-evoked response was significantly different from baseline (P < 0.01). The mean pinch-evoked response was significantly different from baseine (P < 0.001). The mean response to AITC was significantly different from baseline at minute 2 (P < 0.05) but not at minute 1 (P = 0.054) after injection. The mean response to capsaicin was significantly different from baseline at the 1st minute (P < 0.01). Mean responses to saline and Tween-80 were not significantly different from baseline (P > 0.4 for both). Inset: recording sites (format and abbreviations as in A). C: AITC (format as in A and B). Mean response to AITC significant at the 1st minute after injection (P < 0.05). The mean response to histamine was significantly different from baseline out to the 11th minute after injection (P < 0.05 for minutes 1–11). The mean responses of the few units to SLIGRL-NH2 or 5-HT were not significantly different from baseline (P > 0.3 for both). The mean response to brush was significantly greater than baseline (P < 0.01), as was the mean response to pinch (P < 0.05). The mean response to capsaicin was also not significantly different from baseline (P = 0.15). Neither mean responses to saline nor Tween-80 were significantly different from baseline (P > 0.5 for both). Inset: recording sites (format and abbreviations as in A).

Fig. 6.

Schematic diagram of partially overlapping itch- and pain-signaling pathways.